**Salmonellosis in Animals**

[37] Dupray E, Derrien A. Influence of the previous stay of *Escherichia coli* and Salmonella spp. in waste waters on their survival in seawater. *Water Research*. 1995;**4**: 1005–1011. [38] Mimouni R. Microbiological study of wastewater in Greater Agadir: Purification by infil‐ tration‐percolation, impact on the marine environment and reuse in agriculture. Thesis,

[39] Batt AL, Kim S, Aga DS. Comparison of the occurrence of antibiotics in four full‐scale wastewater treatment plants with varying designs and operations. *Chemosphere*. 2007;**68**:

[40] Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy M, Michael I, Fatta‐Kassinos D. Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. *Science of the Total Environment*. 2013;**447**:

[41] Duong HA, Pham NH, Nguyen HT, Hoang TT, Pham HV, Pham VC, Berg M, Giger W, Alder AC. Occurrence, fate and antibiotic resistance of fluoroquinolone antibacterials in hospital wastewaters in Hanoi, Vietnam. *Chemosphere*. 2008;**72**: 968–973. DOI: 10.1016/j.

[42] Novo A, André S, Viana P, Nunes OC, Manaia CM. Antibiotic resistance, antimicrobial residues and bacterial community composition in urban wastewater. *Water Research*.

[43] Adams C, Wang Y, Loftin K, Meyer M. Removal of antibiotics from surface and distilled water in conventional water treatment processes. *Journal of Environmental Engineering*.

[44] Czekalski N, Berthold T, Caucci S, Egli A, Bürgmann H. Increased levels of multiresis‐ tant bacteria and resistance genes after wastewater treatment and their dissemination into Lake Geneva, Switzerland. *Role and prevalence of antibiosis and the related resistance* 

[45] Karthikeyan K, Meyer MT. Occurrence of antibiotics in wastewater treatment facilities in Wisconsin, USA. *Science of the Total Environment*. 2006;**361**: 196–207. DOI: 10.1016/j.

[46] Chen S, Zhao S, White DG, Schroeder CM, Lu R, Yang H, McDermott PF, Ayers S, Meng J. Characterization of multiple‐antimicrobial‐resistant Salmonella serovars isolated from retail meats. *Applied and Environmental Microbiology*. 2004;**70**: 1–7. DOI: 10.1128/

[47] Brands DA, Inman AE, Gerba CP, Maré CJ, Billington SJ, Saif LA, Levine JF, Joens LA. Prevalence of Salmonella spp. in oysters in the United States. *Applied and Environmental* 

*Microbiology*. 2005;**71**: 893–897. DOI: 10.1128/AEM.71.2.893‐897.2005

University of Ibn Zohr, Faculty of Sciences. Agadir Morocco. 2004.

428–435. DOI: 10.1016/j.chemosphere.2007.01.008

345–360. DOI: 10.1016/j.scitotenv.2013.01.032

2013;**47**: 1875–1887. DOI: 10.1016/j.scitotenv.2013.01.032

2002;**128**: 253–260. DOI: 10.1061/(ASCE)0733‐9372(2002)

*genes in the environment*. 2007; **27**: 1–18.

scitotenv.2005.06.030

AEM.70.1.1‐7.2004

chemosphere.2008.03.009

148 Current Topics in Salmonella and Salmonellosis

## **Chapter 9**

**Provisional chapter**

## **Dynamics of** *Salmonella* **Infection**

**Salmonella**

Fathalla A. Rihan **Dynamics of**

Additional information is available at the end of the chapter Fathalla A. Rihan

http://dx.doi.org/10.5772/67284 Additional information is available at the end of the chapter

#### **Abstract**

In this chapter, we propose a mathematical epidemic model, with integer and fractional order to describe the dynamics of *Salmonella* infection in animal herds. We investigate the qualitative behaviors of such model and find the conditions that guarantee the asymptotic stability of disease-free and endemic steady states. To assess the severity of the outbreak, as well as the strength of the medical and/or behavioral interventions necessary for control, we estimate basic reproduction number R0. This threshold parameter specifies the average number of secondary infections caused by one infected individual during his/her entire infectious period at the start of an outbreak. We also provide an unconditionally stable implicit scheme for the fractional-order epidemic model. The theoretical and computational results give insight into the modelers and infectious disease specialists.

**Infection**

**Keywords:** basic reproduction number, *Salmonella* infection, SIRC epidemic model, stability

#### **1. Introduction**

Mathematical epidemic models, for *Salmonella* infections, provide a comprehensive framework for understanding the disease transmission behaviors and for evaluating the effectiveness of different intervention strategies [1, 2]. We recall here that the *Salmonella* infection, a major zoonotic disease, is transmitted between humans and other animals. Reports conducted by the National Center for Emerging and Zoonotic Infectious Diseases (NCEZID) revealed that the number of people infected by *Salmonella*, over the past few years, has remained increasing. The most commonly developed symptoms of *Salmonella* include diarrhea, fever, and abdominal cramps that appear 12–72 hours after infection. The infected people usually recover

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

without medical aid within a period of 4–7 days [3, 4]. However, hospitalization may be needed for some infected people in the case of severe diarrhea. *Salmonella* is found living in the intestinal tracts of not only humans but also other creatures such as birds. The transmission of bacterium to humans occurs through the ingestion of food that has been contaminated with animal feces. These contaminated foods are commonly from an animal source, such as beef, poultry, milk, or eggs [5]. However, vegetables and other foods may also become contaminated. Additionally, foods that have been contaminated are almost impossible to detect while eating, due to their normal taste and smell. Therefore, *Salmonella* is considered as a serious problem for the public health throughout the world. There are no doubts that mathematical modeling of *Salmonella* infection plays an important role in gaining understanding of the transmission of the disease in a specific environment and to predict the behavior of any outbreak. Furthermore, mathematical analysis leads to determining the nature of equilibrium states and to suggest recommended actions to be taken by decision makers to control the spreading of the disease. The objective of this work is to adopt the fractional-order epidemic model to describe the dynamics of *Salmonella* infections in animal herds.

Fractional-order (or free-order) differential models have been successfully applied to system biology, physics, chemistry, and biochemistry, hydrology, medicine, and finance (see, e.g., [6–12] and the references therein). In many cases, they are more contestant with the real phenomena than the integer-order models, because the fractional derivatives and integrals enable the description of the memory and hereditary properties inherent in various materials and processes. Hence, there is a growing need to study and use the fractional-order differential and integral equations in epidemiology and biological systems with memory [13]. However, analytical and closed solutions of these types of fractional equations cannot generally be obtained. As a consequence, approximate and numerical techniques are playing an important role in identifying the solution behavior of such fractional equations and exploring their applications (see, e.g., [14–16] and the references therein).

A large number of work done on modeling biological systems have been restricted to integer-order ordinary (or delay) differential equations (see, e.g., [17–22]). In Ref. [23], the authors proposed the classical *Susceptible-Infected-Recovered* (SIR) model. The authors in Ref. [24] introduced a new compartment into the SIR model, which is called cross-immune compartment to be called SIRC model. The added compartment cross-immune *C*ð*t*Þ describes an intermediate state between the fully susceptible *S*ð*t*Þ and the fully protected *R*ð*t*Þ one. A fractional-order SIRC model of influenza, a disease in human population, was discussed in Ref. [25]. In the present chapter, we consider the fractional-order SIRC model associated with evolution of *Salmonella* infection in animal herds. However, we will take into account the disease-induced mortality rate *m* in the model. Qualitative behavior of the fractional-order SRIC model is then investigated. Numerical simulations of the fractionalorder SRIC model are provided to demonstrate the effectiveness of the proposed method by using implicit Euler's method.

Definitions of fractional-order integration and fractional-order differentiation/integration are given in Appendix.

## **2. Construction of the model**

without medical aid within a period of 4–7 days [3, 4]. However, hospitalization may be needed for some infected people in the case of severe diarrhea. *Salmonella* is found living in the intestinal tracts of not only humans but also other creatures such as birds. The transmission of bacterium to humans occurs through the ingestion of food that has been contaminated with animal feces. These contaminated foods are commonly from an animal source, such as beef, poultry, milk, or eggs [5]. However, vegetables and other foods may also become contaminated. Additionally, foods that have been contaminated are almost impossible to detect while eating, due to their normal taste and smell. Therefore, *Salmonella* is considered as a serious problem for the public health throughout the world. There are no doubts that mathematical modeling of *Salmonella* infection plays an important role in gaining understanding of the transmission of the disease in a specific environment and to predict the behavior of any outbreak. Furthermore, mathematical analysis leads to determining the nature of equilibrium states and to suggest recommended actions to be taken by decision makers to control the spreading of the disease. The objective of this work is to adopt the fractional-order epidemic

Fractional-order (or free-order) differential models have been successfully applied to system biology, physics, chemistry, and biochemistry, hydrology, medicine, and finance (see, e.g., [6–12] and the references therein). In many cases, they are more contestant with the real phenomena than the integer-order models, because the fractional derivatives and integrals enable the description of the memory and hereditary properties inherent in various materials and processes. Hence, there is a growing need to study and use the fractional-order differential and integral equations in epidemiology and biological systems with memory [13]. However, analytical and closed solutions of these types of fractional equations cannot generally be obtained. As a consequence, approximate and numerical techniques are playing an important role in identifying the solution behavior of such fractional equations and exploring their applications (see, e.g., [14–16] and the references

A large number of work done on modeling biological systems have been restricted to integer-order ordinary (or delay) differential equations (see, e.g., [17–22]). In Ref. [23], the authors proposed the classical *Susceptible-Infected-Recovered* (SIR) model. The authors in Ref. [24] introduced a new compartment into the SIR model, which is called cross-immune compartment to be called SIRC model. The added compartment cross-immune *C*ð*t*Þ describes an intermediate state between the fully susceptible *S*ð*t*Þ and the fully protected *R*ð*t*Þ one. A fractional-order SIRC model of influenza, a disease in human population, was discussed in Ref. [25]. In the present chapter, we consider the fractional-order SIRC model associated with evolution of *Salmonella* infection in animal herds. However, we will take into account the disease-induced mortality rate *m* in the model. Qualitative behavior of the fractional-order SRIC model is then investigated. Numerical simulations of the fractionalorder SRIC model are provided to demonstrate the effectiveness of the proposed method by

Definitions of fractional-order integration and fractional-order differentiation/integration are

model to describe the dynamics of *Salmonella* infections in animal herds.

therein).

using implicit Euler's method.

152 Current Topics in Salmonella and Salmonellosis

given in Appendix.

Assume that the *Salmonella* infection spreads in animal herds which are grouped as four compartments, according to their infection status: *S*ð*t*Þ is the proportion of susceptible at time *t* (individuals that do not have the infection), *I*ð*t*Þ is the proportion of infected individuals (that have the infection), *R*ð*t*Þ is the proportion of recovered individuals (that recovered from the infection and have temporary immunity), and *C*ð*t*Þ is the proportion of cross-immune individuals at time *t*. The total number of animals in the herd is given by *N* ¼ *S* þ *I* þ *R* þ *C*. We consider that initially all the animals are susceptible to the infection. Once infected, a susceptible individual leaves the susceptible compartment and enters the infectious compartment where it then becomes infectious. The infected animals pass into the recovered compartment. After recovery from an infection animals, the individuals enter a new class *C*ð*t*Þ. Therefore, we consider the disease transmission model consists of nonnegative initial conditions together with system of equations.

$$\begin{aligned} \dot{S}(t) &= \mu N + \eta \mathbf{C}(t) - (\beta I(t) + \mu) S(t), \\ \dot{I}(t) &= \beta S(t)I(t) + \sigma \beta \mathbf{C}(t)I(t) - (\theta + m + \mu)I(t), \\ \dot{R}(t) &= (1 - \sigma)\beta \mathbf{C}(t)I(t) + \theta I(t) - (\mu + \delta)R(t), \\ \dot{\mathbf{C}}(t) &= \delta R(t) - \beta \mathbf{C}(t)I(t) - (\eta + \mu)\mathbf{C}(t). \end{aligned} \tag{1}$$

Here <sup>0</sup> *:* ′ <sup>¼</sup> *<sup>D</sup>* <sup>¼</sup> *<sup>d</sup> dt*. The parameter *μ* denotes the mortality rate in every compartment and is assumed to equal the rate of newborns in the population. *β* is the contact rate and also called the transmission rate for susceptible to be infected. *η*<sup>−</sup><sup>1</sup> is the cross-immune period, while *θ*<sup>−</sup><sup>1</sup> is the infectious period and *δ*<sup>−</sup><sup>1</sup> is the total immune period. *σ* represents the fraction of the exposed cross-immune individuals who are recruited in a unit time into the infective subpopulation [24, 26]. The presented model (1) differs from existing model, we assume a disease induced mortality rate *m*; see the diagram of **Figure 1**.

**Figure 1.** Schematic diagram of SIRC epidemic model for *Salmonella* infection.

#### **2.1. Fractional-order SIRC epidemic model**

Most of biological systems have long-range temporal memory. Modeling of such systems by fractional-order (or arbitrary order) models provides the systems with long-time memory and gains them extra degrees of freedom [27]. A large number of mathematical models, based on ordinary and delay differential equations with integer-orders, have been proposed in modeling the dynamics of epidemiological diseases [18, 20, 28, 29]. In recent years, it has turned out that many phenomena in different fields can be described very successfully by models using *fractionalorder differential equations* (FODEs) [13, 6, 27]. This is due to the fact that fractional derivatives enable the description of the memory and hereditary properties inherent in various processes. Herein, we replace the integer-order of the model (1) into a fractional-order (or free-order) and assume that *s*ð*t*Þ ¼ *S*ð*t*Þ*=N; i*ð*t*Þ ¼ *S*ð*t*Þ*=N; r*ð*t*Þ ¼ *R*ð*t*Þ*=N; c*ð*t*Þ ¼ *C*ð*t*Þ*=N*, where *N* is the total number of population. Then the model with a fractional-order *α* (0 *< α* ≤ 1) takes the form

$$\begin{aligned} D^a \mathbf{s}(t) &= \mu + \eta \mathbf{c}(t) - (\beta \mathbf{i}(t) + \mu) \mathbf{s}(t), \\ D^a \mathbf{i}(t) &= \beta \mathbf{s}(t) \mathbf{i}(t) + \sigma \beta \mathbf{c}(t) \mathbf{i}(t) - (\theta + m + \mu) \mathbf{i}(t), \\ D^a r(t) &= (1 - \sigma) \beta \mathbf{c}(t) \mathbf{i}(t) + \theta \mathbf{i}(t) - (\mu + \delta) r(t), \\ D^a \mathbf{c}(t) &= \delta r(t) - \beta \mathbf{c}(t) \mathbf{i}(t) - (\eta + \mu) \mathbf{c}(t). \end{aligned} \tag{2}$$

Here,

$$D^a f(t) = \frac{1}{\Gamma(n-a)} \left(\frac{d}{dt}\right)^n \int\_0^t (t-s)^{a-n-1} f(s)ds. \tag{3}$$

When 0 *< α* ≤ 1,

$$D^\alpha f(t) = \frac{1}{\Gamma(1-\alpha)} \int\_0^t \frac{f'(s)}{\left(t-s\right)^\alpha} ds. \tag{4}$$

(The initial conditions *s*ð0Þ ¼ *s*0, *i*ð0Þ ¼ *i*0, *r*ð0Þ ¼ *r*<sup>0</sup> should be given.) We note that the fractional derivatives involve an integration and are nonlocal operators, which can be used for modeling systems with memory; see the Appendix.

#### **2.2. Stability criteria for the epidemic SIRC model (2)**

To find the equilibria of the model (2), we put *<sup>D</sup><sup>α</sup>s*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>i*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>r*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>c*ð*t*Þ ¼ 0. We have disease-free (infection-free) equilibrium state E<sup>0</sup> and endemic equilibrium state Eþ:

$$\mathcal{E}\_0 = (1, 0, 0, 0) \text{ and } \mathcal{E}\_+ = (s^\*, i^\*, r^\*, c^\*), \tag{5}$$

where

$$\begin{split} s^\* &= \frac{\Theta + m + \mu}{\beta} - \sigma (\frac{\delta \Theta i^\*}{(\mu + \delta \sigma) \beta i^\* + (\mu + \delta)(\mu + \eta)}), \\ r^\* &= \frac{\Theta i^\* (\beta i^\* + \eta + \mu)}{(\mu + \delta \sigma) \beta i^\* + (\mu + \delta)(\mu + \eta)}, \\ c^\* &= \frac{\theta \delta i^\*}{(\mu + \delta \sigma) \beta i^\* + (\mu + \delta)(\mu + \eta)}. \end{split} \tag{6}$$

The positive endemic equilibrium E<sup>þ</sup> ¼ ð*s;i ;r;c*Þ satisfies Eq. (2) and *i* is the positive root of *A*1*i* <sup>2</sup> <sup>þ</sup> *<sup>A</sup>*2*<sup>i</sup>* þ *A*3, where

$$\begin{array}{l} A\_{1} = -\beta^{2} [m(\mu + \delta\sigma) + \mu(\theta + \mu + \delta\sigma)], \\ A\_{2} = \beta[\beta\mu(\mu + \delta\sigma) + \eta\theta\delta\gamma(\theta + m + \mu)](\mu + \delta)(\mu + \eta) + (\mu + \delta\sigma)] + \mu\delta\theta], \\ A\_{3} = \beta\mu(\mu + \delta)(\mu + \eta) \left[1 - \left(\frac{\theta + m + \mu}{\beta}\right)\right]. \end{array} \tag{7}$$

The Jacobian matrix of the model (2) is

gains them extra degrees of freedom [27]. A large number of mathematical models, based on ordinary and delay differential equations with integer-orders, have been proposed in modeling the dynamics of epidemiological diseases [18, 20, 28, 29]. In recent years, it has turned out that many phenomena in different fields can be described very successfully by models using *fractionalorder differential equations* (FODEs) [13, 6, 27]. This is due to the fact that fractional derivatives enable the description of the memory and hereditary properties inherent in various processes. Herein, we replace the integer-order of the model (1) into a fractional-order (or free-order) and assume that *s*ð*t*Þ ¼ *S*ð*t*Þ*=N; i*ð*t*Þ ¼ *S*ð*t*Þ*=N; r*ð*t*Þ ¼ *R*ð*t*Þ*=N; c*ð*t*Þ ¼ *C*ð*t*Þ*=N*, where *N* is the total number of popu-

lation. Then the model with a fractional-order *α* (0 *< α* ≤ 1) takes the form

*<sup>D</sup><sup>α</sup>f*ð*t*Þ ¼ <sup>1</sup>

Here,

where

*A*1*i*

<sup>2</sup> <sup>þ</sup> *<sup>A</sup>*2*<sup>i</sup>*

When 0 *< α* ≤ 1,

154 Current Topics in Salmonella and Salmonellosis

systems with memory; see the Appendix.

The positive endemic equilibrium E<sup>þ</sup> ¼ ð*s;i*

þ *A*3, where

**2.2. Stability criteria for the epidemic SIRC model (2)**

*<sup>s</sup>* <sup>¼</sup> *<sup>θ</sup>* <sup>þ</sup> *<sup>m</sup>* <sup>þ</sup> *<sup>μ</sup>*

*<sup>r</sup>* <sup>¼</sup> *<sup>θ</sup><sup>i</sup>*

ð*μ* þ *δσ*Þ*βi*

*<sup>c</sup>* <sup>¼</sup> *θδ<sup>i</sup>*

ð*μ* þ *δσ*Þ*βi*

*<sup>D</sup><sup>α</sup>s*ð*t*Þ ¼ *<sup>μ</sup>* <sup>þ</sup> *<sup>η</sup>c*ð*t*Þ−ð*βi*ð*t*Þ þ *<sup>μ</sup>*Þ*s*ð*t*Þ*;*

*<sup>D</sup><sup>α</sup>c*ð*t*Þ ¼ *<sup>δ</sup>r*ð*t*Þ−*βc*ð*t*Þ*i*ð*t*Þ−ð*<sup>η</sup>* <sup>þ</sup> *<sup>μ</sup>*Þ*c*ð*t*Þ*:*

*Γ*ð*n*−*α*Þ

*<sup>D</sup><sup>α</sup>f*ð*t*Þ ¼ <sup>1</sup>

*<sup>D</sup><sup>α</sup>i*ð*t*Þ ¼ *<sup>β</sup>s*ð*t*Þ*i*ð*t*Þ þ *σβc*ð*t*Þ*i*ð*t*Þ−ð*<sup>θ</sup>* <sup>þ</sup> *<sup>m</sup>* <sup>þ</sup> *<sup>μ</sup>*Þ*i*ð*t*Þ*; <sup>D</sup><sup>α</sup>r*ð*t*Þ¼ð1−*σ*Þ*βc*ð*t*Þ*i*ð*t*Þ þ *<sup>θ</sup>i*ð*t*Þ−ð*<sup>μ</sup>* <sup>þ</sup> *<sup>δ</sup>*Þ*r*ð*t*Þ*;*

> *d dt <sup>n</sup>*ð*<sup>t</sup>*

*Γ*ð1−*α*Þ

(The initial conditions *s*ð0Þ ¼ *s*0, *i*ð0Þ ¼ *i*0, *r*ð0Þ ¼ *r*<sup>0</sup> should be given.) We note that the fractional derivatives involve an integration and are nonlocal operators, which can be used for modeling

To find the equilibria of the model (2), we put *<sup>D</sup><sup>α</sup>s*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>i*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>r*ð*t*Þ ¼ *<sup>D</sup><sup>α</sup>c*ð*t*Þ ¼ 0. We have

 *;i ;r ;c* 

þ ð*μ* þ *δ*Þð*μ* þ *η*Þ

*;*

*:*

Þ*;*

disease-free (infection-free) equilibrium state E<sup>0</sup> and endemic equilibrium state Eþ:

E<sup>0</sup> ¼ ð1*;* 0*;* 0*;* 0Þ and E<sup>þ</sup> ¼ ð*s*

*<sup>β</sup>* <sup>−</sup>*σ*<sup>ð</sup> *δθ<sup>i</sup>*

 ð*βi*

ð*μ* þ *δσ*Þ*βi*

þ *η* þ *μ*Þ

þ ð*μ* þ *δ*Þð*μ* þ *η*Þ

þ ð*μ* þ *δ*Þð*μ* þ *η*Þ

*;r;c*Þ satisfies Eq. (2) and *i*

0 ð*t*−*s*Þ *α*−*n*−1

ð*t* 0 *f* ′ ð*s*Þ ð*t*−*s*Þ

(2)

(6)

*f*ð*s*Þ*ds:* (3)

*<sup>α</sup> ds:* (4)

Þ*;* (5)

is the positive root of

$$J = \begin{pmatrix} -\beta i(t) - \mu & -\beta s(t) & 0 & \eta \\ \beta i(t) & \beta s(t) + \sigma \beta c(t) - (\theta + m + \mu) & 0 & \sigma \beta i(t) \\ 0 & (1 - \sigma)\beta c(t) + \theta & -(\mu + \delta) & (1 - \sigma)\beta i(t) \\ 0 & -\beta c(t) & \delta & -\beta i(t) - (\eta + \mu) \end{pmatrix}. \tag{8}$$

#### **2.3. The reproduction number** R<sup>0</sup>

The basic reproduction number<sup>1</sup> R<sup>0</sup> that includes the indirect transmission may be obtained using next-generation matrix method [30]. The spectral radius of the next generation matrix <sup>ð</sup>*FV*<sup>−</sup><sup>1</sup> Þ, which is the dominant eigenvalue of the same matrix, gives the value of R0. Then, the basic reproductive number R<sup>0</sup> is obtained by the form

$$\mathcal{R}\_0 = \rho(FV^{-1}),\tag{9}$$

where the matrices *<sup>F</sup>* <sup>¼</sup> <sup>∂</sup>ℱ*i*ð*x*<sup>Þ</sup> ∂*xj* h i *x*¼*x*<sup>0</sup> and *<sup>V</sup>* <sup>¼</sup> <sup>∂</sup>V*i*ð*x*<sup>Þ</sup> ∂*xj* h i *x*¼*x*<sup>0</sup> . ℱ*i*ð*x*Þ, where *x* is the set of all diseasefree states in the compartment *i*, is the rate of appearance of new infections in the compartment *i*, and *Vi*ð*x*Þ is the net transfer rate (other than infections) of the compartment *i*. The net transfer rate is given by <sup>V</sup>*<sup>i</sup>* <sup>¼</sup> <sup>V</sup><sup>−</sup> *<sup>i</sup>* −V<sup>þ</sup> *<sup>i</sup> ;* where <sup>V</sup><sup>−</sup> *<sup>i</sup>* is the rate of transfer of individuals out of the compartment *i* and V<sup>þ</sup> *<sup>i</sup>* is the rate of transfer of individuals into the compartment *i* by all other means. Therefore, the disease transmission model consists of nonnegative initial conditions, *xi*ð0Þ, together with the following system of equations:

$$\mathbf{x}'\_{j} = f\_{j}(\mathbf{x}) \mathbf{e} \mathcal{F}\_{j}(\mathbf{x}) - \mathcal{V}\_{j}, \quad j \ge 1. \tag{10}$$

From the model (2), we have

$$\begin{aligned} F &= \begin{pmatrix} \frac{\partial \mathcal{F}\_1}{\partial \dot{t}(t)} & \frac{\partial \mathcal{F}\_1}{\partial r(t)} \\ \frac{\partial \mathcal{F}\_2}{\partial \dot{t}(t)} & \frac{\partial \mathcal{F}\_2}{\partial r(t)} \end{pmatrix} = \begin{pmatrix} \beta s & 0 \\ 0 & 0 \end{pmatrix}, \\\ V &= \begin{pmatrix} \frac{\partial \mathcal{V}\_1}{\partial \dot{t}(t)} & \frac{\partial \mathcal{V}\_1}{\partial r(t)} \\ \frac{\partial \mathcal{V}\_2}{\partial \dot{t}(t)} & \frac{\partial \mathcal{V}\_2}{\partial r(t)} \end{pmatrix} = \begin{pmatrix} \theta + m + \mu & 0 \\ -\theta & \mu + \delta \end{pmatrix}. \end{aligned} \tag{11}$$

Since we have only two distinct stages namely *I*ð*t*Þ and *R*ð*t*Þ; it follows that both *F* and *V* are 2 · 2 square matrices. Furthermore, it can be noticed that *F* is nonnegative and *V* is nonsingular. The

<sup>1</sup> The number of individuals infected by a single infected individual placed in a totally susceptible population.

basic reproductive number R<sup>0</sup> is the dominant eigenvalue of the matrix *FV*<sup>−</sup><sup>1</sup> , which is obtained by solving the characteristic equation <sup>ð</sup>*FV*<sup>−</sup><sup>1</sup> Þ*I*−*ΛI* ¼ 0 where *Λ* is the eigenvalue and *I*ð*t*Þ is the identity matrix. At the disease-free equilibrium, E<sup>0</sup> ¼ ð1*;* 0*;* 0*;* 0Þ, we have

$$\mathcal{R}\_0 = \frac{\beta}{\Theta + m + \mu}.\tag{12}$$

The following theorem states that R<sup>0</sup> is a threshold parameter for the stability of the model (2).

**Theorem 1** *The disease-free equilibrium is locally asymptotically stable and the infection will die out if* R<sup>0</sup> *<* 1 *and is unstable if* R<sup>0</sup> *>* 1*. Conversely, the endemic equilibrium* E<sup>þ</sup> *is stable when* R<sup>0</sup> *>* 1 *and*

$$a\_i > 0, i = 1, 2, 3, 4, \ a\_1 a\_2 \neg a\_3 > 0 \text{ and } \ a\_1 a\_2 a\_3 \neg a\_1^2 a\_4 \neg a\_3^2 > 0,\tag{13}$$

*where*

$$\begin{array}{l} a\_{1} = (D\_{1} + D\_{3} + D\_{5}), \\ a\_{2} = (D\_{1}D\_{3} - D\_{4}\delta + D\_{1}D\_{5} + D\_{3}D\_{5} + \beta^{2}i^{\*}s^{\*} + \sigma\beta^{2}c^{\*}i^{\*}), \\ a\_{3} = (D\_{1}D\_{3}D\_{5} - D\_{1}D\_{4}\delta + D\_{3}\beta^{2}i^{\*}s^{\*} + D\_{5}\beta^{2}i^{\*}s^{\*} + \beta^{2}c^{\*}\eta i^{\*} - D\_{2}\sigma\beta\delta i^{\*} + \\ \sigma\beta^{2}D\_{1}c^{\*}i^{\*} + \sigma D\_{3}\beta^{2}c^{\*}i^{\*}), \\ a\_{4} = D\_{3}D\_{5}\beta^{2}i^{\*}s^{\*} - D\_{2}\beta\delta\eta i^{\*} + D\_{3}\beta^{2}c^{\*}\eta i^{\*} - D\_{4}\beta^{2}\delta i^{\*}s^{\*} - \sigma\beta\delta D\_{1}D\_{2}i^{\*} + \sigma D\_{1}D\_{3}\beta^{2}c^{\*}i^{\*}, \end{array} \tag{14}$$

and

$$\begin{array}{l} D\_1 = \beta i + \mu, \\ D\_2 = (1 - \sigma)\beta c^\* + \theta, \\ D\_3 = (\mu + \delta), \\ D\_4 = (1 - \sigma)\beta i^\*, \\ D\_5 = \beta i^\* + (\eta + \mu), \\ D\_6 = \beta i^\* + \mu. \end{array} \tag{15}$$

Proof The disease-free equilibrium is locally asymptotically stable if all the eigenvalues, *λ<sup>i</sup> i* ¼ 1*;* 2*;* 3*;* 4*:* of the Jacobian matrix, *J*ðE0Þ satisfy the following condition

$$|\text{arg}(\lambda\_i)| > \frac{\alpha \pi}{2}.\tag{16}$$

where

$$f(\mathcal{E}\_0) = \begin{pmatrix} -\mu & -\beta & 0 & \eta \\ 0 & \beta \neg (\theta + m + \mu) & 0 & 0 \\ 0 & 0 & -(\mu + \delta) & 0 \\ 0 & 0 & \delta & -(\eta + \mu) \end{pmatrix}. \tag{17}$$

The eigenvalues of the Jacobian matrix *J*ðE0Þ are

$$
\lambda\_1 = -\mu, \lambda\_2 = \beta \text{--} (\theta + m + \mu), \lambda\_3 = -(\mu + \delta), \lambda\_4 = -(\eta + \mu). \tag{18}
$$

Hence E<sup>0</sup> is locally asymptotically stable if R<sup>0</sup> *<* 1 and is unstable if R<sup>0</sup> *>* 1.

Now, we extend the analysis to endemic equilibrium Eþ. The Jacobian matrix *J*ðEþÞ evaluated at the endemic equilibrium is

$$J(\mathcal{E}\_{+}) = \begin{pmatrix} -\beta i^\* - \mu & -\beta s^\* & 0 & \eta \\ \beta i^\* & \beta s^\* + \sigma \beta c^\* - (\theta + m + \mu) & 0 & \sigma \beta i^\* \\ 0 & (1 - \sigma) \beta c^\* + \theta & -(\mu + \delta) & (1 - \sigma) \beta i^\* \\ 0 & -\beta c^\* & \delta & -\beta i^\* - (\eta + \mu) \end{pmatrix},\tag{19}$$

with characteristic equation

basic reproductive number R<sup>0</sup> is the dominant eigenvalue of the matrix *FV*<sup>−</sup><sup>1</sup>

<sup>R</sup><sup>0</sup> <sup>¼</sup> *<sup>β</sup>*

*ai <sup>&</sup>gt;* <sup>0</sup>*; <sup>i</sup>* <sup>¼</sup> <sup>1</sup>*;* <sup>2</sup>*;* <sup>3</sup>*;* <sup>4</sup>*; <sup>a</sup>*1*a*2−*a*<sup>3</sup> *<sup>&</sup>gt;* <sup>0</sup>   and   *<sup>a</sup>*1*a*2*a*3−*a*<sup>2</sup>

*σβ*<sup>2</sup>*D*1*ci*

<sup>þ</sup> *<sup>D</sup>*3*β*<sup>2</sup>

The following theorem states that R<sup>0</sup> is a threshold parameter for the stability of the model (2). **Theorem 1** *The disease-free equilibrium is locally asymptotically stable and the infection will die out if* R<sup>0</sup> *<* 1 *and is unstable if* R<sup>0</sup> *>* 1*. Conversely, the endemic equilibrium* E<sup>þ</sup> *is stable when* R<sup>0</sup> *>* 1 *and*

> *i <sup>s</sup>* <sup>þ</sup> *σβ*<sup>2</sup>

<sup>þ</sup> *<sup>σ</sup>D*3*β*<sup>2</sup>

 *;*

þ ð*η* þ *μ*Þ*;*

*απ*

*λ*<sup>1</sup> ¼ −*μ; λ*<sup>2</sup> ¼ *β*−ð*θ* þ *m* þ *μ*Þ*; λ*<sup>3</sup> ¼ −ð*μ* þ *δ*Þ*; λ*<sup>4</sup> ¼ −ð*η* þ *μ*Þ*:* (18)

−*μ* −*β* 0 *η* 0 *β*−ð*θ* þ *m* þ *μ*Þ 0 0 0 0 −ð*μ* þ *δ*Þ 0 0 0 *δ* −ð*η* þ *μ*Þ

þ *μ:*

Proof The disease-free equilibrium is locally asymptotically stable if all the eigenvalues, *λ<sup>i</sup>*

jargð*λi*Þj *>*

*<sup>s</sup>* <sup>þ</sup> *<sup>D</sup>*5*β*<sup>2</sup>*<sup>i</sup>*

*cηi* −*D*4*β*<sup>2</sup> *δi* 

*D*<sup>1</sup> ¼ *βi* þ *μ; D*<sup>2</sup> ¼ ð1−*σ*Þ*βc* þ *θ; D*<sup>3</sup> ¼ ð*μ* þ *δ*Þ*; D*<sup>4</sup> ¼ ð1−*σ*Þ*βi*

*D*<sup>5</sup> ¼ *βi*

*D*<sup>5</sup> ¼ *βi*

*i* ¼ 1*;* 2*;* 3*;* 4*:* of the Jacobian matrix, *J*ðE0Þ satisfy the following condition

Hence E<sup>0</sup> is locally asymptotically stable if R<sup>0</sup> *<* 1 and is unstable if R<sup>0</sup> *>* 1.

*ci* Þ*;*

 −*D*2*σβδi* þ

*s*−*σβδD*1*D*2*i*

*<sup>s</sup>* <sup>þ</sup> *<sup>β</sup>*<sup>2</sup>*cη<sup>i</sup>*

*ci* Þ*;*

identity matrix. At the disease-free equilibrium, E<sup>0</sup> ¼ ð1*;* 0*;* 0*;* 0Þ, we have

by solving the characteristic equation <sup>ð</sup>*FV*<sup>−</sup><sup>1</sup>

156 Current Topics in Salmonella and Salmonellosis

*a*<sup>1</sup> ¼ ð*D*<sup>1</sup> þ *D*<sup>3</sup> þ *D*5Þ*;*

*<sup>a</sup>*<sup>4</sup> <sup>¼</sup> *<sup>D</sup>*3*D*5*β*<sup>2</sup>

*<sup>a</sup>*<sup>2</sup> ¼ ð*D*1*D*3−*D*4*<sup>δ</sup>* <sup>þ</sup> *<sup>D</sup>*1*D*<sup>5</sup> <sup>þ</sup> *<sup>D</sup>*3*D*<sup>5</sup> <sup>þ</sup> *<sup>β</sup>*<sup>2</sup>

*s*−*D*2*βδηi*

*<sup>a</sup>*<sup>3</sup> ¼ ð*D*1*D*3*D*5−*D*1*D*4*<sup>δ</sup>* <sup>þ</sup> *<sup>D</sup>*3*β*<sup>2</sup>*<sup>i</sup>*

*i* 

*J*ðE0Þ ¼

The eigenvalues of the Jacobian matrix *J*ðE0Þ are

0

BB@

*where*

and

where

, which is obtained

<sup>3</sup> *>* 0*;* (13)

*ci ;* (14)

(15)

Þ*I*−*ΛI* ¼ 0 where *Λ* is the eigenvalue and *I*ð*t*Þ is the

*<sup>θ</sup>* <sup>þ</sup> *<sup>m</sup>* <sup>þ</sup> *<sup>μ</sup> :* (12)

1*a*4−*a*<sup>2</sup>

<sup>þ</sup> *<sup>σ</sup>D*1*D*3*β*<sup>2</sup>

<sup>2</sup> *:* (16)

1

CCA*:* (17)

$$
\lambda^4 + a\_1 \lambda^3 + a\_2 \lambda^2 + a\_3 \lambda + a\_4 = 0. \tag{20}
$$

Using Routh-Hurwitz stability criteria [31], the endemic equilibrium E<sup>þ</sup> is locally asymptotically stable provided that

$$a\_i > 0, i = 1, 2, 3, 4, \ a\_1 a\_2 - a\_3 > 0 \quad \text{and} \quad a\_1 a\_2 a\_3 - a\_1^2 a\_4 - a\_3^2 > 0. \tag{21}$$

This completes the proof.

#### **3. Numerical method and simulations**

Since most of the FODEs do not have exact analytic solutions, so approximation and numerical techniques must be used. In addition, most of resulting biological systems are stiff,<sup>2</sup> therefore, efficient use of a reliable numerical method for dealing with such problems is necessary. In this section, we provide an implicit scheme to approximate the solutions of the fractional-order epidemic model. We also verify that the approximate solution is stable and convergent.

Consider a biological system, with fractional-order, of the form

$$\begin{aligned} D^a y(t) &= f(t, y(t)), \qquad &t \in [0, T], \\ y^{(k)}(0) &= y^{(k)}(0), \qquad &k = 0, 1, 2, \ldots, m - 1. \end{aligned} \tag{22}$$

Here, *<sup>y</sup>*ð*t*Þ¼½*y*1ð*t*Þ*;y*2ð*t*Þ*;*…*;yn*ð*t*Þ*<sup>T</sup>* and *<sup>f</sup>*ð*t;y*ð*t*ÞÞ satisfy the Lipschitz condition

$$\|f(t, y(t)) - f(t, \mathbf{x}(t))\| \le K \|y(t) - \mathbf{x}(t)\|, \quad K > 0,\tag{23}$$

where *x*ð*t*Þ is the solution of the perturbed system.

**Theorem 2** *The FODE (22) has a unique solution if Lipschitz condition (23) is satisfied and*

<sup>2</sup> One definition of the stiffness is that the global accuracy of the numerical solution is determined by stability rather than local error and implicit methods are more appropriate for it.

$$M = \frac{KT^{\alpha}}{\Gamma(\alpha + 1)} < 1. \tag{24}$$

Proof One can apply the fractional integral operator (given in the Appendix) to the differential Eq. (22) and incorporate the initial conditions. Thus, Eq. (22) can be expressed as

$$y(t) = \sum\_{k=0}^{m-1} y\_0^{(k)} \frac{t^k}{k!} + \frac{1}{\Gamma(\alpha)} \int\_0^t (t \cdot s)^{\alpha - 1} f(s, y(s)) ds. \tag{25}$$

which is a Volterra equation of the second kind. Define the operator ℒ, such that

$$\mathcal{R}y(t) = \sum\_{k=0}^{m-1} y\_0^{(k)} \frac{t^k}{k!} + \frac{1}{\Gamma(\alpha)} \int\_0^t (t \text{-s})^{\alpha - 1} f(s, y(s)) ds. \tag{26}$$

Then, we have

$$\begin{split} \|\|\mathcal{P}y(t) - \mathcal{P}x(t)\|\| &\leq \frac{1}{\Gamma(\alpha)} \int\_{0}^{t} \ell^{(t-s)\alpha - 1} \|f(s, y(s)) - f(s, x(s))\|\| ds \\ &\leq \frac{K}{\Gamma(\alpha)} \int\_{0}^{t} \ell^{(t-s)\alpha - 1} \sup\_{s \in [0, T]} |y(s) - x(s)| ds \\ &\leq \frac{K}{\Gamma(\alpha)} \|y - x\| \int\_{0}^{t} s^{\alpha - 1} ds \\ &\leq \frac{K\Gamma^{\alpha}}{\Gamma(\alpha + 1)} \|y - x\|\Gamma^{\alpha}. \end{split} \tag{27}$$

Then, we have

$$\|\mathcal{J}\mathcal{J}y(t) - \mathcal{J}\mathbf{x}(t)\| \le \mathbf{M} \|y - \mathbf{x}\|. \tag{28}$$

Using the Banach contraction principle, we can prove that that ℒ has a unique fixed point which means that the problem has a unique solution. □

Many efficient numerical methods have been proposed to solve the FODEs [14, 32]. Among them, the so-called predictor-corrector algorithm is a powerful technique for solving the FODEs, and considered as a generalization of the Adams-Bashforth-Moulton method. The modification of the Adams-Bashfourth-Moulton algorithm is proposed by Diethelm [14, 33– 34] to approximate the fractional-order derivative. However, the converted Volterra integral equation (25) is with a weakly singular kernel, such that regularization is not necessary anymore. In our case, the kernel may not be continuous, and therefore the classical numerical algorithms for the integral part of Eq. (25) are unable to handle the solution of Eq. (22). Therefore, we implement the implicit Euler's scheme to approximate the fractional-order derivative.

Given fractional-order model (Eq. (22)) and mesh points T ¼ *{t*0*;t*1*;*…*;tN}*, such that *t*<sup>0</sup> ¼ 0 and *tN* ¼ T. Then a discrete approximation to the fractional derivative can be obtained by a simple

quadrature formula, using the Caputo fractional derivative (42) of order *α*, 0 *< α* ≤ 1, and using *implicit* Euler's approximation as follows (see [15]):

$$\begin{split} D\_{\boldsymbol{x}}^{\boldsymbol{\alpha}}\boldsymbol{x}\_{i}(t\_{n}) &= \frac{1}{\Gamma(1-\alpha)} \Big[ \frac{d\boldsymbol{x}\_{i}(s)}{ds} \Big( \boldsymbol{s}^{(n-s)-\alpha} ds \\ \approx \frac{1}{\Gamma(1-\alpha)} \sum\_{j=1}^{n} \Big[ \int\_{(j-1)h}^{h} \left[ \frac{\boldsymbol{x}\_{i}^{j} - \boldsymbol{x}\_{i}^{j-1}}{h} + O(h) \right] \Big( \boldsymbol{n}^{n-s} \big) \, ds \\ = \frac{1}{(1-\alpha)\Gamma(1-\alpha)} \sum\_{j=1}^{n} \Big[ \frac{\boldsymbol{x}\_{i}^{j} - \boldsymbol{x}\_{i}^{j-1}}{h} + O(h) \Big] [(n-j+1)^{1-\alpha} - (n-j)^{1-\alpha}] \, \big|\, h^{1-\alpha} \\ = \frac{1}{(1-\alpha)\Gamma(1-\alpha)} \frac{1}{h^{\alpha}} \sum\_{j=1}^{n} [\boldsymbol{x}\_{i}^{j} - \boldsymbol{x}\_{i}^{j-1}][(n-j+1)^{1-\alpha} - (n-j)^{1-\alpha}] + \\ \frac{1}{(1-\alpha)\Gamma(1-\alpha)} \sum\_{j=1}^{n} [\boldsymbol{x}\_{i}^{j} - \boldsymbol{x}\_{i}^{j-1}][(n-j+1)^{1-\alpha} - (n-j)^{1-\alpha}]O(h^{2-\alpha}). \end{split} \tag{29}$$

Setting

*<sup>M</sup>* <sup>¼</sup> *KT<sup>α</sup> Γ*ð*α* þ 1Þ

Eq. (22) and incorporate the initial conditions. Thus, Eq. (22) can be expressed as

which is a Volterra equation of the second kind. Define the operator ℒ, such that

1 *Γ*ð*α*Þ

≤ *K Γ*ð*α*Þ

≤ *K Γ*ð*α*Þ

≤

ð*t* 0 ð *t*−*s*Þ*α*−1

ð*t* 0 ð

*K*T*<sup>α</sup> Γ*ð*α* þ 1Þ

∥*y*−*x*∥ ð*t* 0 *s α*−1 *ds*

Using the Banach contraction principle, we can prove that that ℒ has a unique fixed point

Many efficient numerical methods have been proposed to solve the FODEs [14, 32]. Among them, the so-called predictor-corrector algorithm is a powerful technique for solving the FODEs, and considered as a generalization of the Adams-Bashforth-Moulton method. The modification of the Adams-Bashfourth-Moulton algorithm is proposed by Diethelm [14, 33– 34] to approximate the fractional-order derivative. However, the converted Volterra integral equation (25) is with a weakly singular kernel, such that regularization is not necessary anymore. In our case, the kernel may not be continuous, and therefore the classical numerical algorithms for the integral part of Eq. (25) are unable to handle the solution of Eq. (22). Therefore, we implement the implicit Euler's scheme to approximate the fractional-order

Given fractional-order model (Eq. (22)) and mesh points T ¼ *{t*0*;t*1*;*…*;tN}*, such that *t*<sup>0</sup> ¼ 0 and *tN* ¼ T. Then a discrete approximation to the fractional derivative can be obtained by a simple

*<sup>y</sup>*ð*t*Þ ¼ <sup>X</sup>*<sup>m</sup>*−<sup>1</sup>

<sup>ℒ</sup>*y*ð*t*Þ ¼ <sup>X</sup>*<sup>m</sup>*−<sup>1</sup>

∥ℒ*y*ð*t*Þ−ℒ*x*ð*t*Þ∥ ≤

which means that the problem has a unique solution. □

Then, we have

158 Current Topics in Salmonella and Salmonellosis

Then, we have

derivative.

*k*¼0 *y* ð*k*Þ 0 *t k k*! þ 1 *Γ*ð*α*Þ

*k*¼0 *y* ð*k*Þ 0 *t k k*! þ 1 *Γ*ð*α*Þ

Proof One can apply the fractional integral operator (given in the Appendix) to the differential

ð*t* 0 ð*t*−*s*Þ *α*−1

ð*t* 0 ð*t*−*s*Þ *α*−1

*<sup>t</sup>*−*s*Þ*α*−<sup>1</sup> sup *s*∈½0*;*T

∥*y*−*x*∥T*<sup>α</sup>:*

∥*f*ð*s;y*ð*s*ÞÞ−*f*ð*s;x*ð*s*ÞÞ∥*ds*

j*y*ð*s*Þ−*x*ð*s*Þj*ds*

∥ℒ*y*ð*t*Þ−ℒ*x*ð*t*Þ∥ ≤ M∥*y*−*x*∥*:* (28)

*<* 1*:* (24)

*f*ð*s;y*ð*s*ÞÞ*ds:* (25)

*f*ð*s;y*ð*s*ÞÞ*ds::* (26)

(27)

$$\mathcal{G}(\alpha, h) = \frac{1}{(1 - \alpha)\Gamma(1 - \alpha)} \frac{1}{h^{\alpha}}, \text{and } \omega\_j^{\alpha} = j^{1 - \alpha} - (j - 1)^{1 - \alpha}, \quad (\text{where } \omega\_1^{\alpha} = 1), \tag{30}$$

then the first-order approximation method for the computation of Caputo's fractional derivative is then given by the expression

$$D\_\*^\alpha \mathbf{x}\_i(t\_n) = \mathcal{G}(\alpha, h) \sum\_{j=1}^n \omega\_j^\alpha (\mathbf{x}\_i^{n-j+1} - \mathbf{x}\_i^{n-j}) + \mathcal{O}(h). \tag{31}$$

From the above analysis and numerical approximation, one arrives at the following Remark.

**Remark 1** *The presence of a fractional differential order in a differential equation can lead to a notable increase in the complexity of the observed behavior, and the solution continuously depends on all the previous states.*

#### **3.1. Stability and convergence**

Here, we prove that the suggested numerical scheme of implicit difference approximation (Eq. (31)) is unconditionally stable. It follows then that the numerical solution converges to the exact solution as *h* ! 0.

In order to study the stability of the numerical method, let us consider a test problem of linear scaler fractional differential equation

$$D\_\*^
u u(t) = \rho\_0 \mu(t) + \rho\_1, \quad u(0) = u\_0. \tag{32}$$

such that 0 *< α* ≤ 1, and *ρ*<sup>0</sup> *<* 0, *ρ*<sup>1</sup> *>* 0 are constants.

**Theorem 3** *The fully implicit numerical approximation (31), to test problem (32) for all t*≥0*, is consistent and unconditionally stable.*

Proof We assume that the approximate solution of Eq. (32) is of the form *<sup>u</sup>*ð*tn*Þ≈*U<sup>n</sup>* ≡*ζn*, then Eq. (32) can be reduced to

$$\mathbb{P}\left(1-\frac{\rho\_0}{\mathbb{G}\_{a,h}}\right)\mathbb{\zeta}\_n = \mathbb{\zeta}\_{n-1} + \sum\_{j=2}^n w\_j^{(a)}(\mathbb{\zeta}\_{n-j} - \mathbb{\zeta}\_{n-j+1}) + \rho\_1/\mathbb{G}\_{a,h}, \qquad n \ge 2. \tag{33}$$

Or

$$\zeta\_{n-1} = \frac{\zeta\_{n-1} + \sum\_{j=2}^{n} \alpha\_j^{(a)} (\zeta\_{n-j} - \zeta\_{n-j+1}) + \rho\_1 / G\_{a,h}}{(1 - \frac{\rho\_0}{G\_{a,h}})}, \qquad n \ge 2. \tag{34}$$

Since 1− *<sup>ρ</sup>*<sup>0</sup> *Gα;<sup>h</sup>* ≥1 for all *<sup>G</sup><sup>α</sup>;<sup>h</sup>*, then

$$
\mathbb{Q}\_1 \lhd \mathbb{Q}\_0,\tag{35}
$$

$$
\zeta\_n \le \zeta\_{n-1} + \sum\_{j=2}^n \omega\_j^{(a)} (\zeta\_{n-j} - \zeta\_{n-j+1}), \qquad n \ge 2. \tag{36}
$$

Thus, for *n* ¼ 2, the above inequality implies

$$
\zeta\_2 \le \zeta\_1 + \omega\_2^{(\alpha)} (\zeta\_0 - \zeta\_1). \tag{37}
$$

Using the inequality (35) and the positivity of the coefficients *ω*2, one gets

$$
\mathbb{Q}\_2 \le \mathbb{Q}\_1. \tag{38}
$$

Repeating the process, we have from Eq. (36)

$$
\zeta\_n \le \zeta\_{n-1} + \sum\_{j=2}^n \omega\_j^{(a)} (\zeta\_{n-j} - \zeta\_{n-j+1}) \le \zeta\_{n-1}.\tag{39}
$$

Since each term in the summation is negative. Thus *ζ<sup>n</sup>* ≤ *ζn*−<sup>1</sup> ≤ *ζn*−<sup>2</sup> ≤ …≤ *ζ*0. With the assumption that *<sup>ζ</sup><sup>n</sup>* ¼ j*U<sup>n</sup>* <sup>j</sup> <sup>≤</sup> *<sup>ζ</sup>*<sup>0</sup> ¼ j*U*<sup>0</sup> <sup>j</sup>*;* which entails <sup>∥</sup>*U<sup>n</sup>* ∥ ≤ ∥*U*0∥ and we have stability.

The above numerical technique can then be used both for both linear and nonlinear problems, and it may be extended to multiterm FODEs.

#### **3.2. Numerical simulations**

The approximate solutions of epidemic model (2) are displayed in **Figures 2**–**4**, and sensitivity of R<sup>0</sup> to transmission coefficients is displayed in **Figure 5**. The numerical simulations are performed by Euler's implicit scheme discussed in Section 3. We choose different fractionalorder values (0*:*5 *< α <* 1), and parameter values given in **Table 1**. The displayed solutions in **Figure 4** confirm that the fractional order of the derivative plays the role of time-delay (or memory) in the system.

**Theorem 3** *The fully implicit numerical approximation (31), to test problem (32) for all t*≥0*, is*

≡*ζn*, then

*<sup>j</sup>* ð*ζn*−*j*−*ζn*−*j*þ<sup>1</sup>Þ þ *ρ*1*=G<sup>α</sup>;<sup>h</sup> ; n* ≥ 2*:* (33)

<sup>Þ</sup> *; <sup>n</sup>* <sup>≥</sup> <sup>2</sup>*:* (34)

*ζ*<sup>1</sup> ≤ *ζ*0*;* (35)

*<sup>j</sup>* ð*ζn*−*j*−*ζn*−*j*þ<sup>1</sup>Þ*; n* ≥ 2*:* (36)

<sup>2</sup> ð*ζ*0−*ζ*1Þ*:* (37)

*ζ*<sup>2</sup> ≤ *ζ*1*:* (38)

*<sup>j</sup>* ð*ζn*−*j*−*ζn*−*j*þ<sup>1</sup>Þ ≤ *ζn*−1*:* (39)

∥ ≤ ∥*U*0∥ and we have stability.

Proof We assume that the approximate solution of Eq. (32) is of the form *<sup>u</sup>*ð*tn*Þ≈*U<sup>n</sup>*

*ω*<sup>ð</sup>*α*<sup>Þ</sup>

<sup>ð</sup>1<sup>−</sup> *<sup>ρ</sup>*<sup>0</sup> *Gα;<sup>h</sup>*

*<sup>j</sup>* ð*ζn*−*j*−*ζn*−*j*þ<sup>1</sup>Þ þ *ρ*1*=G<sup>α</sup>;<sup>h</sup>*

*j*¼2

*ω*<sup>ð</sup>*α*<sup>Þ</sup>

*j*¼2

Using the inequality (35) and the positivity of the coefficients *ω*2, one gets

*<sup>ζ</sup><sup>n</sup>* <sup>≤</sup> *<sup>ζ</sup>n*−<sup>1</sup> <sup>þ</sup>X*<sup>n</sup>*

*j*¼2

<sup>j</sup>*;* which entails <sup>∥</sup>*U<sup>n</sup>*

*ω*<sup>ð</sup>*α*<sup>Þ</sup>

Since each term in the summation is negative. Thus *ζ<sup>n</sup>* ≤ *ζn*−<sup>1</sup> ≤ *ζn*−<sup>2</sup> ≤ …≤ *ζ*0. With the assump-

The above numerical technique can then be used both for both linear and nonlinear problems,

The approximate solutions of epidemic model (2) are displayed in **Figures 2**–**4**, and sensitivity of R<sup>0</sup> to transmission coefficients is displayed in **Figure 5**. The numerical simulations are

*ω*<sup>ð</sup>*α*<sup>Þ</sup>

*<sup>ζ</sup>*<sup>2</sup> <sup>≤</sup> *<sup>ζ</sup>*<sup>1</sup> <sup>þ</sup> *<sup>ω</sup>*<sup>ð</sup>*α*<sup>Þ</sup>

*<sup>ζ</sup><sup>n</sup>* <sup>¼</sup> *<sup>ζ</sup>n*−<sup>1</sup> <sup>þ</sup>X*<sup>n</sup>*

*<sup>ζ</sup>n*−<sup>1</sup> <sup>þ</sup>X*<sup>n</sup>*

*j*¼2

*<sup>ζ</sup><sup>n</sup>* <sup>≤</sup> *<sup>ζ</sup>n*−<sup>1</sup> <sup>þ</sup>X*<sup>n</sup>*

*consistent and unconditionally stable.*

160 Current Topics in Salmonella and Salmonellosis

<sup>1</sup><sup>−</sup> *<sup>ρ</sup>*<sup>0</sup> *G<sup>α</sup>;<sup>h</sup>* 

*ζ<sup>n</sup>* ¼

≥1 for all *G<sup>α</sup>;<sup>h</sup>*, then

Thus, for *n* ¼ 2, the above inequality implies

Repeating the process, we have from Eq. (36)

and it may be extended to multiterm FODEs.

tion that *<sup>ζ</sup><sup>n</sup>* ¼ j*U<sup>n</sup>*<sup>j</sup> <sup>≤</sup> *<sup>ζ</sup>*<sup>0</sup> ¼ j*U*<sup>0</sup>

**3.2. Numerical simulations**

Eq. (32) can be reduced to

Or

Since 1− *<sup>ρ</sup>*<sup>0</sup>

*Gα;<sup>h</sup>* 

**Figure 2.** Numerical simulation of the fractional-order epidemic model (2), when *α* ¼ 0*:*8, and R<sup>0</sup> *>* 1 (Each infected individual infects more than one other member of the population and a self-sustaining group of infectious individuals will propagate), with parameter values of **Table 1**.

**Figure 3.** Phase plane portrait for the fractional-order endemic model (2), in absence of *C*ð*t*Þ and *R*ð*t*Þ components, when *α* ¼ 0*:*7 (left) and *α* ¼ 0*:*9 (right) with R<sup>0</sup> ¼ 0*:*5 *<* 1. We note that solution paths approach the disease-free equilibrium E<sup>0</sup> ¼ ð1*;* 0*;* 0Þ.

**Figure 4.** Phase plane portrait for the classic fractional-order endemic model (2) when *α* ¼ 1 (left) and *α* ¼ 0*:*9 (right) with R<sup>0</sup> ¼ 1*:*2 *>* 1. We note that solution paths approach the endemic equilibrium E<sup>þ</sup> given by Eq. (5).

**Figure 5.** Sensitivity of R<sup>0</sup> with respect to the transmission coefficients *β* and *θ*.


**Table 1.** List of parameters.

## **4. Discussion and conclusion**

In this chapter, we provided a fractional-order SIRC epidemic model with *Salmonella* infection. The model provides a comprehensive framework for understanding the disease transmission behaviors, as well as for evaluating the effectiveness of different intervention strategies. We derived the sufficient conditions to preserve the asymptotical stability of disease-free and endemic steady states. The threshold parameter (reproduction number) R<sup>0</sup> has been evaluated in terms of contact rate, recovery rate, and other parameters in the model. The threshold parameter R<sup>0</sup> is very sensitive to transmission coefficients *β* and *θ* that reflects that these parameters play an important role to assess the strength of the medical and behavioral interventions necessary for control. We provided an unconditionally stable method, using Euler's implicit method for the fractional-order differential system. The solution of a fractional-order model at any time *t* continuously depends on all the previous states at *t* ≤ *t* .

It has been found that fractional-order dynamical models are more suitable to model biological systems with memory than their integer-orders. The presence of a fractional differential order into a corresponding differential equation leads to a notable increase in the complexity of the observed behavior. However, fractional-order differential models are as stable as their integerorder counterpart.

## **Acknowledgements**

This work was supported by UAE University (NRF Project UAEU-NRF-7-20886).

#### **Appendix**

**Figure 4.** Phase plane portrait for the classic fractional-order endemic model (2) when *α* ¼ 1 (left) and *α* ¼ 0*:*9 (right) with

**Parameter Description Value Reference** *μ* Replacement and exit rate (day<sup>−</sup>1) 0.011 [35] *β* Transmission rate of susceptible to be infected (animal<sup>−</sup><sup>1</sup> day<sup>−</sup>1) 0.15 [35] *θ* Recovery rate of infected animals day<sup>−</sup><sup>1</sup> 0.16 Assumed *m* Disease-induced mortality rate (day<sup>−</sup>1) 0.041 Assumed *η* Cross-immune period 0.5 [36] *σ* The average reinfection probability of *C*ð*t*Þ 0.06 Assumed *δ* The average time of appearance of new dominant clusters 1 Assumed *N* The total number of population 345 Assumed

R<sup>0</sup> ¼ 1*:*2 *>* 1. We note that solution paths approach the endemic equilibrium E<sup>þ</sup> given by Eq. (5).

162 Current Topics in Salmonella and Salmonellosis

**Figure 5.** Sensitivity of R<sup>0</sup> with respect to the transmission coefficients *β* and *θ*.

**Table 1.** List of parameters.

Let *<sup>L</sup>*<sup>1</sup> <sup>¼</sup> *<sup>L</sup>*<sup>1</sup> ½*a;b* be the class of Lebesgue integrable functions on ½*a;b*, *a < b < ∞*.

**Definition 1** *The fractional integral of order β*∈ ℝ<sup>þ</sup> *of the function f*ð*t*Þ, *t >* 0 *(f* : ℝ<sup>þ</sup> ! ℝ*) is defined by*

$$I\_a^\nu f(t) = \int\_a^t \frac{\left(t \because \right)^{\nu - 1}}{\Gamma(\nu)} f(s) ds, \quad t > 0. \tag{40}$$

*The fractional derivative of order α*∈ ð*n*−1*;n*Þ *of f*ð*t*Þ *is defined by two ways:*


$$D\_a^a f(t) = D\_a^n I\_a^{n-a} f(t), \quad D\_\*^n = \frac{d^n}{dt^n}, \quad n = 1, 2, \dots \tag{41}$$

$$D\_a^a f(t) = I\_a^{n-a} D\_a^n f(t), \quad n = 1, 2, \dots \tag{42}$$

We notice that the definition of time-fractional derivative of a function *f*ð*t*Þ at *t* ¼ *tn* involves an integration and calculating time-fractional derivative that requires all the past history, i*:*e*:*, all the values of *f*ð*t*Þ from *t* ¼ 0 to *t* ¼ *tn*. Caputo's definition, which is a modification of the Riemann-Liouville definition, has the advantage of dealing properly with initial value problems. The following Remark addresses some of the main properties of the fractional derivatives and integrals (see [12, 36–39]).

**Remark 2** *Let ν;γ*∈ ℝ<sup>þ</sup> *and α*∈ ð0*;* 1Þ*. Then*

**i.** *If I<sup>ν</sup> <sup>a</sup>* : *<sup>L</sup>*<sup>1</sup> ! *<sup>L</sup>*<sup>1</sup> *and f*ð*t*Þ<sup>∈</sup> *<sup>L</sup>*<sup>1</sup> *, then I<sup>ν</sup> a I γ <sup>a</sup> f*ð*t*Þ ¼ *I ν*þ*γ <sup>a</sup> f*ð*t*Þ*;*


**iv.** *If f*ð*t*<sup>Þ</sup> *is absolutely continuous on* <sup>½</sup>*a;b, then* lim*<sup>α</sup>*!<sup>1</sup>*D<sup>α</sup> <sup>f</sup>*ð*t*Þ ¼ *df*ð*t*<sup>Þ</sup> *dt ;*

**v.** *Thus D<sup>α</sup> <sup>f</sup>*ð*t*Þ ¼ *<sup>d</sup> dt I* 1−*α <sup>f</sup>*ð*t*<sup>Þ</sup> *(Riemann-Liouville sense) and D<sup>α</sup> <sup>f</sup>*ð*t*Þ ¼ *<sup>I</sup>* 1−*α <sup>d</sup> dt f*ð*t*Þ *(Caputo sense).*

The generalized mean value theorem and another property are defined in the following Remark [40].

#### **Remark 3**

**i.** *Suppose f*ð*t*Þ<sup>∈</sup> *<sup>C</sup>*½*a;b and D<sup>α</sup> <sup>f</sup>*ð*t*Þ<sup>∈</sup> *<sup>C</sup>*ð*a;b for* <sup>0</sup> *<sup>&</sup>lt; <sup>α</sup>* <sup>≤</sup> <sup>1</sup>*, then we have*

$$f(t) = f(a) + \frac{1}{\Gamma(\alpha)} D\_\*^a f(\xi) (t \cdot a)^a, \quad \text{with } a < \xi < t \quad \forall \ t \in (a, b]. \tag{43}$$

**ii.** *If (i) holds, and D<sup>α</sup> <sup>f</sup>*ð*t*Þ≥<sup>0</sup> <sup>∀</sup> *<sup>t</sup>*<sup>∈</sup> <sup>½</sup>*a;b, then f*ð*t*<sup>Þ</sup> *is nondecreasing for each t* <sup>∈</sup>½*a;b. If D<sup>α</sup> <sup>f</sup>*ð*t*<sup>Þ</sup> <sup>≤</sup> <sup>0</sup> ∀ *t* ∈½*a;b, then f*ð*t*Þ *is nonincreasing for each t* ∈ ½*a;b*.

## **Author details**

Fathalla A. Rihan

Address all correspondence to: frihan@uaeu.ac.ae

Department of Mathematical Sciences, College of Science, UAE University, Al Ain, United Arab Emirates

## **References**

[1] L. Qin, S. X. Yang, A.-H. Meng, A mathematical model with degree of risk for Salmonella infections, IEEE International Conference on Systems, Man and Cybernetics, Montreal, Que., 2007, pp. 2704–2709.

[2] F. A. Rihan, D. Baleanu, S. Lakshmanan, R. Rakkiyappan, On fractional SIRC model with Salmonella bacterial infection, Abstr. Appl. Anal. 2014 (2014) 1–9.

We notice that the definition of time-fractional derivative of a function *f*ð*t*Þ at *t* ¼ *tn* involves an integration and calculating time-fractional derivative that requires all the past history, i*:*e*:*, all the values of *f*ð*t*Þ from *t* ¼ 0 to *t* ¼ *tn*. Caputo's definition, which is a modification of the Riemann-Liouville definition, has the advantage of dealing properly with initial value problems. The following Remark addresses some of the main properties of the fractional deriva-

> *ν*þ*γ <sup>a</sup> f*ð*t*Þ*;*

> > *<sup>a</sup> f*ð*t*Þ ¼

1−*α <sup>d</sup>*

*;* with *a < ξ < t* ∀ *t*∈ ð*a;b:* (43)

 *<sup>f</sup>*ð*t*Þ ¼ *df*ð*t*<sup>Þ</sup> *dt ;*

*<sup>f</sup>*ð*t*Þ ¼ *<sup>I</sup>*

ð*t* 0 *f*ð*s*Þ*ds;*

*dt f*ð*t*Þ *(Caputo sense).*

*<sup>f</sup>*ð*t*<sup>Þ</sup> <sup>≤</sup> <sup>0</sup>

*<sup>a</sup> <sup>f</sup>*ð*t*<sup>Þ</sup> *uniformly on* <sup>½</sup>*a;b*, *<sup>n</sup>* <sup>¼</sup> <sup>1</sup>*;* <sup>2</sup>*;* <sup>3</sup>*;*…*, where I*<sup>1</sup>

*<sup>f</sup>*ð*t*<sup>Þ</sup> *(Riemann-Liouville sense) and D<sup>α</sup>*

*<sup>f</sup>*ð*ξ*Þð*t*−*a*<sup>Þ</sup>

The generalized mean value theorem and another property are defined in the following

*<sup>f</sup>*ð*t*Þ<sup>∈</sup> *<sup>C</sup>*ð*a;b for* <sup>0</sup> *<sup>&</sup>lt; <sup>α</sup>* <sup>≤</sup> <sup>1</sup>*, then we have*

*<sup>f</sup>*ð*t*Þ≥<sup>0</sup> <sup>∀</sup> *<sup>t</sup>*<sup>∈</sup> <sup>½</sup>*a;b, then f*ð*t*<sup>Þ</sup> *is nondecreasing for each t* <sup>∈</sup>½*a;b. If D<sup>α</sup>*

*α*

Department of Mathematical Sciences, College of Science, UAE University, Al Ain, United

[1] L. Qin, S. X. Yang, A.-H. Meng, A mathematical model with degree of risk for Salmonella infections, IEEE International Conference on Systems, Man and Cybernetics, Montreal,

tives and integrals (see [12, 36–39]).

164 Current Topics in Salmonella and Salmonellosis

**i.** *If I<sup>ν</sup>*

**ii.** lim*<sup>ν</sup>*!*nI*

**iii.** lim*<sup>ν</sup>*!<sup>0</sup>*I*

**v.** *Thus D<sup>α</sup>*

Remark [40].

**Remark 3**

*ν <sup>a</sup> f*ð*x*Þ ¼ *I*

*ν*

 *<sup>f</sup>*ð*t*Þ ¼ *<sup>d</sup> dt I* 1−*α*

**i.** *Suppose f*ð*t*Þ<sup>∈</sup> *<sup>C</sup>*½*a;b and D<sup>α</sup>*

**ii.** *If (i) holds, and D<sup>α</sup>*

**Author details**

Fathalla A. Rihan

Arab Emirates

**References**

**Remark 2** *Let ν;γ*∈ ℝ<sup>þ</sup> *and α*∈ ð0*;* 1Þ*. Then*

*n*

*<sup>a</sup> f*ð*t*Þ ¼ *f*ð*t*Þ *weakly;*

**iv.** *If f*ð*t*<sup>Þ</sup> *is absolutely continuous on* <sup>½</sup>*a;b, then* lim*<sup>α</sup>*!<sup>1</sup>*D<sup>α</sup>*

*<sup>f</sup>*ð*t*Þ ¼ *<sup>f</sup>*ð*a*Þ þ <sup>1</sup>

Address all correspondence to: frihan@uaeu.ac.ae

Que., 2007, pp. 2704–2709.

*Γ*ð*α*Þ *Dα*

∀ *t* ∈½*a;b, then f*ð*t*Þ *is nonincreasing for each t* ∈ ½*a;b*.

*, then I<sup>ν</sup> a I γ <sup>a</sup> f*ð*t*Þ ¼ *I*

*<sup>a</sup>* : *<sup>L</sup>*<sup>1</sup> ! *<sup>L</sup>*<sup>1</sup> *and f*ð*t*Þ<sup>∈</sup> *<sup>L</sup>*<sup>1</sup>


[36] I. Podlubny, *Fractional Differential Equations*, New York: Academic Press, 1999.

[19] D. Kaplan, L. Glass, *Understanding Nonlinear Dynamics*, New York: Springer-Verlag, 1995.

[21] M. Safan, F. A. Rihan, Mathematical analysis of an SIS model with imperfect vaccination

[22] F. A. Rihan, *Numerical Treatment of Delay Differential Equations in Bioscience*, PhD. Thesis,

[23] W. O. Kermack, A. G. McKendrick, Contributions to the mathematical theory of epi-

[24] R. Casagrandi, L. Bolzoni, S. A. Levin, V. Andreasen, The SIRC model and influenza A,

[25] M. El-Shahed, A. Alsaedi, The fractional SIRC model and influenza A, Math. Probl. Eng.

[26] L. Jodar, R. J. Villanueva, A. J. Arenas, G. C. G. alez, Nonstandard finite difference method by nonlocal approximation, Math. Comput. Simul. 79 (2008) 622–633.

[27] F. A. Rihan, S. Lakshmanan, A. Hashish, R. Rakkiyappan, E. Ahmed, Fractional order delayed predator-prey systems with Holling type-II functional response, Nonlinear

[28] H. Smith, *An Introduction to Delay Differential Equations with Applications to the Life Sciences*,

[29] G. Marchuk, *Mathematical Modelling of Immune Response in Infectious Diseases*, Dordrecht:

[30] O. Diekmann, J. Heesterbeek, J. Metz, On the definition and the computation of the basic reproduction ratio R<sup>0</sup> in models for infectious diseases in heterogeneous populations, J.

[31] E. Ahmed, A. El-Sayed, H. A. El-Saka, On some routh–hurwitz conditions for fractional order differential equations and their applications in Lorenz, Rössler, Chua and Chen

[32] R. Anguelov, J. M.-S. Lubuma, Nonstandard finite difference method by nonlocal approx-

[33] K. Diethelm, An algorithm for the numerical solution of differential equations of frac-

[34] K. Diethelm, N. J. Ford, Analysis of fractional differential equations, J. Math. Anal. Appl.

[35] P. Chapagain, J. S. V. Kessel, J. K. Karns, et. al., A mathematical model of the dynamics of salmonella cerro infection in a us dairy herd, Epidemiol. Infect. 136 (2008) 263–272.

New York, Dordrecht, Heidelberg, London: Springer, 2011.

and backward bifurcation, Math. Comput. Simul. 96 (2014) 195–206.

[20] J. Murray, *Mathematical Biology II*, New York: Springer, 2003.

University of Manchester (UK), 2000.

166 Current Topics in Salmonella and Salmonellosis

Math. Biosci. 200 (2006) 152–169.

Dynam. 80 (1) (2015) 777–789.

Kluwer Academic Publishers, 1997.

systems, Phys. Lett. A 356 (2006) 1–4.

imation, Math. Comput. Simul. 61 (2003) 465–475.

tional order, Elec. Trans. Numer. Anal. 5 (1997) 1–6.

Math. Biol. 28 (1990) 365.

265 (2002) 229–48.

2011 (2011) 9.

demics, Proc. R. Soc. Lond. 115 (1927) 700–721.


**Risk Factors and Control Strategies**

## **Interaction between** *Salmonella* **and Plants: Potential Hosts and Vectors for Human Infection Interaction between** *Salmonella* **and Plants: Potential Hosts and Vectors for Human Infection**

Eva Fornefeld, Jasper Schierstaedt, Sven Jechalke, Rita Grosch, Kornelia Smalla and Adam Schikora Sven Jechalke, Rita Grosch, Kornelia Smalla and Adam Schikora

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Eva Fornefeld, Jasper Schierstaedt,

http://dx.doi.org/10.5772/67061

#### **Abstract**

Fruits and vegetables are important for a healthy diet. However, when eaten raw and contaminated with human pathogens (HPs) they may cause a disease outbreak. Contamination with HPs can occur along the entire farm-to-fork production chain and *Salmonella enterica* is one of the most common foodborne pathogens. A range of biotic and abiotic environmental factors can influence the complex interactions between *Salmonella* and plants. Moreover, the outcome of experiments largely depends on the experimental design and parameters or methods employed, and on top, on the accompanying plant microbiome and the genetic equipment of the plant and the *Salmonella* strain. Particularly mobile genetic elements contribute to the diversification and adaptation of *Salmonella* to the plant environment. So far, little is known about the key processes and factors influencing the attachment and potential internalization of *Salmonella* in plants and the plant specific responses. It is therefore important to better understand the ecology of *Salmonella* in the soil and plant environment, in order to propose practicable recommendations for prevention of foodborne diseases. This also requires improved sensitivity and specificity of detection methods. In this chapter, we present the current knowledge, research needs, and methodology regarding the complex interactions between *Salmonella* and plants.

**Keywords:** *Salmonella enterica*, plant, biofilm, colonization mechanisms, interaction

## **1. Introduction**

The natural microbiome of plants includes a wide diversity of microorganisms and is a key determinant of plant health and productivity, e.g., by supporting the uptake

of mineral nutrients in roots or suppressing pathogen growth and inducing the hostimmune system [1–3]. Due to its relevance, the plant microbiome (totality of microorganisms associated with the plant) is even called the second plant genome. Because of the tight interplay between plants and their epiphytic and endophytic microorganisms the terms holobiont and meta-organisms are used as well. The plant microbiome is important not only for plant growth and health, but is also positively influencing human health [4]. However, besides positive effects on human health plants can also be carriers of bacterial HPs.

*Salmonella* is one of the major causal agents of foodborne gastroenteritis and represents a major threat to public health. It is estimated that each year 93.8 million cases of salmonellosis occur globally (86% of which foodborne), with 155,000 deaths [5]. Consumption of raw plants is more and more recognized as a source for HPs and associated with disease outbreaks in several countries. The number of outbreaks linked to fresh produce, spices, and nuts surpassed those linked to foods of animal origin [6]. Sources of HPs in the production chain and factors contributing to the contamination of fruits and vegetables include for example the application of organic fertilizers such as animal manures, contaminated irrigation water, insect and animal vectors but also the use of contaminated seeds [7]. *Enterobacteriaceae* such as *Erwinia*, *Serratia,* and *Pantoea* belong to bacteria typically associated with the phyllosphere [8–10]. However, it is not completely understood how *Salmonella* persists in the plant environment and which environmental factors trigger its survival. In this chapter, we discuss factors influencing the survival of *Salmonella* in the agricultural environment as well as adaptations that allow successful colonization of plants, such as attachment, biofilm formation, and internalization.

## **2. Contamination of fresh produce**

Besides contaminated animal products, *Salmonella* outbreaks are increasingly associated with fruits and vegetables. Already on the field, plants may be contaminated via soil or irrigation, especially if watered with surface water [11–14]. *Salmonella* has been shown to persist in various ecological niches in soil as well as in irrigation water and fertilizers [15–17]. In this context, the watering system and the agricultural practices seem to play a key role in the prevention of contamination with human pathogens. For instance, lettuce plants were more likely to be contaminated with *Escherichia coli* when watered using overhead sprinklers when compared to subsurface drip or surface furrow irrigation [18]. Besides, even noncontaminated rain‐sized water droplets could transfer HPs from contaminated soil or plants to other plants [19].

Organic fertilizers like manure, biogas plant digestates and sewage sludge offer an additional route for contamination of fresh produce. Similarly, animals like birds, game, mice, or insects can contribute to the contamination of fresh produce directly or indirectly via feces or irrigation water [7, 14, 20]. Often underestimated are soil particles, which can be carried by the wind over long distances and contribute to the transient of microbiome between plants [8]. Hence, wind-caused spread of HPs should also be considered. Contaminated plant residues might constitute additional risk if incorporated into soil before the planting of next crop. The infection of plants is essentially dependent on the ability of HPs to survive and persist in the agricultural environment. *Salmonella*, for example, was shown to survive in soil for more than 200 days if the soil was fertilized [21, 22]. The survival of diverse bacteria newly introduced into soil has been subject of research for many years [23], and the mechanisms that govern this process, compared often to microbial invasion, were described in many studies (recently reviewed by [24]). In order to survive in the soil, HPs need to find an adequate ecological niche in which they can establish. Furthermore, their ability to do so and to survive for extended time increased when the indigenous microbial community was reduced as a result of, for example, sterilization [25]. In addition, the survival of microorganisms that successfully invaded the soil is highly dependent on the environmental heterogeneity [26–28].

Contamination of fresh produce with HPs like *Salmonella,* can occur before the harvest and also along the whole production chain [11, 14]. Since the epidemiological investigations start very often long time after the contamination or the harvest, it is very challenging to assess whether the contamination took place in the field or occurred "post‐harvest" during the processing. Consequently in the majority of cases, the information available does not necessarily reveal the real causes of contamination [29].

## **3. Epidemiology of** *Salmonella* **in agricultural systems**

of mineral nutrients in roots or suppressing pathogen growth and inducing the hostimmune system [1–3]. Due to its relevance, the plant microbiome (totality of microorganisms associated with the plant) is even called the second plant genome. Because of the tight interplay between plants and their epiphytic and endophytic microorganisms the terms holobiont and meta-organisms are used as well. The plant microbiome is important not only for plant growth and health, but is also positively influencing human health [4]. However, besides positive effects on human health plants can also be

*Salmonella* is one of the major causal agents of foodborne gastroenteritis and represents a major threat to public health. It is estimated that each year 93.8 million cases of salmonellosis occur globally (86% of which foodborne), with 155,000 deaths [5]. Consumption of raw plants is more and more recognized as a source for HPs and associated with disease outbreaks in several countries. The number of outbreaks linked to fresh produce, spices, and nuts surpassed those linked to foods of animal origin [6]. Sources of HPs in the production chain and factors contributing to the contamination of fruits and vegetables include for example the application of organic fertilizers such as animal manures, contaminated irrigation water, insect and animal vectors but also the use of contaminated seeds [7]. *Enterobacteriaceae* such as *Erwinia*, *Serratia,* and *Pantoea* belong to bacteria typically associated with the phyllosphere [8–10]. However, it is not completely understood how *Salmonella* persists in the plant environment and which environmental factors trigger its survival. In this chapter, we discuss factors influencing the survival of *Salmonella* in the agricultural environment as well as adaptations that allow successful colonization of plants, such as attachment, biofilm formation,

Besides contaminated animal products, *Salmonella* outbreaks are increasingly associated with fruits and vegetables. Already on the field, plants may be contaminated via soil or irrigation, especially if watered with surface water [11–14]. *Salmonella* has been shown to persist in various ecological niches in soil as well as in irrigation water and fertilizers [15–17]. In this context, the watering system and the agricultural practices seem to play a key role in the prevention of contamination with human pathogens. For instance, lettuce plants were more likely to be contaminated with *Escherichia coli* when watered using overhead sprinklers when compared to subsurface drip or surface furrow irrigation [18]. Besides, even noncontaminated rain‐sized water droplets could transfer HPs from contaminated soil or plants to other plants [19].

Organic fertilizers like manure, biogas plant digestates and sewage sludge offer an additional route for contamination of fresh produce. Similarly, animals like birds, game, mice, or insects can contribute to the contamination of fresh produce directly or indirectly via feces or irrigation water [7, 14, 20]. Often underestimated are soil particles, which can be carried by the wind over long distances and contribute to the transient of microbiome between plants [8]. Hence, wind-caused spread of HPs should also be considered. Contaminated plant residues might constitute additional risk if incorporated into soil before the planting of next crop.

carriers of bacterial HPs.

172 Current Topics in Salmonella and Salmonellosis

and internalization.

**2. Contamination of fresh produce**

Fresh produce contaminated with *Salmonella* can easily trigger a salmonellosis outbreak, and despite the difficulties with identification, in the past years fresh produce were repeatedly identified as the outbreak source. Among the outbreaks in the USA, *Salmonella* is the leading cause of the fresh produce‐originated foodborne diseases [30]. The available data are depending on the procedures and records in particular countries. At least 12 large, fresh produce-related *Salmonella* outbreaks have been reported since 2010, an overview of international outbreaks with more than 100 associated cases is presented in **Table 1**.

Although fruits and vegetables were identified as source of human pathogens, it is not clear whether the plants were colonized in the field or during processing. *Salmonella* may live epiphytically or be internalized through wounds, the root system, stomata, or hydathodes (see below). Additionally, *Salmonella* can be entrapped in fruits or seeds after contamination of flowers [31, 32]. Moreover, large outbreaks can be destructive to consumer's confidence which results in economic losses [33, 34]. Therefore, the research on the ecology of HPs like *Salmonella* in relation to farming and harvesting practices is very important for human health and also for the economy.

## **4. Factors influencing the survival of** *Salmonella* **in soil**

Successful establishment of human pathogenic bacteria in soil depends on a variety of biotic and abiotic factors (see **Figure 1** for an overview). Numerous studies, carried out under different conditions, showed that among them are weather or atmospheric conditions like tem-


**Table 1.** International salmonellosis outbreaks associated with fresh produce.

perature, UV radiation, and moisture content of the soil [7, 35]. In general, temperature has an important effect on growth and decay rates of bacteria. Most studies examined the influence of temperature on survival of enteric bacteria under isothermal conditions, showing a generally reduced survival of *Salmonella* in soil with increasing temperature and, accordingly a better persistence in soil at lower temperatures [36, 37]. Semenov et al*.* [38] analyzed how temperature fluctuations affect *Salmonella enterica* serovar Typhimurium (*S.* Typhimurium) in cow manure and demonstrated increased decay rates with increasing amplitudes of daily oscillations. Besides temperature, water availability is a key factor for *Salmonella* to survive in the environment. Humidity in soil depends on rainfall and watering as well as on evaporation. Soil moisture also depends on soil properties like clay content or pore size. In general, it seems that survival of *Salmonella* in soil is promoted by high humidity while water shortage has a detrimental influence on persistence, probably due to drought stress [39–41]. The soil type and its physical and chemical characteristics have a strong influence on the fate of bacterial HPs. Those characteristics include texture and particle size distribution, which affect adsorption of *Salmonella* to soil particles. The soil type determines the extent of *Salmonella* leaching, if the bacteria are applied to the soil surface via contaminated slurry or manure as shown by Bech et al*.* [42]. In this study, percolation of *S.* Typhimurium was more pronounced in loamy

**Figure 1.** Factors influencing the survival of *Salmonella* in soil and its colonization of plants.

perature, UV radiation, and moisture content of the soil [7, 35]. In general, temperature has an important effect on growth and decay rates of bacteria. Most studies examined the influence of temperature on survival of enteric bacteria under isothermal conditions, showing a generally reduced survival of *Salmonella* in soil with increasing temperature and, accordingly a better persistence in soil at lower temperatures [36, 37]. Semenov et al*.* [38] analyzed how temperature fluctuations affect *Salmonella enterica* serovar Typhimurium (*S.* Typhimurium) in cow manure and demonstrated increased decay rates with increasing amplitudes of daily oscillations. Besides temperature, water availability is a key factor for *Salmonella* to survive in the environment. Humidity in soil depends on rainfall and watering as well as on evaporation. Soil moisture also depends on soil properties like clay content or pore size. In general, it seems that survival of *Salmonella* in soil is promoted by high humidity while water shortage has a detrimental influence on persistence, probably due to drought stress [39–41]. The soil type and its physical and chemical characteristics have a strong influence on the fate of bacterial HPs. Those characteristics include texture and particle size distribution, which affect adsorption of *Salmonella* to soil particles. The soil type determines the extent of *Salmonella* leaching, if the bacteria are applied to the soil surface via contaminated slurry or manure as shown by Bech et al*.* [42]. In this study, percolation of *S.* Typhimurium was more pronounced in loamy

*Salmonella* **Serovar Vector Year Country Cases/serovar** 

*S.* Enteriditis Sprouts,

174 Current Topics in Salmonella and Salmonellosis

Unknown Produce-based

*S.* I4,[5],12:i:‐ Vegetables, sprouts,

*S.* Hvittingfoss Vegetables, leafy

beans

*S.* Newport Mung beans 2011 Germany,

salads, broccoli salad

alfalfa sprouts

greens, lettuce, fruit, tomatoes, olives

**Table 1.** International salmonellosis outbreaks associated with fresh produce.

*S*. Newport Tomatoes 2015 USA 115/81 [112] *S.* Poona Cucumbers 2015‐16 USA 907/907 [113] Unknown Onions, tomatoes 2015 USA 200/0 [114]

*S.* Newport Cucumbers 2014 USA 275/0 [113] *S.* Typhimurium Cantaloupe 2012 USA 261/261 [115] *S.* Braenderup Mangoes 2012 USA, Canada 127/0 [113]

*S.* Agona Fruit, papaya 2011 USA 106/0 [113]

Only large outbreaks with more than 100 associated total or confirmed cases since 2010 are shown.

**confirmed**

106/32 [116]

2014 USA 115/0 [113]

The Netherlands

2011 Japan 1500/0 [117]

2010 USA 140/0 [118]

2010 USA 114/108 [119]

**Reference**

than in sandy soil with leaching bacteria reaching 1 m of depth at 105 CFU/ml of leachate. Transport and survival of bacterial pathogens in soil is also influenced by amendment of fertilizers probably because of the presence of organic matter [43]. Leaching of *Salmonella* through soil was observed to reach greater depths after application of slurry than of manure [44]. In the same line, the application method of fertilizers can also have an effect on *Salmonella* survival in soil since an injection of manure or slurry or clumping of the applied fertilizer aboveground protect bacteria in the soil from desiccation, UV, and high temperatures [43, 45, 46]. Agricultural practices like tillage that have an effect on the porosity of soil determine the extent of leaching [47] and the availability of oxygen. While a detrimental influence of aeration on survival of *E. coli* O157:H7 has been demonstrated, the oxygen availability does not influence the survival of *Salmonella* [48]. Soil pH is also an important factor for *Salmonella* survival and *Salmonella* can survive in the environment with neutral to acidic pH while alkaline pH has a detrimental effect on its persistence [49]. Another important determinant of *Salmonella* survival in soil is the availability of nutrients. In this environment, nutrients can only partly be used by bacterial HPs and are generally rather scarce. *Salmonella* is chemoheterotrophic and therefore depends on carbohydrates, lipids, and protein in its environment as sources for energy, nitrogen, and amino acids. Addition of organic fertilizers improves nutrient availability by addition of readily available carbon and nitrogen sources as well as other nutrients. But amendment of fertilizers also changes the microbiological properties of soil by introducing microorganisms to the soil microbial community. Moreover, the additional nutrients stimulate growth of copiotrophic soil bacteria which might compete for the nutrient resources [50]. So far, no clear correlations between the type of fertilizer and survival in soil have been identified [51]. But when survival in manure was compared to survival in manure‐ amended soil, *Salmonella* usually survived better in soil [22]. This could be due to competition by the microbial flora of manure, which is more concentrated than in soil.

The soil microbial community and its composition have a great influence on the survival of *Salmonella* [52]. In the soil ecosystem, *Salmonella* has to compete with the indigenous microbial community for space and nutrients [24]. For example, it was shown that *Salmonella enterica* serovar Newport (*S*. Newport) survived about 10 weeks longer in sterilized soil compared to nonsterilized manure‐amended soil [22]. Similarly, a better survival was found in γ‐irradiated than in untreated soil [53]. These results indicate suppression by the native microbial community. Overall, results demonstrate the importance of the microbial community affecting the fate of *Salmonella* in soil. Plant pathogens, fungi, viruses, and animal pests present in the environment can degrade the plant material and increase the content of available nutrients or provide entry sites facilitating internalization into plants [7, 14, 54]. They may also serve as vectors [7, 55]. Effects of protists have been analyzed using protozoa showing that their presence can foster or reduce survival of different species. For example, *Salmonella enterica* serovar Thompson was accumulated in vesicles of *Tetrahymena* [56], while growth of protozoa can also decimate *S.* Typhimurium populations [37].

In addition to the environment in which *Salmonella* is introduced, the bacterial characteristics are crucial for persistence. Firstly, the genetic disposition of the strain, for example, the presence of type III secretion system (T3SS), the ability to form biofilms, chemotaxis, or motility are important. Studies using strains with mutations influencing these characteristics usually resulted in reduced survival [57–59]. *Salmonella* can also produce an *O*-antigen capsule, which improves survival under desiccation stress [60]. Furthermore, the ability to form biofilms enhances environmental persistence of some *Salmonella* serovars [61]. Similarly, a biofilm‐producing *Salmonella* strain survived chlorination significantly better than the biofilm‐deficient mutant [62]. The conditions under which *Salmonella* are grown before their inoculation in the environment are also important since preadaptation influences the persistence [20]. Finally, many studies on the survival of HPs in soil employed a relatively high inoculum, which under natural conditions occurs only locally, e.g., by fecal point contaminations. Inoculation resulted in a fast initial decline of inoculated bacterial populations with usually low numbers of *Salmonella* that survive for a long time after the inoculation.

In conclusion, studies analyzing the survival of *Salmonella* demonstrated complex interactions with the environment and a network of factors, which might play an important role in the persistence of *Salmonella*. Therefore, the very often contradictory results reflect the variability of strains, their survival strategies in a complex environment as well as differences in experimental setups used.

## **5. Attachment to plant surfaces and biofilm formation**

aboveground protect bacteria in the soil from desiccation, UV, and high temperatures [43, 45, 46]. Agricultural practices like tillage that have an effect on the porosity of soil determine the extent of leaching [47] and the availability of oxygen. While a detrimental influence of aeration on survival of *E. coli* O157:H7 has been demonstrated, the oxygen availability does not influence the survival of *Salmonella* [48]. Soil pH is also an important factor for *Salmonella* survival and *Salmonella* can survive in the environment with neutral to acidic pH while alkaline pH has a detrimental effect on its persistence [49]. Another important determinant of *Salmonella* survival in soil is the availability of nutrients. In this environment, nutrients can only partly be used by bacterial HPs and are generally rather scarce. *Salmonella* is chemoheterotrophic and therefore depends on carbohydrates, lipids, and protein in its environment as sources for energy, nitrogen, and amino acids. Addition of organic fertilizers improves nutrient availability by addition of readily available carbon and nitrogen sources as well as other nutrients. But amendment of fertilizers also changes the microbiological properties of soil by introducing microorganisms to the soil microbial community. Moreover, the additional nutrients stimulate growth of copiotrophic soil bacteria which might compete for the nutrient resources [50]. So far, no clear correlations between the type of fertilizer and survival in soil have been identified [51]. But when survival in manure was compared to survival in manure‐ amended soil, *Salmonella* usually survived better in soil [22]. This could be due to competition

by the microbial flora of manure, which is more concentrated than in soil.

decimate *S.* Typhimurium populations [37].

176 Current Topics in Salmonella and Salmonellosis

The soil microbial community and its composition have a great influence on the survival of *Salmonella* [52]. In the soil ecosystem, *Salmonella* has to compete with the indigenous microbial community for space and nutrients [24]. For example, it was shown that *Salmonella enterica* serovar Newport (*S*. Newport) survived about 10 weeks longer in sterilized soil compared to nonsterilized manure‐amended soil [22]. Similarly, a better survival was found in γ‐irradiated than in untreated soil [53]. These results indicate suppression by the native microbial community. Overall, results demonstrate the importance of the microbial community affecting the fate of *Salmonella* in soil. Plant pathogens, fungi, viruses, and animal pests present in the environment can degrade the plant material and increase the content of available nutrients or provide entry sites facilitating internalization into plants [7, 14, 54]. They may also serve as vectors [7, 55]. Effects of protists have been analyzed using protozoa showing that their presence can foster or reduce survival of different species. For example, *Salmonella enterica* serovar Thompson was accumulated in vesicles of *Tetrahymena* [56], while growth of protozoa can also

In addition to the environment in which *Salmonella* is introduced, the bacterial characteristics are crucial for persistence. Firstly, the genetic disposition of the strain, for example, the presence of type III secretion system (T3SS), the ability to form biofilms, chemotaxis, or motility are important. Studies using strains with mutations influencing these characteristics usually resulted in reduced survival [57–59]. *Salmonella* can also produce an *O*-antigen capsule, which improves survival under desiccation stress [60]. Furthermore, the ability to form biofilms enhances environmental persistence of some *Salmonella* serovars [61]. Similarly, a biofilm‐producing *Salmonella* strain survived chlorination significantly better than the biofilm‐deficient mutant [62]. The conditions under which *Salmonella* are grown before their inoculation in the environment are also important since preadaptation influences the persistence [20]. Finally, Attachment and adhesion of *Salmonella* to plant surfaces are essential steps of plant colonization. Several bacterial elements such as fimbrial structures, nonfimbrial adhesins, flagella, cellulose, and lipopolysaccharides (LPS) are important bacterial factors for colonization [63, 64]. Although previous studies demonstrated that the attachment depends on plant and bacterial factors, no single factor was found to be essential, suggesting that bacteria use several parallel mechanisms to ensure attachment to different plants or to different plant cells under a wide variety of conditions [65]. Furthermore, the attachment of *S*. *enterica* to plant surfaces appears to be serovar‐dependent [66]. For example, the strength of the attachment to basil, lettuce, or spinach leaves differed between *S. enterica* serovars. While *S.* Typhimurium, *Salmonella enterica* serovar Enteritidis, and *Salmonella enterica* serovar Senftenberg were efficient, other serovars including *Salmonella enterica* serovar Agona, *Salmonella enterica* serovar Heidelberg or *Salmonella enterica* serovar *Arizonae* showed less attachment [67]. Clear differences in attachment were also observed in leaves of different age, for example, *S.* Typhimurium showed a better attachment to older compared to younger lettuce leaves [68]. Additionally, *S*. *enterica* serovars were reported to actively move toward plant roots, attracted by root exudates [69]. There, they are able to efficiently attach and to form biofilms at natural openings or wounds [70, 71].

Several other studies provided evidence for biofilm formation by *Salmonella* on plant surfaces [72]. Within biofilms, bacteria are generally well‐protected against environmental stresses, antibiotics, and disinfectants. The importance of biofilms for the attachment of *Salmonella* to plants and their role in the persistence in plants was recently described by Yaron and Romling [65]. Biofilm formation of *Salmonella* is influenced by environmental conditions and is reported to be maximal under reduced nutrient availability, aerobic conditions, low osmolarity, and mid temperatures [73], which are characteristic for the plant surface. In contrast, it was shown *in vitro* that *S*. Typhimurium cells grown at 37°C, the temperature in the animal host, do not produce cellulose and fimbriae [64]. Furthermore, the *red dry and rough* (*rdar*) and the *smooth and white* (*saw*) morphotypes, regulated by the *agfD* promoter and defined by a combination of traits such as the presence of thin aggregative fimbriae (tafi), cellulose, and *O*‐antigen capsule, might affect the dispersal of *Salmonella* in an agricultural environment [74]. In contrast to the *saw* morphotype, the *rdar* morphotype, isolated from tomato, showed better attachment to plant surfaces [74, 75]. Biofilm‐producing *Salmonella* on parsley showed a higher resistance against disinfectants than the biofilm‐deficient mutant. Furthermore, after a storage period of the plant, the cells that were able to produce the biofilm matrix were significantly more resistant to the disinfection treatment [62]. A screening of 6000 transposon mutants of *S*. Newport resulted in the identification of 20 mutants selected for reduced adherence to alfalfa sprouts [70]. Interestingly, these mutants contained insertions associated with genes, for example, for the surface‐exposed aggregative fimbriae nucleator (*agfB*) and the general transcriptional regulator *rpoS*. The respective proteins have been reported to regulate the production of curly, cellulose, and other adhesins such as pili. Two other genes (STM0278 and STM0650) were identified as important factors for the colonization of alfalfa seedlings. Both play an important role in the formation of biofilms [76]. Furthermore, bacterial cellulose and curly were involved in the colonization of parsley with *S.* Typhimurium from irrigation water [77].

Although many factors influencing the colonization of plants were identified by *in vitro* experiments, a more detailed investigation of genes of *Salmonella* that are expressed during the colonization of plants is needed. New techniques for the isolation of mRNA from samples containing both plant and bacterial materials as well as for the quantitative PCR allow the analysis of the transcriptome and the identification of genes with related functions [78].

## **6. Internalization of** *Salmonella* **into plant tissues**

An increasing number of salmonellosis outbreaks associated with plants shows that human pathogenic bacteria use plants as a niche for replication or as hosts and vectors for animal and human infection (**Table 1**). For a long time it was assumed that *Salmonella* rather survives on plant surfaces than colonizes the plant interior. This view has been challenged by recent reports. Today we know that *Salmonella* can actively enter and spread within the plant. Plants offer multiple entry possibilities for HPs; stomata, for example, were identified already a few years ago. Stomata are used for gas exchange between the surroundings and cells of the inner mesophyll layers, this is necessary for proper photosynthetic efficacy. They can close if a pathogen is recognized. Some pathogens, however, produce toxins (coronatine), which reopen stomata and therefore allow their use as gates for colonization of underlying tissues. *Salmonella* was shown to gather around the open stomata and enter the mesophyll tissue of lettuce leaves [58]. Similar to lettuce, a high incidence of internalization was observed in arugula leaves, while romaine and red‐lettuce, as well as basil showed significantly lower internalization rates [79]. Interestingly, in this study parsley and tomato leaves showed only marginal internalization [79]. In addition to stomata, also hydratodes and trichomes allow an internalization of *Salmonella* into leaves [31, 80, 81]. Not only *Salmonella* or phytopathogenic bacteria use stomata as entry points, also other *Enterobacteriaceae,* for example *E. coli,* use similar strategies to access the plant's interior [82, 83]. Importantly to note is the fact that the preference to gather around open stomata manifests only in photosynthetically active leaves, and an artificial opening of the stomata at night has no effect on the bacterial behavior [58]. This observation is in line with the proposed hypothesis that those bacteria are in a direct competition for C‐ and N‐sources with the native leaf microbiome [69, 71], and suggests a chemotaxis toward the newly synthesized products of the photosynthesis.

better attachment to plant surfaces [74, 75]. Biofilm‐producing *Salmonella* on parsley showed a higher resistance against disinfectants than the biofilm‐deficient mutant. Furthermore, after a storage period of the plant, the cells that were able to produce the biofilm matrix were significantly more resistant to the disinfection treatment [62]. A screening of 6000 transposon mutants of *S*. Newport resulted in the identification of 20 mutants selected for reduced adherence to alfalfa sprouts [70]. Interestingly, these mutants contained insertions associated with genes, for example, for the surface‐exposed aggregative fimbriae nucleator (*agfB*) and the general transcriptional regulator *rpoS*. The respective proteins have been reported to regulate the production of curly, cellulose, and other adhesins such as pili. Two other genes (STM0278 and STM0650) were identified as important factors for the colonization of alfalfa seedlings. Both play an important role in the formation of biofilms [76]. Furthermore, bacterial cellulose and curly were involved in the colonization of parsley with *S.* Typhimurium

Although many factors influencing the colonization of plants were identified by *in vitro* experiments, a more detailed investigation of genes of *Salmonella* that are expressed during the colonization of plants is needed. New techniques for the isolation of mRNA from samples containing both plant and bacterial materials as well as for the quantitative PCR allow the analysis of the transcriptome and the identification of genes with related

An increasing number of salmonellosis outbreaks associated with plants shows that human pathogenic bacteria use plants as a niche for replication or as hosts and vectors for animal and human infection (**Table 1**). For a long time it was assumed that *Salmonella* rather survives on plant surfaces than colonizes the plant interior. This view has been challenged by recent reports. Today we know that *Salmonella* can actively enter and spread within the plant. Plants offer multiple entry possibilities for HPs; stomata, for example, were identified already a few years ago. Stomata are used for gas exchange between the surroundings and cells of the inner mesophyll layers, this is necessary for proper photosynthetic efficacy. They can close if a pathogen is recognized. Some pathogens, however, produce toxins (coronatine), which reopen stomata and therefore allow their use as gates for colonization of underlying tissues. *Salmonella* was shown to gather around the open stomata and enter the mesophyll tissue of lettuce leaves [58]. Similar to lettuce, a high incidence of internalization was observed in arugula leaves, while romaine and red‐lettuce, as well as basil showed significantly lower internalization rates [79]. Interestingly, in this study parsley and tomato leaves showed only marginal internalization [79]. In addition to stomata, also hydratodes and trichomes allow an internalization of *Salmonella* into leaves [31, 80, 81]. Not only *Salmonella* or phytopathogenic bacteria use stomata as entry points, also other *Enterobacteriaceae,* for example *E. coli,* use similar strategies to access the plant's interior [82, 83]. Importantly to note is the fact that the preference to gather around open stomata manifests only in photosynthetically active leaves, and

**6. Internalization of** *Salmonella* **into plant tissues**

from irrigation water [77].

178 Current Topics in Salmonella and Salmonellosis

functions [78].

From the consumers' point of view, not only the internalization into leaves but also the translocation within the plant, e.g., toward fruits is important. In some crop plants, e.g., tomato, such translocation was detected [81]. The authors showed internalization into the tomato fruits when the entire plant was systemically colonized. Still, the colonization rates seemed rather low [81]. Nonetheless, in light of the persistent pathogenicity in animals after the passage through a plant host [84], the internalization mechanisms are of high interest. Some detailed mechanisms were already suggested. Erlacher and coworkers proposed one of those possible mechanisms: colonization of the niche below the cuticle layer of the epidermis [9]. Obviously such a behavior protects bacteria from the harsh conditions on the leaf surface (UV light, drought, and quick changes in temperature) but also from surface sterilization agents. Another strategy would be an intracellular lifestyle, which would resemble the strategy in the animal infection model. Until now, this possibility remains unverified, two reports postulated internalization into plant cells using *Arabidopsis* and tobacco systems [84, 85]. Yet, another helpful strategy is the efficient formation of biofilms, this strategy was discussed above and was reviewed by Yaron and Romling [65]. Only recently, it was discovered that particular *Salmonella* strains may avoid the recognition by the plant immune system [86], which would make them very well adapted colonizers (see below).

Many row eaten crop plants plants associated with salmonellosis outbreaks or food poisoning are usually grown in soil (lettuce, basil, parsley, etc.). In such cases the translocation from the potentially contaminated soil (through manure or irrigation water) via roots into the harvested and consumed plant parts is of enormous importance. Several reports assessed already this possibility and pointed at a very diverse picture with regard to pathogenic *E. coli* or *Salmonella*. Here the high heterogeneity with regard to colonization in the plant population is very remarkable [69, 87], usually about 20% of the plant population is colonized, however, this range may vary from 0 to 100% and strongly depends on plant species and bacterial strain [51, 69, 77, 88–90].

## **7. The function of T3SS and the role of plant immune system during the interactions between plant and** *Salmonella*

Bacterial pathogens use T3SS and T4SS to inject so‐called effector proteins directly into the cytoplasm of host cells. Those effectors are able to manipulate the host immune system and suppress the otherwise negative effects of defense responses. *Salmonella* uses two T3SS and more than 40 effectors in order to manipulate the immune system (perception mechanisms and signaling cascades) as well as the cytoskeleton of animal cells at different stages of the infection process [91]. Recent discoveries from others and our group imply that the mechanisms used in animal and plant hosts may resemble each other [59, 85, 86, 92–95]. The inoculation with the wild-type *Salmonella* strains and mutants in one or both of the T3SSs showed that functional secretion systems are required for efficient plant colonization [59, 85, 93]. Two observations allow such a conclusion: (1) The mutants had lower proliferation rates when compared to the respective wild type, which suggests that a functional T3SS helps with the colonization of plants; and (2) T3SS mutants induced stronger immune response of the host plant. Similar to animals or humans, plants respond to colonization of pathogenic bacteria inducing numerous immune responses, among others are oxidative burst and enhanced expression of *Pathogenesis Related* (*PR*) genes. Both were observed after inoculation with *Salmonella* and both were stronger if the inoculation was performed using mutants in T3SS [59, 85, 86]. Those results suggest that the wild‐type strain is able to suppress the immune response. It is very plausible to think that this suppression is due to functional T3SS‐dependent effector proteins. We know only little about their function in plant cells, since only two effectors (SseF and SpvC) were evaluated in this respect. SseF together with SseG are translocated into animal cells and are responsible for the establishment of the reproduction niche [96]. In plants, SseF induces the hypersensitive response (HR) [94]. Important is the fact that silencing of the suppressor of SGT1 eliminates the response to SseF, suggesting that this effector is recognized in R protein‐dependent manner, which is the usual recognition method of pathogen effectors during the effector‐triggered immunity (ETI). SpvC is a phosphothreonin lyase which dephosphorylates activated MAP kinases. Those kinases build a core compound in the signaling cascade leading from the perception of the pathogen on the cell surface to the transcriptional response at the chromatin level. Especially the trio MPK3, MPK4, and MPK6 plays an important role in plants [97], and is activated (phosphorylated) during the response to *Salmonella* [84]. SpvC interacts actively with the MPK6 and dephosphorylates this kinase, consequently abolishing the signal transduction [95]. A comprehensive overview of the reports regarding the plant immune responses to HPs was published only recently and is an excellent compendium of the current knowledge [98].

#### **8.** *Salmonella* **changes its physiology in contact with plant host**

During the interaction between *Salmonella* and crop plants, not only the plant reacts to the presence of the bacteria, also *Salmonella* adapts to the conditions represented by a plant organism. Recent results show that bacteria modify their physiology and motility in order to adjust to the physiological conditions occurring in plants. Several authors evaluated the transcriptional changes of bacteria when in contact with plants or plant‐originated products [99, 100]. Interestingly, the analysis of the transcriptome, revealed a partial overlap between bacteria from macerated lettuce or cilantro leaves and bacteria from intestine, suggesting that those bacteria might be better adapted to the exploitation of plant material than estimated [100]. Similar results were observed for the pathogenic *E. coli* O157:H7, which seem to change its enzymatic and metabolomic equipment in order to utilize plant compounds [101, 102]. In addition, the bacteria upregulate a plethora of genes related to attachment, antimicrobial resistance and response to oxidative stress [101]. Very striking was the fact that although plant filtrates or root exudates contain numerous amino acids, which are available to the bacteria as C and N sources, *E. coli* induced many amino acid synthesis pathways probably to supplement the missing compounds [102].

## **9. Detection, characterization and quantification of** *Salmonella* **in environmental samples**

mechanisms used in animal and plant hosts may resemble each other [59, 85, 86, 92–95]. The inoculation with the wild-type *Salmonella* strains and mutants in one or both of the T3SSs showed that functional secretion systems are required for efficient plant colonization [59, 85, 93]. Two observations allow such a conclusion: (1) The mutants had lower proliferation rates when compared to the respective wild type, which suggests that a functional T3SS helps with the colonization of plants; and (2) T3SS mutants induced stronger immune response of the host plant. Similar to animals or humans, plants respond to colonization of pathogenic bacteria inducing numerous immune responses, among others are oxidative burst and enhanced expression of *Pathogenesis Related* (*PR*) genes. Both were observed after inoculation with *Salmonella* and both were stronger if the inoculation was performed using mutants in T3SS [59, 85, 86]. Those results suggest that the wild‐type strain is able to suppress the immune response. It is very plausible to think that this suppression is due to functional T3SS‐dependent effector proteins. We know only little about their function in plant cells, since only two effectors (SseF and SpvC) were evaluated in this respect. SseF together with SseG are translocated into animal cells and are responsible for the establishment of the reproduction niche [96]. In plants, SseF induces the hypersensitive response (HR) [94]. Important is the fact that silencing of the suppressor of SGT1 eliminates the response to SseF, suggesting that this effector is recognized in R protein‐dependent manner, which is the usual recognition method of pathogen effectors during the effector‐triggered immunity (ETI). SpvC is a phosphothreonin lyase which dephosphorylates activated MAP kinases. Those kinases build a core compound in the signaling cascade leading from the perception of the pathogen on the cell surface to the transcriptional response at the chromatin level. Especially the trio MPK3, MPK4, and MPK6 plays an important role in plants [97], and is activated (phosphorylated) during the response to *Salmonella* [84]. SpvC interacts actively with the MPK6 and dephosphorylates this kinase, consequently abolishing the signal transduction [95]. A comprehensive overview of the reports regarding the plant immune responses to HPs was published only recently and is an excellent compendium of the cur-

**8.** *Salmonella* **changes its physiology in contact with plant host**

During the interaction between *Salmonella* and crop plants, not only the plant reacts to the presence of the bacteria, also *Salmonella* adapts to the conditions represented by a plant organism. Recent results show that bacteria modify their physiology and motility in order to adjust to the physiological conditions occurring in plants. Several authors evaluated the transcriptional changes of bacteria when in contact with plants or plant‐originated products [99, 100]. Interestingly, the analysis of the transcriptome, revealed a partial overlap between bacteria from macerated lettuce or cilantro leaves and bacteria from intestine, suggesting that those bacteria might be better adapted to the exploitation of plant material than estimated [100]. Similar results were observed for the pathogenic *E. coli* O157:H7, which seem to change its enzymatic and metabolomic equipment in order to utilize plant compounds [101, 102]. In addition, the bacteria upregulate a plethora of genes related to attachment, antimicrobial

rent knowledge [98].

180 Current Topics in Salmonella and Salmonellosis

*Salmonella* is rarely detected in crop plants. For example, in a previous study *Salmonella* could not be detected in more than 170 plants but in their environment [103]. This suggests that environmental factors are affecting the prevalence of *Salmonella* in the field, or that the sensitivity of the currently used detection system is not sufficient.

Traditional methods for the detection and identification of HPs often rely on cultivation‐ dependent techniques followed by biochemical and serological identification, which is typically time‐consuming and laborious [104]. Furthermore, in response to environmental stresses *Salmonella* can enter a physiological state where the cells remain viable, but are no longer culturable on typically used growth media. *Salmonella* in this VBNC state are often only detectable by methods depending on nucleic acids. This highlights the importance of the complementary use of cultivation-dependent and -independent detection methods for the diagnosis and prevention of food contamination and foodborne diseases. In the recent decades, there have been increasing efforts to develop and improve molecular methods for the rapid detection and characterization of pathogens in animals and animal products [105–108]. These methods, which include immunological as well as biosensor‐ and nucleic acid‐based assays (e.g., ELISA, PCR, microarrays, next generation sequencing) have an improved sensitivity and specificity but are also time‐, cost‐, and labor‐demanding. Typically, to further increase the sensitivity of these methods nonselective or selective enrichment steps are employed. One of the most challenging problems is the sample preparation, which is strongly depending on the sample matrix, associated inhibitory compounds, and the bacterial load.

So far, knowledge is scarce regarding the specific and reliable detection of *Salmonella* in complex and often heterogenous plant‐ and environmental‐matrices (e.g., vegetables, spices, soil samples, manure, biogas digestates) as well as the appropriate extraction and purification techniques. For iceberg lettuce, carrot‐ and cucumber‐peelings, qPCR detection limits of 10<sup>3</sup> bacterial cells per gram were reported [109]. Since the infectious dose of *Salmonella* was reported to be less than 10<sup>3</sup> cells [110], small numbers have to be detected reliably. Besides direct extraction of total DNA from the sample material, a preceding enrichment step in the respective culture media can be performed. This enrichment has the advantage to increase the sensitivity of detection and additionally to reactivate cells in the dormant VBNC state. After extraction of DNA from the respective samples, *Salmonella* can be detected by qPCR or PCR‐Southern blot hybridization, e.g., by detection of the *invA* gene [104, 111]. Alternatively or additionally to DNA‐based methods, RNA‐based methods can be used. Apart from a more laborious sample preparation, RNA-based methods have the advantage that in contrast to DNA-based methods only living and active *Salmonella* are detected, i.e., *Salmonella* relevant for a potential infection of humans. Especially the detection of mRNA of pathogenicity determinants could be appropriate to prove the viability and potential virulence of HPs.

Microarrays and next‐generation sequencing technologies offer intriguing possibilities regarding the rapid and accurate detection as well as genetic characterization of *Salmonella* in environmental matrices. However, the costs and technical requirements for the analysis of large data sets still limit their practicability in the day-to-day qualitative and quantitative detection. The further development of rapid, reliable, and cost‐effective high‐throughput detection methods will very likely contribute to the understanding of the ecology of *Salmonella* in the plant environment and consequently help to reduce or prevent infections mediated by plant-associated HPs.

## **10. Conclusions**

Today the notion that human pathogenic bacteria such as *Salmonella* might persist on or within plants in low numbers is widely accepted. The research on the interactions between crop or model plants and *Salmonella* is obviously driven by its medical aspects and the need for better prevention methods. We already know various features of the interactions but many are still not fully understood. New techniques that use high-throughput analyses and unbiased approaches are useful. Numerous national survey agencies started to use next‐generation sequencing for the epidemiological analysis of salmonellosis outbreaks and have therefore direct access to the genome sequences of particular serovars. They are also able to monitor the genomic changes, for example, reception of new plasmids or pathogenicity islands, which are important prerequisites in virulence of the bacterial strain. Similarly, the full range of "omic" approaches is being used in model systems providing very detailed data on both partners in the *Salmonella*-plant interaction at biochemical, physiological, and transcriptional levels. The study of those interactions harbors even more potential, it permits the characterization of the different infection mechanisms and the different strategies available for *Salmonella* in contact with diverse hosts. New and more efficient prevention strategies greatly depend on our understanding of these mechanisms. Therefore, the new findings might significantly improve our possibilities to diminish the number of future outbreaks.

## **Acknowledgements**

The authors would like to apologize to all colleagues whose work was not cited due to space limitation. This work was supported by the German Federal Environment Agency (Umweltbundesamt; 371271209), the JKI, and the Federal Office for Agriculture and Food (Bundesanstalt für Landwirtschaft und Ernährung, BLE), Grants 13HS026 and 13HS029.

## **Author details**

can be used. Apart from a more laborious sample preparation, RNA-based methods have the advantage that in contrast to DNA-based methods only living and active *Salmonella* are detected, i.e., *Salmonella* relevant for a potential infection of humans. Especially the detection of mRNA of pathogenicity determinants could be appropriate to prove the viability

Microarrays and next‐generation sequencing technologies offer intriguing possibilities regarding the rapid and accurate detection as well as genetic characterization of *Salmonella* in environmental matrices. However, the costs and technical requirements for the analysis of large data sets still limit their practicability in the day-to-day qualitative and quantitative detection. The further development of rapid, reliable, and cost‐effective high‐throughput detection methods will very likely contribute to the understanding of the ecology of *Salmonella* in the plant environment and consequently help to reduce or prevent infections mediated by

Today the notion that human pathogenic bacteria such as *Salmonella* might persist on or within plants in low numbers is widely accepted. The research on the interactions between crop or model plants and *Salmonella* is obviously driven by its medical aspects and the need for better prevention methods. We already know various features of the interactions but many are still not fully understood. New techniques that use high-throughput analyses and unbiased approaches are useful. Numerous national survey agencies started to use next‐generation sequencing for the epidemiological analysis of salmonellosis outbreaks and have therefore direct access to the genome sequences of particular serovars. They are also able to monitor the genomic changes, for example, reception of new plasmids or pathogenicity islands, which are important prerequisites in virulence of the bacterial strain. Similarly, the full range of "omic" approaches is being used in model systems providing very detailed data on both partners in the *Salmonella*-plant interaction at biochemical, physiological, and transcriptional levels. The study of those interactions harbors even more potential, it permits the characterization of the different infection mechanisms and the different strategies available for *Salmonella* in contact with diverse hosts. New and more efficient prevention strategies greatly depend on our understanding of these mechanisms. Therefore, the new findings might significantly improve our possibilities to diminish the

The authors would like to apologize to all colleagues whose work was not cited due to space limitation. This work was supported by the German Federal Environment Agency (Umweltbundesamt; 371271209), the JKI, and the Federal Office for Agriculture and Food (Bundesanstalt für Landwirtschaft und Ernährung, BLE), Grants 13HS026 and 13HS029.

and potential virulence of HPs.

182 Current Topics in Salmonella and Salmonellosis

plant-associated HPs.

**10. Conclusions**

number of future outbreaks.

**Acknowledgements**

Eva Fornefeld1 , Jasper Schierstaedt2 , Sven Jechalke3 , Rita Grosch2 , Kornelia Smalla1 and Adam Schikora1 \*

\*Address all correspondence to: adam.schikora@julius‐kuehn.de

1 Julius Kühn‐Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany

2 IGZ, Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren, Germany

3 Institute of Phytopathology, JLU Gießen, Gießen, Germany

## **References**


[27] Martiny JB, Bohannan BJM, Brown JH et al*.* Microbial biogeography: Putting microorganisms on the map. *Nat Rev Microbiol* 2006;**4**:102–112.

[12] Allende A & Monaghan J. Irrigation water quality for leafy crops: A perspective of risks

[13] Li B, Jackson SA, Gangiredla J et al*.* Genomic evidence reveals numerous Salmonella enterica serovar Newport reintroduction events in suwannee watershed irrigation

[14] Jacobsen CS & Bech TB. Soil survival of *Salmonella* and transfer to freshwater and fresh

[15] Barak JD & Liang AS. Role of soil, crop debris, and a plant pathogen in Salmonella

[16] Duffy EA, Lucia LM, Kells JM et al*.* Concentrations of *Escherichia coli* and genetic diversity and antibiotic resistance profiling of salmonella isolated from irrigation water, pack-

[17] Miles JM, Sumner SS, Boyer RR et al*.* Internalization of *Salmonella enterica* serovar montevideo into greenhouse tomato plants through contaminated irrigation water or seed

[18] Fonseca JM, Fallon SD, Sanchez CA et al*. Escherichia coli* survival in lettuce fields following its introduction through different irrigation systems. *J Appl Microbiol*

[19] Monaghan JM & Hutchison ML. Distribution and decline of human pathogenic bacteria in soil after application in irrigation water and the potential for soil-splash-mediated

[20] Semenov AM, Kuprianov AA & van Bruggen AH. Transfer of enteric pathogens to suc-

[21] Islam M, Morgan J, Doyle MP et al*.* Persistence of salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water. *Foodborne Pathog Dis*

[22] You Y, Rankin SC, Aceto HW et al*.* Survival of *Salmonella enterica* serovar Newport in manure and manure-amended soils. *Appl Environ Microbiol* 2006;**72**:5777–5783.

[23] Acea MJ, Moore CR & Alexander M. Survival and growth of bacteria introduced into

[24] Mallon CA, Elsas JD & Salles JF. Microbial invasions: the process, patterns, and mecha-

[25] Mallon CA, Poly F, Le Roux X et al*.* Resource pulses can alleviate the biodiversity–invasion relationship in soil microbial communities. *Ecol Soc Am* 2015;**96**:915–926.

[26] Fierer N & Jackson RB. The diversity and biogeography of soil bacterial communities.

ing shed equipment, and fresh produce in texas. *J Food Protect* 2005;**1**:70–79.

and potential solutions. *Int J Environ Res Public Health* 2015;**12**:7457–7477.

enterica contamination of tomato plants. *PLoS One* 2008;**3**:e1657.

dispersal onto fresh produce. *J Appl Microbiol* 2012;**112**:1007–1019.

cessive habitats as part of microbial cycles. *Microb Ecol* 2010;**60**:239–249.

ponds. *Appl Environ Microbiol* 2015;**81**:8243–8253.

produce. *Food Res Int* 2012;**45**:557–566.

184 Current Topics in Salmonella and Salmonellosis

stock. *J Food Protect* 2009;**4**:696–914.

soil. *Soil Biol Biochem* 1988;**20**:509–515.

nisms. *Trends Microbiol* 2015;**23**:719–729.

*Proc Natl Acad Sci USA* 2006;**103**:626–631.

2011;**110**:893–902.

2004;**1**:27–35.


[55] Caldwell KN, Anderson GL, Williams PL et al*.* Attraction of a free‐living nematode, *Caenorhabditis elegans*, to foodborne pathogenic bacteria and its potential as a vector of *Salmonella* Poona for preharvest contamination of cantaloupe. *J Food Protect* 2003;**66**:1964–1971.

[42] Bech TB, Johnsen K, Dalsgaard A et al*.* Transport and distribution of *Salmonella enterica* serovar Typhimurium in loamy and sandy soil monoliths with applied liquid manure.

[43] Horswell J, Hewitt J, Prosser J et al*.* Mobility and survival of *Salmonella Typhimurium* and human adenovirus from spiked sewage sludge applied to soil columns. *J Appl Microbiol*

[44] Semenov AV, van Overbeek L & van Bruggen AH. Percolation and survival of *Escherichia coli* O157:H7 and *Salmonella enterica* serovar Typhimurium in soil amended with con-

[45] Hutchison ML, Walters LD, Moore T et al*.* Fate of pathogens present in livestock wastes

[46] Nicholson FA, Groves SJ & Chambers BJ. Pathogen survival during livestock manure

[47] Hruby CE, Soupir ML, Moorman TB et al*.* Effects of tillage and poultry manure application rates on *Salmonella* and fecal indicator bacteria concentrations in tiles draining Des

[48] Semenov AV, van Overbeek L, Termorshuizen AJ et al*.* Influence of aerobic and anaerobic conditions on survival of *Escherichia coli* O157:H7 and *Salmonella enterica* serovar Typhimurium in Luria‐Bertani broth, farm‐yard manure and slurry. *J Environ Manage*

[49] Bennett DD, Higgins SE, Moore RW et al*.* Effects of lime on *Salmonella enteritidis* survival

[50] Ding GC, Radl V, Schloter‐Hai B et al*.* Dynamics of soil bacterial communities in response to repeated application of manure containing sulfadiazine. *Plos One*

[51] Franz E, van Diepeningen AD, de Vos OJ et al*.* Effects of cattle feeding regimen and soil management type on the fate of *Escherichia coli* O157:H7 and *Salmonella enterica* serovar Typhimurium in manure, manure‐amended soil, and lettuce. *Appl Environ Microbiol*

[52] Moynihan EL, Richards KG, Brennan FP et al*.* Enteropathogen survival in soil from different land-uses is predominantly regulated by microbial community composition. *Appl* 

[53] Goberna M, Podmirseg SM, Waldhuber S et al*.* Pathogenic bacteria and mineral N in soils following the land spreading of biogas digestates and fresh manure. *Appl Soil Ecol*

[54] Ge C, Lee C, Nangle E et al*.* Impact o f phytopathogen infection and extreme weather stress on internalization of *Salmonella* Typhimurium in lettuce. *Int J Food Microbiol*

taminated dairy manure or slurry. *Appl Environ Microbiol* 2009;**75**:3206–3215.

storage and following land application. *Bioresour Technol* 2005;**96**:135–143.

spread onto fescue plots. *Appl Environ Microbiol* 2005;**71**:691–696.

Moines Lobe soils. *J Environ Manage* 2016;**171**:60–69.

in vitro. *J Appl Poult Res* 2003;**12**:65–68.

*Appl Environ Microbiol* 2010;**76**:710–714.

2010;**108**:104–114.

186 Current Topics in Salmonella and Salmonellosis

2011;**92**:780–787.

2014;**9(3)**:e92958.

2005;**71**:6165–6174.

*Soil Ecol* 2015;**89**:76–84.

2011;**49**:18–25.

2014;**168**–**169**:24–31.


[85] Shirron N & Yaron S. Active suppression of early immune response in tobacco by the human pathogen *Salmonella Typhimurium*. *PLoS One* 2011;**6**:e18855.

[70] Barak JD, Gorski L, Naraghi‐Arani P et al*. Salmonella enterica* virulence genes are required for bacterial attachment to plant tissue. *Appl Environ Microbiol* 2005;**71**:5685–5691. [71] Klerks MM, van Gent‐Pelzer M, Franz E et al*.* Physiological and molecular responses of *Lactuca sativa* to colonization by *Salmonella enterica* serovar Dublin. *Appl Environ Microbiol*

[72] Steenackers H, Hermans K, Vanderleyden J et al*. Salmonella* biofilms: An overview on occurrence, structure, regulation and eradication. *Food Res Int* 2012;**45**:502–531.

[73] Gerstel U & Romling U. The *csgD* promoter, a control unit for biofilm formation in

[74] Cevallos-Cevallos JM, Gu G, Danyluk MD et al*.* Adhesion and splash dispersal of *Salmonella enterica* Typhimurium on tomato leaflets: effects of rdar morphotype and tri-

[75] Cevallos-Cevallos JM, Gu G, Danyluk MD et al*. Salmonella* can reach tomato fruits on plants exposed to aerosols formed by rain. *Int J Food Microbiol* 2012;**158**:140–146.

[76] Barak JD, Gorski L, Liang AS et al*.* Previously uncharacterized *Salmonella enterica* genes required for swarming play a role in seedling colonization. *Microbiology*

[77] Lapidot A & Yaron S. Transfer of *Salmonella enterica* serovar Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose, the biofilm

[78] Holmes A, Birse L, Jackson RW et al*.* An optimized method for the extraction of bacterial mRNA from plant roots infected with *Escherichia coli* O157:H7. *Front Microbiol* 2014;**5**:286.

[79] Golberg D, Kroupitski Y, Belausov E et al*. Salmonella Typhimurium* internalization is variable in leafy vegetables and fresh herbs. *Int J Food Microbiol* 2011;**145**:250–257.

[80] Barak JD, Kramer LC & Hao LY. Colonization of tomato plants by *Salmonella enterica* is cultivar dependent, and type 1 trichomes are preferred colonization sites. *Appl Environ* 

[81] Gu G, Hu J, Cevallos-Cevallos JM et al*.* Internal colonization of *Salmonella enterica* serovar

[82] Berg G, Erlacher A, Smalla K et al*.* Vegetable microbiomes: Is there a connection among opportunistic infections, human health and our 'gut feeling'? *Microb Biotechnol*

[83] Erlacher A, Cardinale M, Grosch R et al*.* The impact of the pathogen *Rhizoctonia solani* and its beneficial counterpart *Bacillus amyloliquefaciens* on the indigenous lettuce micro-

[84] Schikora A, Carreri A, Charpentier E et al*.* The dark side of the salad: *Salmonella Typhimurium* overcomes the innate immune response of Arabidopsis thaliana and shows

*Salmonella* Typhimurium. *Res Microbiol* 2003;**154**:659–667.

chome density. *Int J Food Microbiol* 2012;**160**:58–64.

matrix components. *J Food Prot* 2009;**72**:618–623.

Typhimurium in tomato plants. *PLoS One* 2011;**6**:e27340.

an endopathogenic lifestyle. *PLoS One* 2008;**3**:e2279.

2007;**73**:4905–4914.

188 Current Topics in Salmonella and Salmonellosis

2009;**155**:3701–3709.

*Microbiol* 2011;**77**:498–504.

biome. *Front Microbiol* 2014;**5**:175.

2014;**7**:487–495.


[100] Goudeau DM, Parker CT, Zhou Y et al*.* The *Salmonella* transcriptome in lettuce and cilantro soft rot reveals a niche overlap with the animal host intestine. *Appl Environ* 

[101] Kyle JL, Parker CT, Goudeau D et al*.* Transcriptome analysis of *Escherichia coli* O157:H7 exposed to lysates of lettuce leaves. *Appl Environ Microbiol* 2010;**76**:1375–1387.

[102] Crozier L, Hedley PE, Morris J et al*.* Whole‐transcriptome analysis of verocytotoxigenic *Escherichia coli* O157:H7 (Sakai) suggests plant‐species‐specific metabolic responses on

[103] Micallef SA, Rosenberg Goldstein RE, George A et al*.* Occurrence and antibiotic resistance of multiple Salmonella serotypes recovered from water, sediment and soil on

[104] Hein I, Flekna G, Krassnig M et al*.* Real-time PCR for the detection of *Salmonella* spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD‐

[105] Park SH, Aydin M, Khatiwara A et al*.* Current and emerging technologies for rapid detection and characterization of *Salmonella* in poultry and poultry products. *Food* 

[106] Mandal PK, Biswas AK, Choi K et al*.* Methods for rapid detection of foodborne patho-

[107] Law JWF, Ab Mutalib NS, Chan KG et al*.* Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. *Front* 

[108] Zhao X, Lin CW, Wang J et al*.* Advances in rapid detection methods for foodborne

[109] Vojkovska H, Kubikova I & Kralik P. Evaluation of DNA extraction methods for PCR-based detection of *Listeria monocytogenes* from vegetables. *Lett Appl Microbiol*

[110] Blaser MJ & Newman LS. A review of human salmonellosis: I. Infective dose. *Rev Infect* 

[111] Nastasi A, Mammina C & Mioni R. Detection of *Salmonella* spp. in food by a rapid PCR-

[112] MDH. Update: Tomatoes identified as source of Salmonella outbreak in restaurant

[113] CDC. *Salmonella* outbreaks. Available at http://www.cdc.gov/Salmonella/outbreaks.

exposure to spinach and lettuce extracts. *Front Microbiol* 2016;**7:**1088.

mid-Atlantic tomato farms. *Environ Res* 2012;**114**:31–39.

PCR project. *J Microbiol Methods* 2006;**66**:538–547.

gens: an overview. *Am J Food Technol* 2011;**6**:87–102.

pathogens. *J Microbiol Biotechnol* 2014;**24**:297–312.

hybridization procedure. *Microbiologica* 1999;**22**:195–202.

[114] CDHD. Health department investigating recent salmonella cases. 2015.

*Microbiol* 2013;**79**:250–262.

190 Current Topics in Salmonella and Salmonellosis

*Microbiol* 2014;**38**:250–262.

*Microbiol* 2015;**5**:19.

2015;**60**:265–272.

chain. 2015.

*Dis* 1982;**4**:1096–1106.

html. Accessed July 2016:177–188.

[119] IDPH. Summary of *S*. ser. Hvittingfoss outbreak April‐June 2010. 2010.

#### **Preharvest** *Salmonella* **Risk Contamination and the Control Strategies Preharvest** *Salmonella* **Risk Contamination and the Control Strategies**

Rebeca Zamora-Sanabria and Rebeca Zamora-Sanabria and Andrea Molina Alvarado

Andrea Molina Alvarado

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67399

#### **Abstract**

*Salmonella* is present in most food production environments and can enter the food supply at any stage of food production from farm to fork. Control strategies for *Salmonella* include preharvest and postharvest aspects. Preharvest approach is very important because as a result of large-scale production, many animals could be infected with *Salmonella* serotypes during the primary production, causing human salmonellosis by consuming meat, milk, and eggs or foods containing ingredients of animal origin. The first step for prevention approaches is to determinate the source of infection; *Salmonella* serovars should be founded, and control strategies must be executed. Infection sources include vertical transmission, feed, pest (rodents and insects), wild birds, water, humans, manure, transportation coops, tractors or vehicles, and farm environment. Preventive and control strategies involve many factors, including hygiene, biosecurity procedures, animal feed surveillance, litter, manure and carcasses disposed, cleaning and disinfection programs, food interventions, diagnostic, and vaccination.

**Keywords:** *Salmonella*, preharvest, farm to fork approach, surveillance, sources of infection, biosecurity, feedstuffs, cleaning and disinfection, pest control, water safety, vaccination, litter and carcasses disposal

## **1. Introduction**

Salmonellosis is one of the most common food-borne bacterial diseases in the world. In most food animal species, *Salmonella* can establish a clinically unapparent infection of variable duration, which is significant as a potential zoonosis.

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Human food-borne salmonellosis has increased in association with the development of food industry. Food industry is based on large-scale animal production. Food processing plants have grown larger, and when there is a salmonellosis outbreak, it will infect many more people than in the past. In addition, there has been a change in dining habits of consumers, and a high proportion of meals are eaten at institutions, restaurants, and fast food places. These establishments are often a significant link and amplifier of *Salmonella* infections.

*Salmonella* is present in most food production environments and can enter the food supply at any stage of food production from *farm to fork*. Control strategies for *Salmonella* include preharvest and postharvest aspects. Most control strategies for *Salmonella* are focused on specific aspects of food production or processing and are generally assessed on their ability to reduce levels of *Salmonella* spp. at the processing stage.

Nevertheless, preharvest approach is very important because as a result of large-scale production, many animals are placed in small area producing a lot of feces. Several *Salmonella* serovars that are not host specific may colonize the digestive tract of animals, provoking human salmonellosis by consuming meat, milk, and egg or food containing ingredients of animal origin.

Animal feed (and ingredients therein) has been described as a source of *Salmonella* infection for animals and humans, through the contamination of food products of animal origin. This threat is aggravated due to the bacteria capability to persist for long periods in a wide variety of feedstuffs. Therefore, animal feed may serve as vehicle to introduce *Salmonella* serovars into the food chain and could contribute to the circulation and spreading of antimicrobial-resistant bacteria or antimicrobial-resistant genes.

At the farm, level food safety programs involve many factors such as hygiene, biosecurity procedures, animal feed surveillance, litter and carcasses disposal, depopulation, cleaning, disinfection programs, food interventions, diagnostic, and vaccination. The source of infection should be determined. At the end of the production, animals should be sent to slaughter with special precaution, and they should be healthy to prevent contamination during the processing.

Other strategies should be taken during the transport and time of slaughter to decrease *Salmonella* contamination. A good food safety program should include the entire food chain of production; however, the aim of this chapter is to describe preharvest *Salmonella* risk contamination factors including *Salmonella* prevalence in animal feedstuffs and the control strategies and interventions.

#### **1.1. Farm-to-fork concept**

"Farm to fork" is a strategy to prevent food-borne hazards. This approach is based in many measures to trace the different stages of the food chain. "Farm-to-fork" system examines the practices and procedures that ensure food safety.

The procedures to prevent *Salmonella* contamination in the food chain comprise many events, from the primary production to the final consumer. *Salmonella* contamination events can occur during different parts of the food chain which included primary production, processing, distribution, preparation, and dining habits of consumers.

In 2003, Food and Agriculture Organization (FAO) of the United Nations [1] showed the importance about a new approach in food-borne hazards which it had called "food chain approach." Its objective is to ensure that the food is free from borne hazards: pesticides, chemicals, bacteria, and others contaminants. Every food chain step has to be analyzed: growing, raising, production, collecting, processing, packing, commercialization, and consumption.

The FAO and World Health Organization (WHO) [2] have produced guidance documents for use by governmental authorities on food-borne outbreak investigation [3]. They suggest that good control measurements at the farm level are likely to correspond with lower prevalence of *Salmonella* infection and, subsequently, a reduction of cross contamination of carcasses processed at the slaughterhouse and a reduction in human salmonellosis.

#### **1.2. One health**

Human food-borne salmonellosis has increased in association with the development of food industry. Food industry is based on large-scale animal production. Food processing plants have grown larger, and when there is a salmonellosis outbreak, it will infect many more people than in the past. In addition, there has been a change in dining habits of consumers, and a high proportion of meals are eaten at institutions, restaurants, and fast food places. These

*Salmonella* is present in most food production environments and can enter the food supply at any stage of food production from *farm to fork*. Control strategies for *Salmonella* include preharvest and postharvest aspects. Most control strategies for *Salmonella* are focused on specific aspects of food production or processing and are generally assessed on their ability to reduce

Nevertheless, preharvest approach is very important because as a result of large-scale production, many animals are placed in small area producing a lot of feces. Several *Salmonella* serovars that are not host specific may colonize the digestive tract of animals, provoking human salmonellosis by consuming meat, milk, and egg or food containing ingredients of animal origin. Animal feed (and ingredients therein) has been described as a source of *Salmonella* infection for animals and humans, through the contamination of food products of animal origin. This threat is aggravated due to the bacteria capability to persist for long periods in a wide variety of feedstuffs. Therefore, animal feed may serve as vehicle to introduce *Salmonella* serovars into the food chain and could contribute to the circulation and spreading of antimicrobial-resistant

At the farm, level food safety programs involve many factors such as hygiene, biosecurity procedures, animal feed surveillance, litter and carcasses disposal, depopulation, cleaning, disinfection programs, food interventions, diagnostic, and vaccination. The source of infection should be determined. At the end of the production, animals should be sent to slaughter with special precaution, and they should be healthy to prevent contamination during the processing. Other strategies should be taken during the transport and time of slaughter to decrease *Salmonella* contamination. A good food safety program should include the entire food chain of production; however, the aim of this chapter is to describe preharvest *Salmonella* risk contamination factors including *Salmonella* prevalence in animal feedstuffs and the control strate-

"Farm to fork" is a strategy to prevent food-borne hazards. This approach is based in many measures to trace the different stages of the food chain. "Farm-to-fork" system examines the

The procedures to prevent *Salmonella* contamination in the food chain comprise many events, from the primary production to the final consumer. *Salmonella* contamination events can occur during different parts of the food chain which included primary production, processing,

establishments are often a significant link and amplifier of *Salmonella* infections.

levels of *Salmonella* spp. at the processing stage.

194 Current Topics in Salmonella and Salmonellosis

bacteria or antimicrobial-resistant genes.

gies and interventions.

**1.1. Farm-to-fork concept**

practices and procedures that ensure food safety.

distribution, preparation, and dining habits of consumers.

Also, Codex Alimentarius (CA) standards and risk analysis methodologies are recognized in the area for food safety. The CA and the World Organization for Animal Health (OIE) are working together to develop their respective standards for food-borne zoonosis so that they are non-duplicative, cohesive, and will cover the whole food chain [3].

Primary production is focused in animal health, livestock, housing management, animal food quality, animal welfare, and transportation regarding for food processing.

Farming practices or primary production vary widely according to soil and climatic conditions, social conditions, cost of the feedstuffs, potential marketability of specific farm products, and the economic objectives of the farmer. However, there are general control strategies to prevent the entrance of *Salmonella* in primary production.

In spite of those production measurements, bacteria can enter anywhere in the food chain, causing animal disease and food contamination. One of the major sources of *Salmonella* in the food chain has been animal feed, especially swine and poultry. It is a major cause of economic loss in swine production [4] and has a great economic significance to the poultry industry around the world.

*Salmonella* could be a risk to public health through consumption of contaminated eggs and meat. These bacteria causes diarrheal diseases in humans [5] and high mortality in animals, like chickens. Other farm animals as cattle and sheep suffer disease, could become *Salmonella* reservoirs, and contribute as vector in the transmission.

## **2. Sources of infection**

*Salmonella* genus is a group of microorganisms that are successfully adapted to live in very different environmental conditions [6]. For this reason, it is easy to find many potential sources of contamination, and control could be complicated. These sources include vertical transmission, feed, pest (rodents and insects), wild birds, water, humans, manure, transportation coops, tractors or vehicles, and farm environment. There are also some variables that contribute *Salmonella* contamination, such as age of the animal, survival of the bacteria through the gastric barrier, competing bacteria in the intestinal tract, availability of a hospitable colonization site, the diet, physiological status, health, disease, and medications [7].

Identifying animal sources of infection, target interventions, and control measurements is the correct approach for preventing *Salmonella*; every source should be considered. Risk assessment studies have recommended an intervention for a productive overall approach.

#### **2.1. Transmission**

*Salmonella* is extremely widespread and very persistent in the environment. It is recovered from many vertebrates which included many farm animal species. Serovars of *Salmonella enterica* have varied hosts and reservoirs, cause disease in animals and humans, and can move between host species [5] because most of them are nonhost specific (**Table 1**).


**Table 1.** *Salmonella enterica* subsp. *enterica* common serovars in farm animals.

In farm animals *Salmonella* cause clinical disease, and there are also asymptomatic animals called carriers, e.g., *Salmonella* subclinical infections persist in hens more than 22 weeks [8]. Carrier pigs are important as the initial source of contamination of the environment, other animals, and carcasses in the harvest [9]. Monitoring programs in the USA suggest that 20% of broiler chickens are contaminated with harmful *Salmonella* strains [6] and 27% incidence was found in feces in organic pig farms [10]. They are very important in the transmission because they can shed *Salmonella* in feces continuously and intermittently in the absence of clinical signs. Pets such as dogs and cats [11] show asymptomatic infections and could shed *Salmonella* and contaminated food-producing animals.

There is a different *Salmonella* susceptibility in farm animals. Stressors can aggravate *Salmonella* shedding, including mixing, climate, transportation, and food deprivation. Some results suggest that the duration of *Salmonella* shedding might depend on serotypes, strain, animal age, farm, or others risk factors [10].

Horizontal transmission also occurs by fecal-oral route or by aerogenous transmission. In pigs oropharyngeal secretions can contaminate and spread the disease via nose to nose [12]. *Salmonella* can be introduced in a herd through new purchased and infected pigs. There is evidence of bacterial spread by feed, drinking water, fomites, asymptomatic carriers, and dry feces from infected animals with clinical disease.

Vertical transmission is crucial in poultry related infected with *S. enterica* subspecies *enterica* serovars Enteritidis, Typhimurium, Gallinarum, Heidelberg, and Infantis [13]. *Salmonella* produces persistent infection in birds, located in the ovary [13]. Transmission to progeny occurs by transovarian infection, when the ovary and the developing eggs became infected in the oviduct. Bacteria migrate inside the yolk before shell deposition. *Salmonella* enteritidis can also get access to eggs by migrating from the cloaca to the reproductive organs. *S. enterica* subsp. *enterica* serovar Heidelberg was the most common serovar founded in ovaries in layers in Canada [8]; there is evidence supporting vertical transmission of *Salmonella* in dairy cattle [14]. *Salmonella* might be transmitted vertically from the dam to her fetus in utero. Calves might be infected with *Salmonella* at birth or post birth.

If progeny persists infected, there is no chance of eradication, and the control becomes complicated. From a public health point of view, the number of eggs and animals affected by *Salmonella* is a risk for a human disease or infection.

#### **2.2. Feed**

competing bacteria in the intestinal tract, availability of a hospitable colonization site, the diet,

Identifying animal sources of infection, target interventions, and control measurements is the correct approach for preventing *Salmonella*; every source should be considered. Risk assess-

*Salmonella* is extremely widespread and very persistent in the environment. It is recovered from many vertebrates which included many farm animal species. Serovars of *Salmonella enterica* have varied hosts and reservoirs, cause disease in animals and humans, and can move

polyarthritis

septicemia

depressed milk yield Calves: enteritis, arthritis,

Adults: abortion, gastroenteritis, pneumonia Lams: gastroenteritis, pneumonia,

Frequently is a subclinical disease Adults: diarrhea, enteritis abortion,

Frequently is a subclinical disease Gallinarum and pullorum (nonmotile):

Abortion, diarrhea, typhlitis, colitis, arthritis, nosocomial infections

Septicemia and enterocolitis, pigs 6–8 weeks [10]

Others strains: asymptomatic

meningoencephalitis, respiratory signs

[59]

[59] [60] [61]

[62] [63]

[26] [59] [64]

[19] [64]

ment studies have recommended an intervention for a productive overall approach.

between host species [5] because most of them are nonhost specific (**Table 1**).

**Farm animal** *S. enterica* **subsp.** *enterica* **serovar Clinical signs Authors**

physiological status, health, disease, and medications [7].

**2.1. Transmission**

196 Current Topics in Salmonella and Salmonellosis

Sheep Brandenburg

Cattle Dublin

Abortusovis Dublin Arizonae Typhimurium

Typhimurium Montevideo Brandenburg Enteritidis Panamá Heidelberg Kentucky

Poultry Enteritidis, Typhimurium Paratyphi B Heidelberg Kentucky Infantis

Pig Typhimurium

Gallinarum, pullorum

**Table 1.** *Salmonella enterica* subsp. *enterica* common serovars in farm animals.

Choleraesuis Derby Enteritidis Istanbul Mbandaka Agona Heidelberg

Horse Typhimurium Newport Enteritidis St Paul Agona Anatum Heidelberg

Animal feed is a recognized source of *Salmonella* for farm livestock. Bacteria can be introduced into the feed by contaminated feedstuffs, processing, transport, storage, distribution, and administration due to dirty feeders.

*Salmonella* can be isolated frequently from animal-feed ingredients, such as meat bone meals and fish meals. Few quantities of *Salmonella* cause infection, less than one *Salmonella* per gram of feed has been shown to establish colonization in 1- to 7-day-old chicks [15].

*Salmonella* could be isolated from feedstuff in 17.6% of pig herds among five EU countries and from 6.9% of all feed samples [16], and also it can survive at least 26 months in artificially contaminated poultry food [17].

#### **2.3. Farm environment**

Farm *Salmonella* eradication is a complicated strategy, and its control could be difficult because there are numerous potential source environment. It is able to grow between 7 and 45°C, is destroyed at 65°C during 10–15 minutes, and resists every acid pH and salt added in food up to 20% [6].

Animals are the major reservoir of *Salmonella*; dissemination into environment has resulted from the human practices and animal behavior. *Salmonella* may be present in any waste from human or animal activities; it survives in frozen food and remains viable during years in the environment. In broiler houses, microorganisms could persist for at least 1 year [18]. *Salmonella* is shed efficiently in feces, persists within the environment, and is spread readily between food-producing animals in the farm environment. *Salmonella* can survive desiccation and persist for many months in association with dust particles on fans, floors, and feed deposits.

This microorganism can survive and replicate for long periods in different environments, although the original fecal source may be remote in time. For instance, *S. enterica* subsp. *enterica* serovar Choleraesuis persists in dry feces 13 months post shedding and after disinfection process and survives in soil between 25 and 200 days [5].

Bailey et al. [7] found that the environment was the primary source of contaminating *Salmonella* in chicken houses not treated with competitive exclusion microflora. They recovered high rates of *Salmonella* from feces, litter, and near the entrance doors to the poultry houses. Hatchery transport paper pads were the most frequently observed *Salmonella* positive in this research. Salmonellosis is also commonly observed in contaminated facilities in veterinary hospitals [19]. Barns, pens, dust, egg belts, feeders, fans, feed bins, vehicles, and equipment can be contaminated.

Survival capacity, environment persistence, and infection may be influenced by different genetic, productive, and environmental factors such as intensification of handling practices, reduction in genetic diversity of breeding stock, and increasing standardization of food types [5].There is a differential distribution of specific serovars and genotypes between animals and environments. Certain serovars have a greater ability to establish infection, shedding patterns, and concentration. In pigs, *S. enterica* subsp. *enterica* serovar Typhimurium was more frequently isolated from the manure compared to other bacteria [20].

#### **2.4. Water**

Contaminated water supplies have been implicated in the introduction and persistence of *Salmonella*. Contaminated waters might contribute through direct ingestion of the water or via indirect contamination of the surfaces. In a review [21], they found *Salmonella* in different countries and in very diverse water sources. *Salmonella* contamination occurred in surface water used for recreational purposes, as source of drinking water and for irrigation. They detected a mixed of human and animal origin of *Salmonella* serovars in drinking water sources.

In artificial freshwater systems, *Salmonella* and *Escherichia coli* survived for at least 56 days [5]. Factor contributes to *Salmonella* resistance, and persistence in water is its capacity to attach to different types of plastic, glass, cement, rubber, and stainless steel or biotic surfaces (plant surfaces, epithelial cells, and gallstones) [6]. *Salmonella* forms a complex called biofilm inside drinkers and pipes. This biofilm is a bacteria surface-associated formation that allows bacteria to resist against different stress factors such as desiccation, disinfectants, and antibiotics [22].

#### **2.5. Pest**

*Salmonella* could be isolated from feedstuff in 17.6% of pig herds among five EU countries and from 6.9% of all feed samples [16], and also it can survive at least 26 months in artificially

Farm *Salmonella* eradication is a complicated strategy, and its control could be difficult because there are numerous potential source environment. It is able to grow between 7 and 45°C, is destroyed at 65°C during 10–15 minutes, and resists every acid pH and salt added in food up

Animals are the major reservoir of *Salmonella*; dissemination into environment has resulted from the human practices and animal behavior. *Salmonella* may be present in any waste from human or animal activities; it survives in frozen food and remains viable during years in the environment. In broiler houses, microorganisms could persist for at least 1 year [18]. *Salmonella* is shed efficiently in feces, persists within the environment, and is spread readily between food-producing animals in the farm environment. *Salmonella* can survive desiccation and persist for many months in association with dust particles on fans, floors, and feed

This microorganism can survive and replicate for long periods in different environments, although the original fecal source may be remote in time. For instance, *S. enterica* subsp. *enterica* serovar Choleraesuis persists in dry feces 13 months post shedding and after disinfec-

Bailey et al. [7] found that the environment was the primary source of contaminating *Salmonella* in chicken houses not treated with competitive exclusion microflora. They recovered high rates of *Salmonella* from feces, litter, and near the entrance doors to the poultry houses. Hatchery transport paper pads were the most frequently observed *Salmonella* positive in this research. Salmonellosis is also commonly observed in contaminated facilities in veterinary hospitals [19]. Barns, pens, dust, egg belts, feeders, fans, feed bins, vehicles, and

Survival capacity, environment persistence, and infection may be influenced by different genetic, productive, and environmental factors such as intensification of handling practices, reduction in genetic diversity of breeding stock, and increasing standardization of food types [5].There is a differential distribution of specific serovars and genotypes between animals and environments. Certain serovars have a greater ability to establish infection, shedding patterns, and concentration. In pigs, *S. enterica* subsp. *enterica* serovar Typhimurium was more

Contaminated water supplies have been implicated in the introduction and persistence of *Salmonella*. Contaminated waters might contribute through direct ingestion of the water or via indirect contamination of the surfaces. In a review [21], they found *Salmonella* in different

tion process and survives in soil between 25 and 200 days [5].

frequently isolated from the manure compared to other bacteria [20].

contaminated poultry food [17].

198 Current Topics in Salmonella and Salmonellosis

equipment can be contaminated.

**2.3. Farm environment**

to 20% [6].

deposits.

**2.4. Water**

#### *2.5.1. Rodents*

Mice and rats are involved in the transmission and the perpetuation of the infection in the farm buildings and facilities. Rodents can be long-term sources of *Salmonella* infection. Their droppings can be contaminated for up to 3 months for infection. A study found that 3 weeks old chicks became infected via mice artificially contaminated with *S. enterica subsp. enterica* serovar Enteritidis 5 months before [23].

Mice travel from one farm to another; they leave empty farms or facilities and return after cleaning and disinfection activities. They have also good reproductive capacity and can spread *Salmonella* for one flock to other flocks or herds. They contribute to perpetuate infections. Rodents are important vectors and amplifiers of *Salmonella* infection in farm animals, e.g., mouse fecal pellets have been shown to contain up to 104 CFU of *Salmonella* [23, 24]. One single mouse can shed 100 fecal pellets per day [24]. Fecal pellets are seed shaped; pigs and chicken eat these pellets and become infected. On a clean pig farm, 5–10% rodents can be found infected with *Salmonella* [25, 26]. Isolates from contaminated mice contained three times more *Salmonella* than isolates from environment of contaminated house samples [24]. The presence of a mouse-infected population is an important risk for animal and product contamination. Layer farms with high rodent densities showed more *S. enterica* subsp. *enterica* serovar Enteritidis and serovar Infantis isolations and hens infected than farms with low rodent densities [27].

Rats, mice, and cats are associated with contamination of water, food, and grains stored. They carry bacteria in their intestinal tracts without clinical symptoms and disease and cause transmission of pathogens to farm animal feed and environment. Rodents acquire the infection from feces of sick animals, wild animals, and members of their family [23]; they also get infected from outdoor paddocks and inaccessible feces-contaminated parts of the livestock houses.

The environment conditions around facilities attract rodent, e.g., waste, spilled food and feedstuffs, sources of water, and abilities to build dens. Dead mice also can be a contamination source if they remain in the barns of houses after cleaning and disinfection procedures.

#### *2.5.2. Darkling beetles, flies, mites, ticks, and cockroaches*

*Salmonella* is widely distributed in flies and less in beetle and mites of affected livestock units. Farms offer great and suitable niches as manure, dust, spilled food, and long production periods of time without cleaning.

Flies act as mechanical vector; the *Musca domestica* is most prevalent in farms and associates with zoonosis. They perform diurnal excursions around animal houses and can fly many miles from the farms contributing with *Salmonella* dissemination. Heavy fly populations have been identified as a risk factor for *Salmonella* in poultry, dairy cattle, swine, and feedlot cattle [28]. Authors report that flies carry *S. enterica* subsp. *enterica* serovar Typhimurium for up to 10 days [26]. Flies become contaminated from environment, and animals ingesting contaminated flies get infected. There is not enough evidence of flies as biological vector (*Salmonella* multiplication inside the flies).

Darkling beetle *Alphitobius diaperinus* is a very common pest in poultry houses. They carry and shed by defecation variety of microorganisms which included *Salmonella*. Beetles survive cleaning and disinfection because they hide in inaccessible poultry house structures and outside of the poultry buildings. They drill wall cavities complicating insecticides access. Chickens can ingest contaminated beetle larvae and adults and become infected. *Salmonella* isolates from beetles are usually lower than isolates from flies [29].

Mites can acquire and transmit *Salmonella*. The most frequently mites founded in poultry are *Dermanyssus gallinae* (red mite), *Ornithonyssus sylviarum*, and *Ornithonyssus bursa.* They are usually present in manure, litter, and feed. Adults and nymphs of ticks visit poultry houses only to feed; adults can survive for months or years at swine or poultry facilities. A *Salmonella* vector role for ticks remains speculative.

Cockroaches will opportunistically colonize animal facilities and carry bacteria. They have been reported to carry *Salmonella* [30] and can transmit these bacteria to other cockroaches and to eggshells.

#### **2.6. Wild animals**

Wild bird and little mammals are regarded as the main reservoir for *Salmonella* in the environment. Wildlife vectors may be responsible for the introduction of some Salmonellae to farms.

Birds as pigeons, sparrows [5], foxes [31], shrews, reptiles, and other wild animals have a potential role in the *Salmonella* dissemination [29]. The spread or recycling of *Salmonella* infection among livestock may occur through the contamination of water or feed or the direct contamination of the environment. Building, houses, and barns should be constructed to block wild animal access. Birds cannot nest and reproduce in the houses to prevent bacterial contamination.

#### **2.7. Humans**

Human traffic on the farm increases the risk of infection in pigs, chickens, and hens. The entrance of visitors was associated with higher *Salmonella* prevalence [32].

People transport pathogens from their nose, hair, throat, pharynx, clothes, and shoes. They also could have *Salmonella* in their intestine; therefore, having access to toilets and washing facilities have a protective effect against *Salmonella* [9].

## **3. Surveillance and prevalence of** *Salmonella* **in animal feedstuffs**

*2.5.2. Darkling beetles, flies, mites, ticks, and cockroaches*

ods of time without cleaning.

200 Current Topics in Salmonella and Salmonellosis

*Salmonella* is widely distributed in flies and less in beetle and mites of affected livestock units. Farms offer great and suitable niches as manure, dust, spilled food, and long production peri-

Flies act as mechanical vector; the *Musca domestica* is most prevalent in farms and associates with zoonosis. They perform diurnal excursions around animal houses and can fly many miles from the farms contributing with *Salmonella* dissemination. Heavy fly populations have been identified as a risk factor for *Salmonella* in poultry, dairy cattle, swine, and feedlot cattle [28]. Authors report that flies carry *S. enterica* subsp. *enterica* serovar Typhimurium for up to 10 days [26]. Flies become contaminated from environment, and animals ingesting contaminated flies get infected. There is not enough evidence of flies as biological vector (*Salmonella* multiplication inside the flies).

Darkling beetle *Alphitobius diaperinus* is a very common pest in poultry houses. They carry and shed by defecation variety of microorganisms which included *Salmonella*. Beetles survive cleaning and disinfection because they hide in inaccessible poultry house structures and outside of the poultry buildings. They drill wall cavities complicating insecticides access. Chickens can ingest contaminated beetle larvae and adults and become infected. *Salmonella*

Mites can acquire and transmit *Salmonella*. The most frequently mites founded in poultry are *Dermanyssus gallinae* (red mite), *Ornithonyssus sylviarum*, and *Ornithonyssus bursa.* They are usually present in manure, litter, and feed. Adults and nymphs of ticks visit poultry houses only to feed; adults can survive for months or years at swine or poultry facilities. A *Salmonella*

Cockroaches will opportunistically colonize animal facilities and carry bacteria. They have been reported to carry *Salmonella* [30] and can transmit these bacteria to other cockroaches

Wild bird and little mammals are regarded as the main reservoir for *Salmonella* in the environment. Wildlife vectors may be responsible for the introduction of some Salmonellae to farms. Birds as pigeons, sparrows [5], foxes [31], shrews, reptiles, and other wild animals have a potential role in the *Salmonella* dissemination [29]. The spread or recycling of *Salmonella* infection among livestock may occur through the contamination of water or feed or the direct contamination of the environment. Building, houses, and barns should be constructed to block wild animal access. Birds cannot nest and reproduce in the houses to prevent bacterial contamination.

Human traffic on the farm increases the risk of infection in pigs, chickens, and hens. The

entrance of visitors was associated with higher *Salmonella* prevalence [32].

isolates from beetles are usually lower than isolates from flies [29].

vector role for ticks remains speculative.

and to eggshells.

**2.6. Wild animals**

**2.7. Humans**

Animal feedstuff could serve as vehicle for *Salmonella* serovars into the farm environment and cause animal infection that could reach the human consumer through animal food products. As we already mentioned above, *Salmonella* has the capability to survive in a vast variety of commodities and to resist desiccation among other adverse conditions. During our work and research in *Salmonella* surveillance in animal feed, we have seen that *Salmonella* has the ability to remain in different animal feedstuffs for long time periods; this has been also confirmed by other research groups [33, 34].

Animal feedstuffs have been found to be a cause of *Salmonella* infection in animals and humans [17, 35, 36]. In spite of this, there is controversy in the roll or relevance of animal feed in food-borne infections since the serovars frequently isolated from animal feed do not correlate with the serovars frequently associated with human infections. Through animal feed new *Salmonella* serovars and resistance bacteria could enter and spread into the food chain [37, 38]. The surveillance and control of *Salmonella* in animal feed and feed ingredients should be an important part of animals and food safety programs aimed to counteract *Salmonella* food-borne infections.

In many countries around the world, *Salmonella* surveillance feedstuff programs are being executed; each program has its own specific objectives and specifications. For example, in Costa Rica all finished feed and feed ingredients must be registered and inspected by the Ministry of Agriculture and Livestock. These feedstuffs are also analyzed for *Salmonella*, and this must be absent regardless the serovar. In contrast with the FDA guidance for control of *Salmonella* in food for animals, the FDA recommended regulatory actions depending on the serovar found and the animal species that would receive the feed [39]. The serovars that have been reported to cause disease in the animal species for which the feed is for should be absent, for example [39]:

Poultry feed: *S. enterica* subsp. *enterica* serovar Gallinarum and Enteritidis

Swine feed: *S. enterica* subsp. *enterica* serovar Choleraesuis

Sheep feed: *S. enterica* subsp. *enterica* serovar Abortusovis

Horse feed: *S. enterica* subsp. *enterica* serovar Abortusequi

Dairy and beef feed: *S. enterica* subsp. *enterica* serovar Newport or Dublin

These differences between the *Salmonella* control programs could hamper international trade. Furthermore, in a previous research [40] in which we analyzed 1725 samples of feed and feed ingredient between the years 2009 and 2014, we found *Salmonella* serovars which do not frequently cause disease in animals but have been involved in food-borne outbreaks.

In our study, the overall *Salmonella* prevalence in animal feedstuff was 6.4%. Finished feeds such as: poultry, pet, and swine and feed ingredients such as: meat and bone meal (MBM), fish meal and poultry meal were tested.

Meat and bone meal and poultry feed presented the higher *Salmonella* relative prevalence 26,7 and 5,4%, respectively [40]. **Figure 1** shows the most frequently found serovars in MBM and poultry feed in this study [40, 41]: in MBM: *S. enterica* subsp. *enterica* serovar Give (13.8%) and serovar Rissen (4.6%) and in poultry feed: *S. enterica* subsp. *enterica* serovar Havana (10.8%), serovar Rissen, serovar Soerenga, and serovar Schwarzengrund (6.2%). These serovars have been associated with animal and human infections and outbreaks [42–44].

**Figure 1.** Distribution of *Salmonella enterica* serovars among the isolates found in feed and feed ingredients in Costa Rica [41].

The high *Salmonella* prevalence found in MBM in our previous study [40] is worrying given that MBM is used in some countries as a relative cheap protein source to feed pets and monogastric animals [38, 45].

In the EU, there is also no common sampling plan for *Salmonella* surveillance in animal feed; in the EFSA report for 2014, the overall level of *Salmonella* contamination in feedstuff was 3.8% [46] similar to our previously reported prevalence for Costa Rica [40].

## **4. Detection and surveillance of** *Salmonella* **in food production systems**

Owing to the fact that *Salmonella* is ubiquitous and has the capability to survive in a great variety of commodities, it is important to control it in each step of the food chain in order to minimize the risk of human infections and food-borne outbreaks and achieve safer food to consumers. It is crucial to maintain a *Salmonella* surveillance program in food-producing animals in order to reduce food-borne Salmonellosis and infections in animals causing economic loss to the livestock sector. The fact that *Salmonella* in animals causes frequently subclinical infections that could go unnoticed favors the *Salmonella* spread in a herd or flock [47].

In our study, the overall *Salmonella* prevalence in animal feedstuff was 6.4%. Finished feeds such as: poultry, pet, and swine and feed ingredients such as: meat and bone meal (MBM),

Meat and bone meal and poultry feed presented the higher *Salmonella* relative prevalence 26,7 and 5,4%, respectively [40]. **Figure 1** shows the most frequently found serovars in MBM and poultry feed in this study [40, 41]: in MBM: *S. enterica* subsp. *enterica* serovar Give (13.8%) and serovar Rissen (4.6%) and in poultry feed: *S. enterica* subsp. *enterica* serovar Havana (10.8%), serovar Rissen, serovar Soerenga, and serovar Schwarzengrund (6.2%). These serovars have

The high *Salmonella* prevalence found in MBM in our previous study [40] is worrying given that MBM is used in some countries as a relative cheap protein source to feed pets and mono-

**Figure 1.** Distribution of *Salmonella enterica* serovars among the isolates found in feed and feed ingredients in Costa

In the EU, there is also no common sampling plan for *Salmonella* surveillance in animal feed; in the EFSA report for 2014, the overall level of *Salmonella* contamination in feedstuff was 3.8%

**4. Detection and surveillance of** *Salmonella* **in food production systems**

Owing to the fact that *Salmonella* is ubiquitous and has the capability to survive in a great variety of commodities, it is important to control it in each step of the food chain in order to minimize the risk of human infections and food-borne outbreaks and achieve safer food to consumers. It is crucial to maintain a *Salmonella* surveillance program in food-producing animals

[46] similar to our previously reported prevalence for Costa Rica [40].

been associated with animal and human infections and outbreaks [42–44].

fish meal and poultry meal were tested.

202 Current Topics in Salmonella and Salmonellosis

gastric animals [38, 45].

Rica [41].

**Table 2** shows the *Salmonella* prevalence in farm animals, and the serovars most commonly found in animals and in their meat according to the last EFSA and ECDC [46] report. In this report, the authors demonstrated that the most prevalent serovars were shared between food producing animals and the meat for consumption. In contrast, other researchers (including ourselves) found no relation among the strains encountered in feed, live animals and processed meat [40].


**Table 2.** *Salmonella* prevalence in farm animals and their meat in the Europe Union [46].

## **5. Control measures for** *Salmonella* **in food production**

The objective of preharvest approach is to minimize opportunities for the introduction, persistence, and transmission of *Salmonella* infections and other animal pathogens. Strategies should be directed against all *Salmonella* serovars, but sometimes more specific strategies against particular *Salmonella* serovars are required when one of them has high public health impact or economic significance.

Most of the time, general strategies are sufficient to control all *Salmonella* serovars; nevertheless, sometimes it is necessary to apply specific tools, e.g., vaccination against specific serovars. Prevention programs or strategies included risk reduction, risk management, and verification by implementation of biosecurity programs.

Biosecurity is known as a group of procedures or prevention measurements to protect farm animals against biological agents, such as bacteria, viruses, fungi, parasites, protozoa, and any other agents able to induce infectious diseases into a farm. Biosecurity programs identify risk, origin, reservoirs, vector, and carriers, preventing the access to the farm. It includes strategies as control of wild birds and flies, obligatory disinfection of boots, clothes, and equipment for farm workers and visitors. Cleaning and disinfection of houses, litter, and dead animal's management and vaccination are also important in a prevention program.

#### **5.1. Cleaning and disinfection**

High level of *Salmonella* persisting for months in surfaces and contaminated facilities demonstrates the importance of cleaning organic matter and dust from the environment and animal houses. Empty houses should be cleaned and disinfected between flocks and herds.

Cleaning has to be detailed, using water and appropriate detergents. In poultry houses, cleaning should be focused in difficult access places as ceilings, cages, egg-conveyor belts, egggrading equipment, manure belts, feed troughs, hoppers, feed bins, louvers, curtains, brush blades, air inlets, fans, and other ventilation equipment. Feather removal is an important measure in poultry facilities. Also, frequently visited rooms should be cleaned; anterooms, egg-packing rooms, and egg-storage rooms, offices, storage rooms, and restrooms can be contaminated.

After washing and cleaning, administration of disinfectants by high-pressure spray, foam, and fumigation reduce environmental contamination. Disinfectant dilutions and application directions should be strictly followed. A suitable disinfectant against *Salmonella* should have residual properties and activity in the presence of organic matter. Drying of houses immediately after application of disinfectants is highly advisable to reduce water activity, which allows *Salmonella* multiplication.

Disinfectants as sodium hypochlorite or quaternary ammonium compounds are able to eliminate *Salmonella* bacteria. Other studies showed that the use of glutaraldehyde, formaldehyde, and peroxygen at a concentration of 1% in field conditions was inadequate for the elimination of *Salmonella* in the farm [48]. Higher doses should be used. Povidone-iodine, potassium permanganate, ethanol, chlorhexidine digluconate, and hydrogen peroxide exhibited high efficacy in other studies [49, 50].

Recontamination after cleaning and disinfecting may occur. Houses recently cleaned should be closed before animals arrive to prevent organic matter and dust contamination. Equipment should be washed and disinfected before entering a house to prevent recontamination.

#### **5.2. Vaccination**

Vaccination is a specific control tool against *Salmonella*. Vaccines are used to increase the infection resistance. It can enhance the short-term responsiveness of control programs but does not completely eliminate problems. A combination of biosecurity procedures, *Salmonella*-free replacement of flocks and herds, and vaccination should be a suitable control approach. Farm management programs need integrated interventions to be satisfactory.

Immunization has been shown to significantly reduce the number of hens infected by *S. enterica* subsp. *enterica* serovar Enteritidis and the rate of egg transmission [51]. Live-attenuated vaccines and nonliving vaccines (bacterins) of *S. enterica* subsp. *enterica* serovar Enteritidis vaccines are used to immunize chickens. Live vaccines are used against *S. enterica* subsp. *enterica* serovar Gallinarum and Typhimurium.

Live vaccines reduce intestinal and internal organ (spleen, liver, ovary, and oviducts) infection and stimulate mucosal immunity in the digestive tract [52]. Bacterins (killed vaccines) induce high levels of circulation antibodies and reduce colonization of internal organs and the number of bacteria in egg content [53]. However, they have a limited effect in feces shedding; for this reason, they may not contribute to prevent environment contamination. Therefore, a combination of both lives and bacterins are commonly used in layers and showed to be effective in *Salmonella* control in poultry [51].

Vaccination of sows and piglets can be helpful. Both vaccines are used, live and bacterins. Live vaccines are considered to provide good protection in pigs. However, some live vaccines in pigs show risks as reversion to virulence and excretion to the environment. And also, there is no differentiation between naturally infected and vaccinated animals [12]. Inactivated vaccines in sows could reduce transmission to the progeny and enhance maternal immunity. An effective, safe, and efficient vaccine program should prevent clinical symptoms, colonization, and development of carriers and reduce shedding.

#### **5.3. Pest control**

#### *5.3.1. Rodents*

other agents able to induce infectious diseases into a farm. Biosecurity programs identify risk, origin, reservoirs, vector, and carriers, preventing the access to the farm. It includes strategies as control of wild birds and flies, obligatory disinfection of boots, clothes, and equipment for farm workers and visitors. Cleaning and disinfection of houses, litter, and dead animal's

High level of *Salmonella* persisting for months in surfaces and contaminated facilities demonstrates the importance of cleaning organic matter and dust from the environment and animal

Cleaning has to be detailed, using water and appropriate detergents. In poultry houses, cleaning should be focused in difficult access places as ceilings, cages, egg-conveyor belts, egggrading equipment, manure belts, feed troughs, hoppers, feed bins, louvers, curtains, brush blades, air inlets, fans, and other ventilation equipment. Feather removal is an important measure in poultry facilities. Also, frequently visited rooms should be cleaned; anterooms, egg-packing rooms, and egg-storage rooms, offices, storage rooms, and restrooms can be

After washing and cleaning, administration of disinfectants by high-pressure spray, foam, and fumigation reduce environmental contamination. Disinfectant dilutions and application directions should be strictly followed. A suitable disinfectant against *Salmonella* should have residual properties and activity in the presence of organic matter. Drying of houses immediately after application of disinfectants is highly advisable to reduce water activity, which

Disinfectants as sodium hypochlorite or quaternary ammonium compounds are able to eliminate *Salmonella* bacteria. Other studies showed that the use of glutaraldehyde, formaldehyde, and peroxygen at a concentration of 1% in field conditions was inadequate for the elimination of *Salmonella* in the farm [48]. Higher doses should be used. Povidone-iodine, potassium permanganate, ethanol, chlorhexidine digluconate, and hydrogen peroxide exhibited high

Recontamination after cleaning and disinfecting may occur. Houses recently cleaned should be closed before animals arrive to prevent organic matter and dust contamination. Equipment should be washed and disinfected before entering a house to prevent

Vaccination is a specific control tool against *Salmonella*. Vaccines are used to increase the infection resistance. It can enhance the short-term responsiveness of control programs but does not completely eliminate problems. A combination of biosecurity procedures, *Salmonella*-free replacement of flocks and herds, and vaccination should be a suitable control approach. Farm

management programs need integrated interventions to be satisfactory.

houses. Empty houses should be cleaned and disinfected between flocks and herds.

management and vaccination are also important in a prevention program.

**5.1. Cleaning and disinfection**

204 Current Topics in Salmonella and Salmonellosis

allows *Salmonella* multiplication.

efficacy in other studies [49, 50].

recontamination.

**5.2. Vaccination**

contaminated.

Reduction or elimination of these vectors is an important part of the prevention strategies or control. An effective control program should be keep rodents number to the lowest level possible.

Chemicals and baits are the most common methods of rodent control. Farmers use frequently traps and cats. The use of cats as exterminator is not recommended. A study in a pig farm founded 12% of farm cat *Salmonella* [20] and *Toxoplasma gondii* positive [25].

A rodent control should have an integral approach, and it should include:


It has been demonstrated that rodent integral control programs that follow these guidelines, has effectively decreased *Salmonella* in livestock animal houses.

Sometimes when high rodent densities are found, a program such as the mentioned is required.

#### *5.3.2. Insects*

For a successful insect control is required to keep litter dry and well ventilated, preventing wet areas and leaks is a must. Frequent removal of litter and replacement of fresh shavings in poultry houses can help to reduce beetle populations [54].

The use of insecticides such as :pyrethrins, carbamates and phosphates is a common practice. Sometimes, mite control could be complicated, because of the resistance from the insects to the acaricide; and also technical limitations like the usage in the lay period in hens. Rotation of insecticides reduces development of resistance.

Biological control methods should be used especially in animal production periods. Fly parasites, depredators, and insect growth regulators could be good options. Wettable powders are used with chemical insecticides in the beetle control.

#### **5.4. Water safety**

Water sanitation at the farm is essential in a biosecurity program. Drinking water sanitation can prevent initial contamination and recontamination of animals with *Salmonella.* Water filtration is a critical component of a water sanitation program. Dirty water cannot be effectively sanitized. Frequent washed and cleaned tanks are also required.

Chlorine is the most common disinfectant used in drinking water. It is a strong oxidizing agent and used to sanitize drinking water in farms. It is effective against Gram-positive and Gram-negative bacteria, viruses, fungal, and protozoa. When added to the water, a chemical reaction occurs, formation of Hypochlorous acid (HOCL) (weak acid) and Hypochlorite ion (OCL- ). Both are referred as free chlorine or available free chlorine. HOCL is more efficient as sanitizer. HOCL is necessary to keep low water pH, under 6.5 [55]. Chlorine is available in liquid form as sodium hypochlorite and in solid form as calcium hypochlorite. Sodium hypochlorite is usually available at a concentration of 10–12%.

Other halogens as iodine and bromine are used. Hydrogen peroxide, peracetic acid, ozone, and ultraviolet light showed to be successful to sanitize drinking water. Addition of organic acids to the drinking water showed variable results [56]. The antibacterial effects of acids depend on the type of organic acid, the bacterial species, the concentration used, and the physical form in which it is administered to the animals.

Strategies to reduce drinkers and pipe biofilm should be implemented. Biofilm causes resistance to free chlorine residuals, which can lead to persistence of bacteria in chlorine-treated water. Surfactin, glucose, halogenated furanones, 4(5)-aryl 2-aminoimidazoles, furocoumarins, and salicylates are used as biofilm inhibitors and disinfectant combinations of triclosan and quaternary ammonium salts or halogenated furanones and treatment antibiotics/disinfectants and microemulsions such as soybean oil in water [6]. It is essential that the effectiveness of sanitization program can be monitored.

#### **5.5. Litter and carcasses disposed**

• Cleaning of outdoor paddocks.

206 Current Topics in Salmonella and Salmonellosis

• The use of effective rodenticides.

• Do not maintain spilled feed.

required.

*5.3.2. Insects*

**5.4. Water safety**

(OCL-

• Follow strict biosecurity procedures.

• Removal of vegetation around the houses.

• Secure disposal of died animals, litter, and waste.

has effectively decreased *Salmonella* in livestock animal houses.

poultry houses can help to reduce beetle populations [54].

of insecticides reduces development of resistance.

used with chemical insecticides in the beetle control.

sanitized. Frequent washed and cleaned tanks are also required.

chlorite is usually available at a concentration of 10–12%.

It has been demonstrated that rodent integral control programs that follow these guidelines,

Sometimes when high rodent densities are found, a program such as the mentioned is

For a successful insect control is required to keep litter dry and well ventilated, preventing wet areas and leaks is a must. Frequent removal of litter and replacement of fresh shavings in

The use of insecticides such as :pyrethrins, carbamates and phosphates is a common practice. Sometimes, mite control could be complicated, because of the resistance from the insects to the acaricide; and also technical limitations like the usage in the lay period in hens. Rotation

Biological control methods should be used especially in animal production periods. Fly parasites, depredators, and insect growth regulators could be good options. Wettable powders are

Water sanitation at the farm is essential in a biosecurity program. Drinking water sanitation can prevent initial contamination and recontamination of animals with *Salmonella.* Water filtration is a critical component of a water sanitation program. Dirty water cannot be effectively

Chlorine is the most common disinfectant used in drinking water. It is a strong oxidizing agent and used to sanitize drinking water in farms. It is effective against Gram-positive and Gram-negative bacteria, viruses, fungal, and protozoa. When added to the water, a chemical reaction occurs, formation of Hypochlorous acid (HOCL) (weak acid) and Hypochlorite ion

). Both are referred as free chlorine or available free chlorine. HOCL is more efficient as sanitizer. HOCL is necessary to keep low water pH, under 6.5 [55]. Chlorine is available in liquid form as sodium hypochlorite and in solid form as calcium hypochlorite. Sodium hypoManure is one of the most important sources of *Salmonella* contamination. Pig slurry and poultry litter should not be spread, sprayed, or reused before a disinfection treatment. Land spreading of manure can lead to contamination of soil and water, which can potentially lead to bacteria transmission to animals and humans.

Transportation and disposal of slurry and manure from pig and poultry houses and barns, the transportation of slaughter offal to rendering plants, the cross contamination of rendered meat meal, and other poultry and animal byproducts contribute to spreading *Salmonella* in the environment [5].

If *Salmonella* is present in the litter and manure, the birds and pigs could be exposed at a time when they are highly susceptible and get sick. Well-designed facilities should avoid contact between animals and their feces. There are many manure treatments or disinfection procedures. Manure methods can be physical, chemical, biological, or a combination of all three and include technologies such as anaerobic digestion, composting, and separation. It has been shown that stored separate pig manure fractions under controlled conditions (10.5°C for 84–112 days) reduced *Salmonella* [57].

*Salmonella* may also be introduced into soil and the adjacent environment by decomposition of infected carcasses [5]. Dead animals should be disposed into a secured container, which is regularly washed and disinfected. Burying, composting, incineration, and dropping off at designated sites are the most commonly recommended and utilized methods for carcass disposed [58].

#### **5.6. Transportation**

Pigs and chickens and other animals increased shedding of *Salmonella* during transport from the farm to the slaughterhouse. Long transportation duration, high stock density, weather conditions, and long feed withdrawal are causes of bacteria increase shedding.

Feed tracks can also act as mechanical vectors and can transfer bacteria from one farm to another. Pig and poultry vehicles and drivers represent a considerable risk; therefore, they should not be allowed into the clean areas of the farm. Transport vehicles, feed trucks, and chicken coops should to be cleaned and appropriately disinfected to prevent *Salmonella* contamination in harvest. In layers decontaminated and sanitized coops or cages and vehicles should be used to transport pullets from grow-out houses to the layer farm.

#### **5.7. Feed additives and heat treatment**

Organic acids and their salts, essential oils, formaldehyde, bacteriophages, probiotics, prebiotics, and symbiotics can be used to modify the gut environment to prevent *Salmonella* colonization, invasion, multiplication, and shedding. Probiotics consist of single or multiple beneficial bacteria strains that colonize intestinal tract; they compete with pathological bacteria as *Salmonella* for attachment sites, nutrients in the luminal surface of enterocytes. Probiotics also produce antibacterial compounds as bacteriocins and volatile fatty acids. Prebiotics are food ingredients as oligosaccharides that stimulate intestinal bacteria and probiotic growth. Symbiotics are products that contain both prebiotics and probiotics. Bacteriophages are viruses that infect and replicate in bacteria and have an effect against *Salmonella*.

Organic acids reduce *Salmonella* in contaminated feed. Formic and propionic acids and their salts are commonly included in feed, but the effect varies by the inclusion rate, food level contamination, feed´s moisture and the type of acid. Formaldehyde is permitted in some countries; therefore, it is corrosive and potentially harmful for humans and animals.

Appropriate pelleting process can eliminate *Salmonella* by heat treatment; it is performed at 93°C for 90 s [17]. Combinations of several of these treatments have been shown effective in recontaminated feed. Measures to prevent recontamination of finished feed should to be taken.

## **Author details**

Rebeca Zamora-Sanabria and Andrea Molina Alvarado\*

\*Address all correspondence to: andrea.molina@ucr.ac.cr

Center for Research in Animal Nutrition (CINA), University of Costa Rica, Ciudad Universitaria Rodrigo Facio, San José, Costa Rica

## **References**


[3] Slorach S. Coordinating surveillance policies in animal health and food safety "from farm to fork". Scientific and Technical Review OIE. 2013;32(2):313-317.

should not be allowed into the clean areas of the farm. Transport vehicles, feed trucks, and chicken coops should to be cleaned and appropriately disinfected to prevent *Salmonella* contamination in harvest. In layers decontaminated and sanitized coops or cages and vehicles

Organic acids and their salts, essential oils, formaldehyde, bacteriophages, probiotics, prebiotics, and symbiotics can be used to modify the gut environment to prevent *Salmonella* colonization, invasion, multiplication, and shedding. Probiotics consist of single or multiple beneficial bacteria strains that colonize intestinal tract; they compete with pathological bacteria as *Salmonella* for attachment sites, nutrients in the luminal surface of enterocytes. Probiotics also produce antibacterial compounds as bacteriocins and volatile fatty acids. Prebiotics are food ingredients as oligosaccharides that stimulate intestinal bacteria and probiotic growth. Symbiotics are products that contain both prebiotics and probiotics. Bacteriophages are

Organic acids reduce *Salmonella* in contaminated feed. Formic and propionic acids and their salts are commonly included in feed, but the effect varies by the inclusion rate, food level contamination, feed´s moisture and the type of acid. Formaldehyde is permitted in some coun-

Appropriate pelleting process can eliminate *Salmonella* by heat treatment; it is performed at 93°C for 90 s [17]. Combinations of several of these treatments have been shown effective in recontaminated feed. Measures to prevent recontamination of finished feed should to be

Center for Research in Animal Nutrition (CINA), University of Costa Rica, Ciudad Universitaria

[1] FAO. www.fao.org [Internet]. 2003. Available from: www.fao.org/english/newsroom/

[2] FFAO/WHO Food and Agriculture Organization of the United Nations/World Health Organization. *Salmonella* and *Campylobacter* in chicken meat: Meeting report.

Microbiological Risk Assessment Series. 2009. No.19. Rome, 56 pp.

should be used to transport pullets from grow-out houses to the layer farm.

viruses that infect and replicate in bacteria and have an effect against *Salmonella*.

tries; therefore, it is corrosive and potentially harmful for humans and animals.

Rebeca Zamora-Sanabria and Andrea Molina Alvarado\* \*Address all correspondence to: andrea.molina@ucr.ac.cr

**5.7. Feed additives and heat treatment**

208 Current Topics in Salmonella and Salmonellosis

taken.

**Author details**

**References**

Rodrigo Facio, San José, Costa Rica

news/2003/15903-en.html


[32] Kich J.D., Mores N., Piffer I.A., Coldebella A., Amaral A., Ramminger L.,Cardoso M. Factor associated with seroprevalence of *Salmonella* in commercial pig herds. Ciencia Rural. 2005;35:398-405.

[17] Jones F.T. A review of practical *Salmonella* control measures in animal feed. Journal of

[18] Aphis. www.aphis.usda.gov [Internet]. 25 March 2005. Available from: www.aphis.

[19] Ekiri A.B., Morton A.J., Long M.T., MacKay R.J., Hernandez J.A. Review of the epidemiology and infection control aspects of nosocomial *Salmonella* infections in hospitalized

[20] Barber D.A., Bahnson P.B., Jones C.J., Weigel R.M. Distribution of *Salmonella* in swine

[21] Levantesi C., Bonadonna L., Briancesco R., Grohmann E., Toze S., Tandoi V. *Salmonella* in surface and drinking water: Occurrence and water-mediated transmission. Food

[22] Jacques M., Aragon V., Tremblay Y.D. Biofilm formation in bacterial pathogens of veteri-

[23] Davies R.H., Wray C. Mice and carriers of *Salmonella* enteritidis on persistently infected

[24] Henzler D.J., Opitz H.M. The role of mice in the epizootiology of *Salmonella* enteritidis

[25] Meeburg, B.G., Van Riel J.W., Cornelissen J.B., Kijlstra A., Mul M.F.. Cats and goat whey associated with *Toxoplasma gondii* infection in pigs. Vector Borne Zoonot. 2006;6:266-274.

[26] Wales A.D., Carrique-Mas J.J., Ranquin M., Bell B., Thind B.B., Davies R.H. Review of the carriage of zoonotic bacteria by Arthropods, with special reference to *Salmonella* in

[27] Lapuz R.R.S., Umali D., Suzuki T., Shirota K., Katoh H. Comparison of prevalence of *Salmonella* infection in layer hens from commercial layer farms with high and low rodent

[28] Vanselow B.A., Hornitzky M.A., Walker K.H., Eamens G.J., Bailey G.D., Gill P.A., Coates K., Corney B., Cronin J.P., Renilson S. *Salmonella* on-farm risk factors in healthy slaughterage cattle and sheep in eastern Australia. Australian Veterinary Journal. 2007;85:498-502.

[29] Tessier C., Parama A.L., Lagadec E., Le Minter G., Denis M., Cardinale E. Wild fauna as a carrier of Salmonella in Reunion Island: Impact on pig farms. Acta Tropica. 2016;158:

[30] Fathpour H., Emtiazi G., Ghasemi E.. Cockroaches as reservoirs and vectors of drug

[31] Handeland K., Nesse L.L., Lillehaugh A., Vokore T., Djonne B., Bergsjo B. Natural and experimental *Salmonella* Typhimurium infections in foxes. Veterinary Microbiology.

resistant *Salmonella* spp. Fresenius Environmental Bulletin. 2003;12:724-727.

mites, flies and litter beetles. Zoonosis and Public Health. 2010;57:299-314.

production ecosystems. Journal of Food Protection. 2002;65(12):1861-1868.

nary importance. Animal Health Research Review. 2010;11(2):97-121.

infection on chicken layer farms. Avian Diseases. 1992;336:625-631.

Applied Poultry Research. 2011;20:102-113.

210 Current Topics in Salmonella and Salmonellosis

Research International. 2012;45:587-602.

densities. Avian Diseases. 2012;56:29-34.

6-12. doi:10.1016/j.actatropica.2016.01.027

2008;132:129-134.

usda.gov/vs/ceah/ncahs/nahms/poultry/Layers99/lay99se.pdf

horses. Equine Veterinary Education. 2010;22:631-641.

poultry units. Veterinary Record. 1995;137(14):337-341.


[57] Mccarthy G., Lawflor P., Gutiérrez M., OSulivan L., Muphy A., Zhan X., Gardiner G. An assessment of *Salmonella* survival in pig manure and its separated solid and liquid fractions during storage. Journal of Enviromental Science and Health. Part B. 2015;50:1532-4109.

[45] Adedokun S.A., Adeola O. Metabolizable energy value of meat and bone meal for pigs.

[46] EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). The European Union Summary Report on Antimicrobial Resistance in Zoonotic and Indicator Bacteria from Humans, Animals and Food in 2014.

[47] Hugas M., Beloeil P.A. Controlling *Salmonella* along the food chain in the European Union – progress over the last ten years. Eurosurveillance. 2014;19(19):20804. Doi:

[48] Marin C., Hernandez A., Lainez M. Biofilm development capacity of *Salmonella* Stains isolated in poultry risk factors and their resistance against disinfectants. Poultry Science.

[49] Martínez-Martínez S.,Yubero-Delgado S., Rodríguez-Ferri E.F., Frandoloso R., Alvarez-Estrada A., Gutiérrez-Martín C.B. In vitro efficacy of several disinfectants against *Salmonella enterica* serovar Enteritidis and *Escherichia coli* strains from poultry. Ciencia

[50] Soliman E. Taha E.G., Sobiehand M.A.A., Reddy P.G. Efficacy of some commercial chemical disinfectants on *Salmonella enterica* serovar Typhimurium. American Journal of

[51] Dorea F., Cole D.J., Hofacre C., Zamperini K., Mathis D., Doyle M.P., Lee M., Maurer J.J. Effect of *Salmonella* vaccination of breeder chickens on contamination of broiler chicken carcasses in integrated poultry operations. Applied and Environmental Microbiology.

[52] Horacio T.. Model of *Salmonella* live vaccine safety management program. In: Alberto R., editor. International Seminar on Avian Salmonellosis; 28-30 June 2011; Rio Janeiro.

[53] Villareal Laura. Model of *Salmonella* Killed vaccine (bacterins) safety management programme. In: Alberto R., editor. International Seminar on Avian Salmonellosis; 28-30 June

[54] Dunford J., Kaufman P. University of Florida. Lesser Mealworm, Litter Beetle, *Alphitobius diaperinus* [Internet]. March 2006. Available from: http://edis.ifas.ufl.edu [Accessed:

[55] Tahseen A. Chlorinating drinking water on poultry farms. World Poultry. 2005;21(5):24-25. [56] De Ridder L., Maes D., Dewulf J., Pasmans F., Boyen F., Méroc E., Butaye P., Van der Stede Y. Transmission study of *Salmonella* in pigs with 3 intervention strategies. Proceedings of the 9th International Conference on the Epidemiology and Control of Biological, Chemical and Physical Hazards in Pigs and Pork. 2011. Iowa State University, June 19–22, Maastricht, Netherlands; 30-33. Available from: www.lib.dr.iastate.edu/safepork/

2016; Rio Janeiro. Brazil: Latin America Poultry Association; 2011.

EFSA Journal. 2016;14(2):4380,207p. doi:10.2903/j.efsa.2016.4380

Journal of Animal Science. 2005;83:2519-2526.

10.2807/1560-7917.ES2014.19.19.20804

Rural, Santa María. 2016;45:1438-1442.

Animal and Veterinary Sciences. 2009;4:58-64.

Brazil: Latin America Poultry Association; 2011.

2009; 88:424-431.

212 Current Topics in Salmonella and Salmonellosis

2010;76:7820-7825.

August 2016]


#### **Prevalence, Risks and Antibiotic Resistance of Salmonella in Poultry Production Chain Prevalence, Risks and Antibiotic Resistance of Salmonella in Poultry Production Chain**

Niki Mouttotou, Shakeel Ahmad, Zahid Kamran and Niki Mouttotou, Shakeel Ahmad, Zahid Kamran and Konstantinos C. Koutoulis

Konstantinos C. Koutoulis

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67438

#### **Abstract**

*Salmonella* spp. are bacteria that cause salmonellosis, a common form of foodborne illness with major impact on human health and huge financial losses in poultry industry. The incidence of notified cases of salmonellosis has declined from a peak of 24 per 100,000 in 2009 to 20.4 reported cases per 100,000 population in 2013, with *S. enteritidis* and *S. typhimurium* being the most commonly reported serovar in EU. *Salmonella* spp. has been detected in a range of foods, and outbreaks have predominantly been associated with animal products such as eggs, poultry and dairy products, but also with plant origin food such as salad dressing, fruit juice and sesame. At the time of slaughter, *Salmonella*-infected poultry may have high numbers of organisms in their intestines as well as on the outside of the bird and are therefore an important source of contamination. Nowadays, food safety has become an important concern for the European society and governments; therefore, more strict and harmonized regulations are being implemented throughout the poultry production chain with the aim to guarantee and increase the consumer confidence in foodstuffs of animal origin. Furthermore, increasing antimicrobial resistance in non-typhoid *Salmonella* species has been a serious problem for public health worldwide.

**Keywords:** salmonellosis, foodborne, prevalence, poultry, antibiotic resistance

## **1. Introduction**

*Salmonella* has been declared by the World Health Organization (WHO) and the Food Agriculture Organization (FAO) as the most common and important zoonosis since 1950.

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

This has led to its inclusion in the terrestrial animal health code of the World Animal Health Organization In humans, typhoid disease manifests one to 2 weeks following bacterial inoculation with generalized fever and malaise, abdominal pain with or without other symptoms including headache, myalgias, nausea, anorexia and constipation [1]. An estimation of the annual non-typhoid *Salmonella* gastroenteritis suggests that there are around 94 million cases, resulting in 155,000 deaths, and that the majority of the disease burden, according to this study, is in the South-East Asian Region and the Western Pacific Region [2]. Most human salmonellosis cases are foodborne, but each year, infections are also acquired through direct or indirect animal contact in homes, veterinary clinics, zoos, farm environments or other public, professional or private settings. It has been estimated that approximately 80.3 million of 93.8 million human *Salmonella*-related gastroenteritis cases—that are diagnosed globally each year—are foodborne, thus representing approximately 86% of human salmonellosis cases [2]. Another study estimated that approximately 55% of human *Salmonella* cases were foodborne, 14% were travel-related, 13% are acquired through environmental sources, 9% occurred due to direct human-to-human transmission and 9% were attributable to direct animal contact [3, 4].

## **2.** *Salmonella* **species classification**

The bacteria of the genus *Salmonella* are responsible for illnesses in human beings and animals. The genus is divided into two species: *Salmonella enterica* and *Salmonella bongori* [5]. *S. enterica* is divided into six subspecies (*enterica, salamae, arizonae, diarizonae, houtenae* and *indica*) and each one of them has several serovars or serotypes. Nowadays, more than 2500 serotypes are known and almost 1500 of them belong to the subspecies enterica [6]. Most pathogenic isolates from humans and other mammals belong to *S. enterica* subspecies enterica. Other *S. enterica* subspecies and *S. bongori* are more common in cold-blooded animals and the environment, with lower pathogenicity to humans and livestock [7, 8].

A few serotypes are host specific; i.e. *S. typhi* is implicated in typhoid fever in human beings, while *Salmonella pullorum* and *gallinarum* are responsible for bacillary white diarrhoea and fowl typhoid in poultry, respectively [9]. *Salmonella choleraesuis* is host restricted to pigs, *Salmonella* ser. *abortusovis* is involved in sheep abortions and *Salmonella dublin* infects bovines [10]. There are a number of non-host-specific serotypes that may infect several animal species, including humans, and these are generally responsible for foodborne diseases with foods of animal origin being the main source. From the early years, the most common agent of human foodborne disease was *Salmonella typhimurium*, but in the last few decades the frequency of *Salmonella enteritidis* has dramatically increased [11]. Almost 80 out of 2500 serovars are thought to be frequently involved in animal and human salmonellosis. *S. typhimurium* and *S. enteritidis* are the most common agents of disease in human beings and animals, but lately, there is also increasing concern about *S. typhimurium monophasic, S. derby, infantis, agona, hadar, heidelberg* and *virchow* serotypes.

## **3. Transmission routes, public health and economic cost associated with** *Salmonella* **infection**

This has led to its inclusion in the terrestrial animal health code of the World Animal Health Organization In humans, typhoid disease manifests one to 2 weeks following bacterial inoculation with generalized fever and malaise, abdominal pain with or without other symptoms including headache, myalgias, nausea, anorexia and constipation [1]. An estimation of the annual non-typhoid *Salmonella* gastroenteritis suggests that there are around 94 million cases, resulting in 155,000 deaths, and that the majority of the disease burden, according to this study, is in the South-East Asian Region and the Western Pacific Region [2]. Most human salmonellosis cases are foodborne, but each year, infections are also acquired through direct or indirect animal contact in homes, veterinary clinics, zoos, farm environments or other public, professional or private settings. It has been estimated that approximately 80.3 million of 93.8 million human *Salmonella*-related gastroenteritis cases—that are diagnosed globally each year—are foodborne, thus representing approximately 86% of human salmonellosis cases [2]. Another study estimated that approximately 55% of human *Salmonella* cases were foodborne, 14% were travel-related, 13% are acquired through environmental sources, 9% occurred due to direct human-to-human transmission

The bacteria of the genus *Salmonella* are responsible for illnesses in human beings and animals. The genus is divided into two species: *Salmonella enterica* and *Salmonella bongori* [5]. *S. enterica* is divided into six subspecies (*enterica, salamae, arizonae, diarizonae, houtenae* and *indica*) and each one of them has several serovars or serotypes. Nowadays, more than 2500 serotypes are known and almost 1500 of them belong to the subspecies enterica [6]. Most pathogenic isolates from humans and other mammals belong to *S. enterica* subspecies enterica. Other *S. enterica* subspecies and *S. bongori* are more common in cold-blooded animals and the environ-

A few serotypes are host specific; i.e. *S. typhi* is implicated in typhoid fever in human beings, while *Salmonella pullorum* and *gallinarum* are responsible for bacillary white diarrhoea and fowl typhoid in poultry, respectively [9]. *Salmonella choleraesuis* is host restricted to pigs, *Salmonella* ser. *abortusovis* is involved in sheep abortions and *Salmonella dublin* infects bovines [10]. There are a number of non-host-specific serotypes that may infect several animal species, including humans, and these are generally responsible for foodborne diseases with foods of animal origin being the main source. From the early years, the most common agent of human foodborne disease was *Salmonella typhimurium*, but in the last few decades the frequency of *Salmonella enteritidis* has dramatically increased [11]. Almost 80 out of 2500 serovars are thought to be frequently involved in animal and human salmonellosis. *S. typhimurium* and *S. enteritidis* are the most common agents of disease in human beings and animals, but lately, there is also increasing concern about *S. typhimurium monophasic, S. derby, infantis, agona, hadar,* 

and 9% were attributable to direct animal contact [3, 4].

ment, with lower pathogenicity to humans and livestock [7, 8].

**2.** *Salmonella* **species classification**

216 Current Topics in Salmonella and Salmonellosis

*heidelberg* and *virchow* serotypes.

The gastrointestinal tracts of humans and animals are the primary sources of *Salmonella*. The bacteria are carried asymptomatically in the intestines or gall bladder of many animals and are continuously or intermittently shed in the faeces. Also, these can be carried latently in the mesenteric lymph nodes or tonsils [12], which are not then shed, but can become reactivated after stress or immunosuppression [13]. Although most infections cause mild to moderate self-limiting disease, serious infections leading to deaths do occur [14]. Its widespread presence in the environment is considered to be due to the direct or indirect faecal contamination [15]. The transmission to humans usually occurs through the consumption of food or water contaminated with animal faeces, but it can also happen through direct contact with infected animals or their environment and directly between humans. In the same way, animals can become infected from contaminated feed (including pastures), drinking water or close contact with an infected animal (including humans).

Transovarian (vertical transmission) or trans-shell (horizontal transmission) occurs in poultry. In the first case, a contamination of the vitelline membrane, albumen and possibly the yolk of eggs occurs. Following this route, *Salmonellae* are introduced from infected reproductive tissues to eggs prior to shell formation. *Salmonella* serotypes with high importance to public health, associated with poultry reproductive tissues, include *S. enteritidis, S. typhimurium* and *Salmonella heidelberg*. Among all the different serotypes, *S. enteritidis* may be more invasive and, consequently, may be found more frequently in reproductive tissues. Faecal contamination of egg shell is the primary cause of horizontal transmission [16]. This can also include contamination through environmental vectors, such as farmers, pets and rodents, feed, water, fluff, dust, shavings and straw, insects, equipment, and thus, many different serotypes of the genus *Salmonella* can be involved [17, 18]. Bacteria can contaminate egg contents by migration through the egg shell and membranes. Such a route is facilitated by factors such as moist egg shells, storage at ambient temperature and shell damage. Faecal shedding of *S. enteritidis* was detected for up to 8-week post-inoculation by hens housed in enriched colony cages and 10 weeks by hens housed in conventional cages, which were experimentally infected with *S. enteritidis* [19]. Studies on the survival of *S. enteritidis* in poultry units and food were carried out over a 2-year period and showed that the organism persisted for at least 1 year in an empty trial house at the laboratory in which naturally infected broiler breeder birds had previously been housed [20]. In the same study, a similar survival period was found in a building which had housed an infected layer breeder flock, although infection was not detected in a subsequent pullet flock. *Salmonella* contamination appeared to persist preferentially in association with dust particles swept from the floor and in food troughs, and *S. enteritidis* survived at least 26 months in artificially contaminated poultry food [20].

*Salmonella* spp. can also be transmitted *in utero* in other mammals. Wild birds, rodents, fomites and mechanical vectors (insects) can spread *Salmonella* to livestock. In general, *Salmonella* serotypes have a broad host range and clinical manifestations that result from the combination between serotype and host species involved [21] are prevalent in a whole range of warm-blooded animal population [22] but also in snakes [23], and free-living terrestrial and aquatic turtles [24].

*Salmonella* spp. can survive for long periods in the environment, particularly, where it is wet and warm. They can be isolated from many sources including farm effluents, human sewage and water. Persistence of *Salmonella* in acid soils is facilitated by their ability to adapt to lowpH environments [25]. There is also some evidence that *Salmonella* may survive in soils in a viable but non-culturable state [26], although significance of this state is not yet understood. *S. choleraesuis* has been isolated for up to 450 days from pig meat and for several months from faeces or faecal slurries [27]. *Salmonella typhimurium* and *Salmonella dublin* have been found for over a year in the environment.

Plant origin material can be contaminated through direct deposition of *Salmonella*-containing animal faeces or through deposition of soil or dust previously contaminated with animal faecal material. In some circumstances, there has been an increasing evidence that *Salmonella* may be internalized in plant tissues [28]. This, however, was quite uncertain whether it was relevant to crops commonly used as components of animal feed.

Person-to-person transmission of *Salmonella* is well-recognized, and secondary transmission of *Salmonella* in outbreaks has been demonstrated [29]. Carriage in faeces in convalescent cases can be quite substantial with numbers approximating 106 to 107 Salmonella/g persisting up to 10 days after initial diagnosis according to the authors. Reduction in numbers with time seems to be variable; most people will have count of less than 100 Salmonella/g after 35–40 days, but a count of 6 × 103 /g has been recorded in one patient 48 days post-illness [30]. Asymptomatic carriage may also occur, as it was mentioned for a British outbreak of hospital-acquired infection [31] and another case where asymptomatic food handlers have been responsible for an outbreak in a catering establishment in Jerusalem [32].

Non-typhoidal *Salmonella* are a leading cause of bacterial diarrhoea worldwide; they are estimated to cause 94 million cases of gastroenteritis and 115,000 deaths globally each year [2]. Of these, 80.3 million cases were estimated as foodborne origin. In one analysis [33] using data from the Foodborne Diseases Active Surveillance Network (FoodNet), the risk of *Salmonella* infection among travellers returning to the USA varied by region of the world visited. Travellers with salmonellosis were most likely to report visiting the following countries: Mexico (38% of travel-associated salmonellosis), India (9%), Jamaica (7%), the Dominican Republic (4%), China (3%) and the Bahamas (2%). Travel-associated infections were related to *Salmonella* in 36.7% of the cases reported, of which non-typhoidal *Salmonella* accounted for 88.3%, typhoidal *Salmonella* 7.7%, and paratyphoidal *Salmonella* 3.9%.

In the latest EFSA's report, a total of 82,694 confirmed salmonellosis cases were reported by 27 European Union (EU) member states in 2013, resulting in an EU notification rate of 20.4 cases per 100,000 population [11].

A decrease of 7.9% in the EU notification rate compared with 2012 was shown in the above report, which supports the declining trend of salmonellosis in the EU/European Economic Area (EEA) in the 5-year period of 2009–2013 (**Figure 1**). However, the above was not statistically significant when analysed by month. Nine out of 14 EU member states reported a total of 59 fatal cases, which gave an EU case-fatality rate of 0.14% among the 40,976 confirmed cases. Some researchers claim that human salmonellosis represents a considerable economic impact and the estimated costing can be as €3 billion/year [34]. As in previous years, *S. enteritidis* and *S. typhimurium* represented 39.5 and 20.2%, respectively, in confirmed human cases, and they were the two most commonly reported *Salmonella* serovars in 2013 [11]. An interesting finding in the same report was that in the 2-year period from 2011 to 2013, cases of *S. typhimurium*, including the variant monophasic *S. typhimurium* 1,4,[5],12:i:-, decreased by 11.1%, while cases of *S. infantis* (which was the fourth most common serovar observed), increased by 26.5%. The fifth most common serovar observed in 2013, was *S. derby*, and this could partly be explained by a local outbreak in Berlin, Germany and surrounding areas in December 2013/January 2014. The outbreak occurred in hospitals and nursing homes with 145 elderly patients affected and one fatal case. The suspected vehicle of infection was rawfermented pork spread ('teewurst') [11].

serotypes have a broad host range and clinical manifestations that result from the combination between serotype and host species involved [21] are prevalent in a whole range of warm-blooded animal population [22] but also in snakes [23], and free-living terrestrial and aquatic turtles [24].

*Salmonella* spp. can survive for long periods in the environment, particularly, where it is wet and warm. They can be isolated from many sources including farm effluents, human sewage and water. Persistence of *Salmonella* in acid soils is facilitated by their ability to adapt to lowpH environments [25]. There is also some evidence that *Salmonella* may survive in soils in a viable but non-culturable state [26], although significance of this state is not yet understood. *S. choleraesuis* has been isolated for up to 450 days from pig meat and for several months from faeces or faecal slurries [27]. *Salmonella typhimurium* and *Salmonella dublin* have been found for

Plant origin material can be contaminated through direct deposition of *Salmonella*-containing animal faeces or through deposition of soil or dust previously contaminated with animal faecal material. In some circumstances, there has been an increasing evidence that *Salmonella* may be internalized in plant tissues [28]. This, however, was quite uncertain whether it was

Person-to-person transmission of *Salmonella* is well-recognized, and secondary transmission of *Salmonella* in outbreaks has been demonstrated [29]. Carriage in faeces in convalescent

ing up to 10 days after initial diagnosis according to the authors. Reduction in numbers with time seems to be variable; most people will have count of less than 100 Salmonella/g after

[30]. Asymptomatic carriage may also occur, as it was mentioned for a British outbreak of hospital-acquired infection [31] and another case where asymptomatic food handlers have

Non-typhoidal *Salmonella* are a leading cause of bacterial diarrhoea worldwide; they are estimated to cause 94 million cases of gastroenteritis and 115,000 deaths globally each year [2]. Of these, 80.3 million cases were estimated as foodborne origin. In one analysis [33] using data from the Foodborne Diseases Active Surveillance Network (FoodNet), the risk of *Salmonella* infection among travellers returning to the USA varied by region of the world visited. Travellers with salmonellosis were most likely to report visiting the following countries: Mexico (38% of travel-associated salmonellosis), India (9%), Jamaica (7%), the Dominican Republic (4%), China (3%) and the Bahamas (2%). Travel-associated infections were related to *Salmonella* in 36.7% of the cases reported, of which non-typhoidal *Salmonella* accounted for

In the latest EFSA's report, a total of 82,694 confirmed salmonellosis cases were reported by 27 European Union (EU) member states in 2013, resulting in an EU notification rate of 20.4 cases

A decrease of 7.9% in the EU notification rate compared with 2012 was shown in the above report, which supports the declining trend of salmonellosis in the EU/European Economic

been responsible for an outbreak in a catering establishment in Jerusalem [32].

88.3%, typhoidal *Salmonella* 7.7%, and paratyphoidal *Salmonella* 3.9%.

to 107

/g has been recorded in one patient 48 days post-illness

Salmonella/g persist-

relevant to crops commonly used as components of animal feed.

cases can be quite substantial with numbers approximating 106

over a year in the environment.

218 Current Topics in Salmonella and Salmonellosis

35–40 days, but a count of 6 × 103

per 100,000 population [11].

**Figure 1.** Trend in reported confirmed cases of human non-typhoidal salmonellosis in the EU/EEA, 2009–2013. Source: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Lithuania, Luxembourg, Malta, Netherlands, Norway, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden and United Kingdom. Bulgaria, Croatia, Italy, Latvia, Poland and Romania did not report data over the whole period in the level of detail needed for the analysis.

In a recent report published by USDA in 2015 [35], a comparison of the economic burden showed that *Salmonella* ranks first among the 15 pathogens included in the study and sixth on a per-case basis. It imposes an estimated \$3.7 billion in economic burden in a typical year. Almost 90% of this burden, thus \$3.3 billion, is due to deaths; 8%, \$294 million, is due to hospitalization and the remaining 2% is due to non-hospitalized cases (hospitalization rate of 27.2% and a death rate of 0.5%).

According to Decision No. 2119/98/EC and 2000/96/EC, surveillance of foodborne salmonellosis in humans is mandatory in the EU member states as well as setting up a network for the epidemiological surveillance and control of communicable diseases in the Community [36, 37]. Data on humans, animals and food are compiled and analysed jointly by the European Food Safety Agency (EFSA) and the European Centre for Disease Prevention and Control (ECDC) and presented annually in the EU Summary Report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks [37].

## **4.** *Salmonella* **spp. in poultry and poultry products**

*Salmonella* species that colonize the intestinal tract of poultry can persist throughout the bird's lifespan in a poultry-producing environment and are shed with faeces [9, 16]. Faecal shedding allows *Salmonella* to be transmitted among birds in a flock. *Salmonella* spp. is widespread in poultry production in Europe. Prevalence varies considerably depending on country and type of production as well as the detection methods applied. Results showed that prevalence is at the lowest level at the top of the production pyramid, i.e. the breeding stock. As mentioned above poultry meat and eggs represent an important source of human infection with *Salmonella* spp. *with S. enteritidis* and *S. typhimurium* been the most commonly reported serovars involved.

In the primary production, there are numerous activities that influence the introduction, growth or elimination of *Salmonella* species for poultry and poultry products, and therefore, many opportunities are given to *Salmonella* to enter the food chain, even though other steps will prevent growth or inactivate the pathogen. Several studies have studied the risk factors [38–40] associated with *Salmonella* contamination in broiler chickens. The most important risk factors included contaminated chicks, size of the farm (>3 poultry sheds—presumably related to increased human traffic among multiple sheds) and contaminated feed (the risk of Salmonella contamination of the flock was increased when feed trucks were parked near the entrance of the workers' change room and when feed meal, instead of small pellets). A systematic review of the risk factors associated with *Salmonella* in laying hens [41] concluded that the presence of previous *Salmonella* infection, absence of larger flock size (>30,000 hens), multi-age management, cage housing systems, rearing pullets on the floor, induced molting and in-line egg processing were factors associated with Salmonella infection. Also, cleaning and disinfection, presence of rodents, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses and trucks near farms and air inlets were risks identified to be associated with *Salmonella* contamination of laying hen premises. However, high level of manure contamination, middle and late phase of production, high degree of egghandling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with *Salmonella* contamination of shell eggs. These were risks which showed strong to moderate evidence of association with *Salmonella* contamination of laying hens and shell eggs. In the same study, eggshells testing positive for *Salmonella* were 59 times higher when faecal samples were positive and nine times higher when floor dust samples were positive. Furthermore, the presence of *Salmonella enteriditis* infection in laying hens was associated with risk factors such as flock size, housing system and farms with hens of different ages.

hospitalization and the remaining 2% is due to non-hospitalized cases (hospitalization rate of

According to Decision No. 2119/98/EC and 2000/96/EC, surveillance of foodborne salmonellosis in humans is mandatory in the EU member states as well as setting up a network for the epidemiological surveillance and control of communicable diseases in the Community [36, 37]. Data on humans, animals and food are compiled and analysed jointly by the European Food Safety Agency (EFSA) and the European Centre for Disease Prevention and Control (ECDC) and presented annually in the EU Summary Report on trends and sources of zoonoses, zoo-

*Salmonella* species that colonize the intestinal tract of poultry can persist throughout the bird's lifespan in a poultry-producing environment and are shed with faeces [9, 16]. Faecal shedding allows *Salmonella* to be transmitted among birds in a flock. *Salmonella* spp. is widespread in poultry production in Europe. Prevalence varies considerably depending on country and type of production as well as the detection methods applied. Results showed that prevalence is at the lowest level at the top of the production pyramid, i.e. the breeding stock. As mentioned above poultry meat and eggs represent an important source of human infection with *Salmonella* spp. *with S. enteritidis* and *S. typhimurium* been the most commonly reported

In the primary production, there are numerous activities that influence the introduction, growth or elimination of *Salmonella* species for poultry and poultry products, and therefore, many opportunities are given to *Salmonella* to enter the food chain, even though other steps will prevent growth or inactivate the pathogen. Several studies have studied the risk factors [38–40] associated with *Salmonella* contamination in broiler chickens. The most important risk factors included contaminated chicks, size of the farm (>3 poultry sheds—presumably related to increased human traffic among multiple sheds) and contaminated feed (the risk of Salmonella contamination of the flock was increased when feed trucks were parked near the entrance of the workers' change room and when feed meal, instead of small pellets). A systematic review of the risk factors associated with *Salmonella* in laying hens [41] concluded that the presence of previous *Salmonella* infection, absence of larger flock size (>30,000 hens), multi-age management, cage housing systems, rearing pullets on the floor, induced molting and in-line egg processing were factors associated with Salmonella infection. Also, cleaning and disinfection, presence of rodents, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses and trucks near farms and air inlets were risks identified to be associated with *Salmonella* contamination of laying hen premises. However, high level of manure contamination, middle and late phase of production, high degree of egghandling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with *Salmonella* contamination of shell eggs.

27.2% and a death rate of 0.5%).

220 Current Topics in Salmonella and Salmonellosis

serovars involved.

notic agents and foodborne outbreaks [37].

**4.** *Salmonella* **spp. in poultry and poultry products**

The Panel on Biological Hazards [42] recommended that the application of hazard analysis critical control point (HACCP) principles, including good manufacturing practices and general hygiene procedures are recognized as important measures for *Salmonella* control in feed production. However, prevalence data for Salmonella in feed ingredients or compounded feed are usually very difficult to compare between different studies due to differences in sampling and analytical methods applied. The existing community legislation on food hygiene and control of zoonosis [43] constitutes a number of provisions that aim to control and prevent the *Salmonella* contamination of foodstuffs. Targets for *Salmonella* spp. were set progressively in different animal populations: breeding flocks of Gallus gallus, laying hens, broilers and turkeys. As an obligation, member states have developed and submitted national control programs to the commission which include recommendation on establishing strict biosecurity measures at farm level (including *Salmonella*-free poultry feed and water), vaccination programs in the parent flocks [44] as well as testing and removal of positive flocks from production. Except of encouraging immunity or resistance to *Salmonella* infection in birds through the use of antibodies, other strategies to prevent infection include the use of feed additives or acidified food. It is expected that acid treatments have a residual protective effect on feed, which reduces both the recontamination of feed as well as the contamination of milling and feeding equipment and the general environment [45]. However, the efficacy of organic acids against *Salmonella* depends on the level of bacterial contamination [46]. The same author recommends that, except feed treatment, water acidification can help prevent *Salmonella*, as the supplementation of acids in drinking water reduces the pH level and bacterial counts.

Nowadays, the trend seems to be towards production becoming more integrated, and many small farms will be replaced in the future by fewer, bigger farms, which will allow a greater integration and consequently to a better control of *Salmonella*. Furthermore, comparisons of *Salmonella* species contamination of free range or organic production systems with 'conventional' systems have produced varied results and more statistically valid surveys are required to ascertain if differences do occur [47]. In addition, the transportation of poultry between farms and from the farm to the processing plant offers an environment where *Salmonella* species might be spread between birds [48–50]. Shedding of large numbers of pathogens in faecal material during transport is believed to be related to increased stress in birds [48, 49].

Sewage and farm effluents, which can contaminate pasture, soil and water with *Salmonella*, tend to be handled more consciously lately, due to the pressure of environmental law requirements. However, the breeding stocks used all over the world are produced by a small number of companies, meaning that these sell to purchasers worldwide and this can lead to the wide-scale spread of *Salmonella*, if the breeding stocks are infected. One should also take into consideration that where the aim is to control specific serotypes, a zero-tolerance policy with respect to these organisms may give a false sense of security, because the predominant serotypes in poultry flocks are likely to change over time.

## **5. Primary and secondary poultry processing and retail**

The most important control measure at primary production, apart from those focusing in the elimination of *Salmonella* in grandparent and parent flocks by vaccination and an all-inall-out production at the broiler farm, is to avoid any carry over during processing which is achieved by a logistic slaughter planning scheduled to avoid pathogens being transferred from contaminated processing equipment to another flock, and finally the satisfactory cleaning of transport crates. The operations that are thought to increase the contamination while in the processing line are scalding, plucking and evisceration. The most important critical control point in the process in relation to contamination is the feather plucker. Also, evisceration can be considered as an important risk due to a consequence of gut rupture. The evisceration machinery may play a role in damaging poultry carcasses while these are not entirely uniform in size. Most studies so far have shown that the prevalence of *Salmonella* species is usually higher on poultry carcasses at the end of primary processing than at the start [51, 52], although the concentrations of organisms on carcasses tend to decrease [17].

To reduce carcass contamination, decontamination measures can be applied. Many countries after the adoption of the 'Code of hygienic practice recommended for poultry processing' by the Codex Alimentarius in 1994, adopted their own code of practices for poultry processing. The requirements for cleaning of de-feathering equipment and recommended list of used disinfectants and practices of physical separation of de-feathering from later primary processing steps, requirements for processors to define acceptable levels of visible faecal contamination following evisceration and monitoring requirements for faecal contamination and practices of spaying or rinsing/dipping are included in this code. As far as these decontamination measures is concerned, one should take into consideration that, there are some regional differences, since chemical treatment is not accepted in the EU at the moment, but is widely used in other parts of the world, e.g. in the USA and New Zealand.

Poultry secondary processing includes portioning and processing of carcasses or portions into value-added products. During secondary processing, *Salmonella* prevalence may increase due to cross-contamination, while concentrations of *Salmonella* may increase if temperature control is not properly maintained [53]. Both poultry muscle and skin are excellent substrates for a wide variety of microorganisms [54], but the potential shelf life of raw poultry is quite short (e.g. chicken samples had spoiled after 4 days at 9°C) [55]. Unless frozen, raw poultry has a rapid turnover at retail, often 24–48 hours with a best before date of 3–4 days [56].

*Salmonella* species can survive well at refrigeration temperatures and will grow on fresh poultry under warmer, more favourable, temperatures (e.g. during transportation from a retail outlet to a consumer's home). *Salmonella* numbers are reduced under frozen storage conditions but salmonellosis outbreaks from 1998 to 2008 due to consumption of frozen products showed that bacteria can survive freezing and *Salmonella* may pose an infection risk if the product is improperly cooked [57]. Thus, freezing cannot be considered as an adequate control step.

wide-scale spread of *Salmonella*, if the breeding stocks are infected. One should also take into consideration that where the aim is to control specific serotypes, a zero-tolerance policy with respect to these organisms may give a false sense of security, because the predominant sero-

The most important control measure at primary production, apart from those focusing in the elimination of *Salmonella* in grandparent and parent flocks by vaccination and an all-inall-out production at the broiler farm, is to avoid any carry over during processing which is achieved by a logistic slaughter planning scheduled to avoid pathogens being transferred from contaminated processing equipment to another flock, and finally the satisfactory cleaning of transport crates. The operations that are thought to increase the contamination while in the processing line are scalding, plucking and evisceration. The most important critical control point in the process in relation to contamination is the feather plucker. Also, evisceration can be considered as an important risk due to a consequence of gut rupture. The evisceration machinery may play a role in damaging poultry carcasses while these are not entirely uniform in size. Most studies so far have shown that the prevalence of *Salmonella* species is usually higher on poultry carcasses at the end of primary processing than at the start [51, 52],

To reduce carcass contamination, decontamination measures can be applied. Many countries after the adoption of the 'Code of hygienic practice recommended for poultry processing' by the Codex Alimentarius in 1994, adopted their own code of practices for poultry processing. The requirements for cleaning of de-feathering equipment and recommended list of used disinfectants and practices of physical separation of de-feathering from later primary processing steps, requirements for processors to define acceptable levels of visible faecal contamination following evisceration and monitoring requirements for faecal contamination and practices of spaying or rinsing/dipping are included in this code. As far as these decontamination measures is concerned, one should take into consideration that, there are some regional differences, since chemical treatment is not accepted in the EU at the moment, but is widely used in

Poultry secondary processing includes portioning and processing of carcasses or portions into value-added products. During secondary processing, *Salmonella* prevalence may increase due to cross-contamination, while concentrations of *Salmonella* may increase if temperature control is not properly maintained [53]. Both poultry muscle and skin are excellent substrates for a wide variety of microorganisms [54], but the potential shelf life of raw poultry is quite short (e.g. chicken samples had spoiled after 4 days at 9°C) [55]. Unless frozen, raw poultry has a rapid turnover at retail, often 24–48 hours with a best before date of 3–4 days [56].

*Salmonella* species can survive well at refrigeration temperatures and will grow on fresh poultry under warmer, more favourable, temperatures (e.g. during transportation from a retail outlet to a consumer's home). *Salmonella* numbers are reduced under frozen storage conditions but salmonellosis outbreaks from 1998 to 2008 due to consumption of frozen

types in poultry flocks are likely to change over time.

222 Current Topics in Salmonella and Salmonellosis

**5. Primary and secondary poultry processing and retail**

although the concentrations of organisms on carcasses tend to decrease [17].

other parts of the world, e.g. in the USA and New Zealand.

In a New Zealand consumer survey, the times and temperatures of purchased poultry products during transportation by consumers were examined [58]. It showed that thawing poultry at room temperature for up to 12 hours was a common practice and that any *Salmonella* present on the surface of the poultry could be able to grow once the surface reached room temperature [59]. Other studies have shown that the time required for frozen poultry (–18°C) to reach minimum growth temperature (7°C) would be in the range 3–16 hours, depending on the freezer temperature and ambient (air) temperatures [60]. As growth is greatly reduced up to 15°C (requiring another 3 hours thawing), and not optimal until 35–37°C, normal thawing periods before cooking are unlikely to permit much growth, although situations involving warm freezer temperatures (–7°C) and high ambient temperatures may increase the amount of growth that occurs.

The detection of *Salmonella* in poultry products leads to rejection of large consignments of raw poultry meat, thus affecting poultry trade with huge economic impacts as a consequence. Of course, on top of that, the impact on human health and the associated costs, the trade disruptions and the cost of implementing effective control measures explains why the Codex Alimentarius Commission (CAC) in 2010 [61] agreed that the development of guidelines for the control of *Salmonella* in poultry was a priority. Even though information on the prevalence of *Salmonella* on poultry meat at the end of processing or at retail were available, very few surveys have been undertaken where the number of organisms has been quantified [62] because enumeration of *Salmonella* proved to be very laborious.

Furthermore, interventions at the processing stages are assessed using growth models. These take into consideration several factors such as the levels of contamination when carcasses leave the processing plant, storage time in retail stores, transport time, storage times in homes and the temperatures carcasses were exposed to during each of these periods. It should be mentioned that the presence and level of *Salmonella* in this step is very much country specific, since the level of infection when leaving the processing step varies between the countries in relation to the methods which are used at the processing plant. In any case, national data must be used when estimating levels of contamination therefore [16].

## **6. Food of animal and plant origin as a source of** *Salmonella* **serovars for humans**

Both plant and animal product-based animal feed ingredients may be contaminated with *Salmonella*. Red and white meat, meat products, milk, cheese and eggs are considered the major food sources of human salmonellosis, although a wide variety of other foods have been associated with outbreaks [8]. Other researchers reported that lamb's liver was responsible for an outbreak of *S. typhimurium* phage-type 197 in Australia [63]. In Germany, from 2001 to 2005, microbiological testing, trace-back investigations and epidemiological studies showed that pork and pork products were involved in human salmonellosis outbreaks [64]. In Italy, an outbreak of *S. typhimurium* phage-type DT 104A involving 63 cases suggested that the consumption of pork salami was associated with this outbreak, underlining the importance of good manufacturing practices for ready-to-eat foods [65]. Many other reports involving human salmonellosis outbreaks associated with consumption of red meat have been recorded in the literature [66, 67], and in most of cases, the disease was associated with the consumption of contaminated meat or was a result of incorrect or inadequate cooking.

In the European Union (EU), contaminated foodstuffs serving as a source of *Salmonella* infection for humans include table eggs closely followed by pig meat, whereas the risks associated with broiler and turkey meat are similar and approximately two-fold lower [68]. As far as the distribution of serovars is concerned, in the EU, *S. enteritidis* and *S. typhimurium* are the serovars most commonly associated with human illness. Human cases of *S. enteritidis* are most frequently associated with the consumption of contaminated eggs and poultry meat, while *S. typhimurium* cases are associated with the consumption of contaminated pig meat or bovine meat [69]. It is estimated that around 10.6, 17, 56.8 and 2.6% of the human salmonellosis cases in the EU are attributable to broilers, laying hens (eggs), pigs and turkeys, respectively [70]. Of the broiler-associated human salmonellosis cases, around 82 and 6.5% are estimated to be due to the serovars *S. enteritidis* and *S. infantis*, respectively [71]. In the EU, approximately 9% of turkey carcasses are *Salmonella*-positive and the top six serovars that contribute to human cases are *S. enteritidis, S. kentucky, S. typhimurium, S. newport, S. virchow* and *S. saintpaul* [70]. While there are few data on the prevalence of pathogens on trimmings and meat cuts used for minced meat products, in [71] *Salmonella* spp. was detected on up to 5.3% of beef trimmings. The highest levels of non-compliance with *Salmonella* criteria generally occurred in foods of meat origin that are intended to be cooked before consumption in 2014, as in the last years [11]. Minced meat and meat preparations from poultry intended to be eaten cooked showed the highest level of non-compliance (category 1.5; 8.7% of single samples and 5.7% of batches). One should consider that growth of *Salmonella* should be absent or very slow in correctly chilled meat intended for preparation of mince since the organism show a reported minimum growth temperature of 5°C and an optimum temperature of 35–43°C [72], a pH growth range of 4.5–9.0.

A long list of foods that have been contaminated by *Salmonella* includes: seafood (shellfish, salmon), cereal and cereal products (barley, cereal powder), oilseeds and oilseed products (cottonseed, soybean sauce, sesame seeds), nuts and nut products (desiccated coconut, peanut butter), spices (white and black pepper, paprika), vegetables (watercress, tomatoes, lettuce, potato and other salads and bean sprouts), fruit and fruit products (watermelon, melon and cider) and other miscellaneous products (chocolate, cocoa powder, dried yeast and candy). *Salmonella* contaminated tahini (a product made from crushed sesame seeds) has caused a number of outbreaks worldwide, including New Zealand and Australia [73]. In 2002, an outbreak of *S. montevideo* occurred in New South Wales, Australia showing that imported 'tahini' was rapidly identified as the source of infection.

In foods from vegetable origin, detection of *Salmonella* serovars is a matter of increasing concern. Recent literature highlights the importance of foods of vegetable origin as potential vehicles of gastrointestinal infection nowadays. *Salmonella* serovars may contaminate vegetables during production, storage or even in retail outlets. Furthermore, fruits and juices, as they are usually consumed raw, may also be implicated in human salmonellosis.

In 2002, tomatoes, grown and packed in Virginia state (USA), contaminated with *S. newport*, caused illness in 510 patients in 26 other states [74]. Later, in July–November 2005, the same strain (confirmed by PFGE) caused illness in at least 72 patients in 16 states of the USA. *S. newport* strain was responsible for the outbreak which was isolated from pond water used to irrigate tomato fields, suggesting persistent contamination of the fields [75]. Also, during 2005–2006, in the USA and Canada three more outbreaks of *Salmonella* infections associated with eating tomatoes were detected. These outbreaks resulted in 387 culture-confirmed cases of salmonellosis, with isolation of *S. newport*, *S. braenderup* and *S. typhimurium* [76].

that pork and pork products were involved in human salmonellosis outbreaks [64]. In Italy, an outbreak of *S. typhimurium* phage-type DT 104A involving 63 cases suggested that the consumption of pork salami was associated with this outbreak, underlining the importance of good manufacturing practices for ready-to-eat foods [65]. Many other reports involving human salmonellosis outbreaks associated with consumption of red meat have been recorded in the literature [66, 67], and in most of cases, the disease was associated with the consump-

In the European Union (EU), contaminated foodstuffs serving as a source of *Salmonella* infection for humans include table eggs closely followed by pig meat, whereas the risks associated with broiler and turkey meat are similar and approximately two-fold lower [68]. As far as the distribution of serovars is concerned, in the EU, *S. enteritidis* and *S. typhimurium* are the serovars most commonly associated with human illness. Human cases of *S. enteritidis* are most frequently associated with the consumption of contaminated eggs and poultry meat, while *S. typhimurium* cases are associated with the consumption of contaminated pig meat or bovine meat [69]. It is estimated that around 10.6, 17, 56.8 and 2.6% of the human salmonellosis cases in the EU are attributable to broilers, laying hens (eggs), pigs and turkeys, respectively [70]. Of the broiler-associated human salmonellosis cases, around 82 and 6.5% are estimated to be due to the serovars *S. enteritidis* and *S. infantis*, respectively [71]. In the EU, approximately 9% of turkey carcasses are *Salmonella*-positive and the top six serovars that contribute to human cases are *S. enteritidis, S. kentucky, S. typhimurium, S. newport, S. virchow* and *S. saintpaul* [70]. While there are few data on the prevalence of pathogens on trimmings and meat cuts used for minced meat products, in [71] *Salmonella* spp. was detected on up to 5.3% of beef trimmings. The highest levels of non-compliance with *Salmonella* criteria generally occurred in foods of meat origin that are intended to be cooked before consumption in 2014, as in the last years [11]. Minced meat and meat preparations from poultry intended to be eaten cooked showed the highest level of non-compliance (category 1.5; 8.7% of single samples and 5.7% of batches). One should consider that growth of *Salmonella* should be absent or very slow in correctly chilled meat intended for preparation of mince since the organism show a reported minimum growth temperature of 5°C and an optimum temperature of 35–43°C [72], a pH growth range of 4.5–9.0.

A long list of foods that have been contaminated by *Salmonella* includes: seafood (shellfish, salmon), cereal and cereal products (barley, cereal powder), oilseeds and oilseed products (cottonseed, soybean sauce, sesame seeds), nuts and nut products (desiccated coconut, peanut butter), spices (white and black pepper, paprika), vegetables (watercress, tomatoes, lettuce, potato and other salads and bean sprouts), fruit and fruit products (watermelon, melon and cider) and other miscellaneous products (chocolate, cocoa powder, dried yeast and candy). *Salmonella* contaminated tahini (a product made from crushed sesame seeds) has caused a number of outbreaks worldwide, including New Zealand and Australia [73]. In 2002, an outbreak of *S. montevideo* occurred in New South Wales, Australia showing that imported 'tahini'

In foods from vegetable origin, detection of *Salmonella* serovars is a matter of increasing concern. Recent literature highlights the importance of foods of vegetable origin as potential vehicles of gastrointestinal infection nowadays. *Salmonella* serovars may contaminate vegetables during production, storage or even in retail outlets. Furthermore, fruits and juices, as they are

usually consumed raw, may also be implicated in human salmonellosis.

was rapidly identified as the source of infection.

tion of contaminated meat or was a result of incorrect or inadequate cooking.

224 Current Topics in Salmonella and Salmonellosis

Unpasteurized orange juice was responsible for foodborne salmonellosis in 152 people in six states in the USA between May and July 2005 [77]. From 1995 to 2005, some researchers reviewed fruit juice-associated outbreaks of illness reported to Centres for Disease Control and Prevention (CDC), in Atlanta, USA [78]. Twenty-one juice-associated outbreaks were reported to CDC; 10 implicated apple juice or cider, eight were linked to orange juice and three implicated other types of fruit juice. These outbreaks caused 1366 illnesses, with an average of 21 cases per outbreak (range, 2–398 cases). Five out of 13 outbreaks of known aetiology, were caused by *Salmonella* serovars.

Human salmonellosis due to *S. thompson* infection were reported in Norway as a result of the consumption of rucola lettuce and mixed salad [79]. Prepared salads were also responsible for infectious intestinal disease outbreaks in England and Wales from 1992 to 2006 [80] as a result of international trade. Cross-contamination, infected food handler or inappropriate storage were the most common factors associated with this vegetable contamination.

## **7. Antibiotic resistance in** *Salmonella* **serovars: a serious problem in public health**

Since 2003, according to the U.S. Food and Drug Administration, antimicrobial resistance in *Salmonella* spp., as well as in other bacterial species, has been recognized as a global threat and an increasing public health matter. Salmonellae have evolved not only virulence mechanisms to interact with host defence mechanisms at various tissues in different stages of infection resulting in significant host immunopathology, morbidity and mortality [1] but have evolved resistance mechanisms against antimicrobial agents, thus triggering host responses.

Individual organisms may transfer mutations that render antibiotics ineffective, passing on a survival advantage to the mutated strain, resulting in a normal genetic variation in bacterial populations. Advantageous mutations can also be conveyed via plasmid exchange within the bacterial colony, in the presence of antibiotics, resulting in proliferation of the resistance trait in the bacterial populations. Natural selection leads to an inherent consequence of exposure to antibiotic compounds and then antibiotic resistance arises.

On the other hand, the spread of particularly resistant clones and the occurrence of resistance genes within these clones can be exacerbated by the use of antimicrobials in human and animal populations and its selective pressure [81]. Many factors may also influence the spread of resistant clones, such as foreign travel by humans, international food trade, animal movements, farming systems, animal husbandry and the pyramidal structure of some types of animal primary production. During the late 1990s and early 2000s, several clones of multi-drug-resistant (MDR) *Salmonella* emerged, and since then, they have expanded worldwide. Multi-drug-resistant *S. enterica* serotype *typhimurium* has been associated with a higher risk of invasive infection, higher frequency and duration of hospitalization, longer illness and increased risk of death as compared to infections caused by susceptible strains [82]. The spread of this resistance in other serotypes is of great concern as well. A very characteristic example is the behaviour of *S. typhimurium*, the genomic element that carries resistance to five antimicrobials (ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline), which can be spread horizontally among other serotypes and acquire additional resistance determinants. Genes conferring antimicrobial resistance in *Salmonella* are often carried on integrons and plasmids and could be transmitted through conjugation. These are mobile DNA elements and play an important role in transmission and dissemination of antimicrobial resistance determinants among *Salmonella* strains [83].

2013/652/EU Commission Decision sets an enhanced monitoring of antibiotic resistance (AMR) in bacteria from food and food-producing animals, which has been successfully implemented in all reporting and non-reporting member states. In accordance with the above legislation, the AMR monitoring started in 2014 and collected data referred on food and food-producing animals specifically targeted in different poultry populations and meat derived thereof. Two agents are responsible in performing the analyses of the data: EFSA and ECDC. The results are published in the first EU Summary Report on AMR [81] derived from 28 member states which reported data on AMR in zoonotic bacteria to the EFSA and 21 member states which submitted data to the ECDC. In the above report, the results showed that high proportions of human *Salmonella* isolates were resistant to tetracyclines (30.3%), sulphonamides (28.6%) and ampicillin (28.2%) and more than half (54.8%) of all isolates from humans were susceptible to the complete range of antimicrobial classes tested. A total of 8.8 and 1.1% of *Salmonella* isolates were resistant to ciprofloxacin to cefotaxime, respectively, which is thought to be an overall relatively low proportion of resistance to these clinically important antimicrobials. In the same report, resistance to third-generation cephalosporins was more common in *S. infantis* and *S. kentucky* with particularly high levels observed in Italy, most likely due to the circulation of a multiresistant and ESBL-producing (cefotaximase (CTX-M) type) clone of S. infantis. Also, an extremely high proportion (84.0%) of *S. kentucky* which showed high resistance to ciprofloxacin was mentioned. This is consistent with the dissemination of the ciprofloxacin-resistant *S. kentucky* ST198 strain in Europe and elsewhere since 2010 [84]. Overall, MDR in the EU was high (26.0%), with very high prevalence in some countries. It must be mentioned that some serovars exhibited very high to extremely high MDR. These were *S. kentucky* (74.6%), monophasic *S. typhimurium* 1,4,[5],12:i:- (69.4%) and *S. infantis* (61.9%). Another interesting observation derived from this study was the resistance to colistin which was commonly detected in *S. enteritidis* (67.5%, two member states) and it is thought that could be due to intrinsic resistance in this serovar.

In another study [85], it was reported that over 80% of strains from both human and animal sources that were tested for their antimicrobial resistance, showed that resistance patterns were similar among strains from humans and animals: the commonest phenotype comprised resistance to ampicillin, sulphonamides, streptomycin, chloramphenicol, and tetracycline and was found in 76% of human and 73% of animal strains. Between 1972 and 1974, almost 50,000 *Salmonella* isolates from several sources (humans, animals, animal products, sewage, etc.) were tested for resistance to ampicillin, chloramphenicol, kanamycin and tetracycline in the Netherlands. The incidence of resistance to at least one of the above drugs ranged from 39.2 to 45.6%. An interesting finding was that multidrug-resistant strains of *S. typhimurium* and *S. dublin* were isolated from calves and cattle. A total of 64.4% of all strains of *S. typhimurium* from these animals appeared to be resistant to ampicillin, tetracycline, chloramphenicol and kanamycin, and 25.5% of *S. dublin* were found to be resistant to chloramphenicol and tetracycline in the latest year of the study [86]. In NARMS's last report which presents data for 2013 in the USA [87], *Salmonella*, antimicrobial resistance varies by serotype: 3% (61/2178) of non-typhoidal *Salmonella* isolates were resistant to nalidixic acid. The most common serotypes among the 55 ceftriaxone-resistant isolates were *S. newport, dublin, typhimurium, heidelberg* and *infantis*.

Overall, antimicrobial resistance varies among different serotypes of non-typhoidal *Salmonella*, and in some of them is considerably significant. It is well-recognized that the emergence of antimicrobial resistance in bacteria, which can be transferred to humans, is attributable to antimicrobial use in animals; therefore the effectiveness of antimicrobial drugs for treating human disease has been reduced extensively. The resistance to certain antimicrobials, especially fluoroquinolones and cephalosporins, are of particular concern with major consequences, since therapy of human systemic bacterial infections are critically dependent on their effectiveness. In face of this public health concern, it is highly recommended to follow a very careful prescription of antimicrobial agents during veterinary practice, regardless of the purpose of this prescription (prophylaxis or therapy) and a prudent use of antimicrobial agents after microbiological identification of the causative pathogen. Last but not least, it is very important to highlight that good hygiene practices and, wherever possible, alternative management methods should be sought and used and should not be substituted by the use of any antimicrobial agent.

## **Author details**

types of animal primary production. During the late 1990s and early 2000s, several clones of multi-drug-resistant (MDR) *Salmonella* emerged, and since then, they have expanded worldwide. Multi-drug-resistant *S. enterica* serotype *typhimurium* has been associated with a higher risk of invasive infection, higher frequency and duration of hospitalization, longer illness and increased risk of death as compared to infections caused by susceptible strains [82]. The spread of this resistance in other serotypes is of great concern as well. A very characteristic example is the behaviour of *S. typhimurium*, the genomic element that carries resistance to five antimicrobials (ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline), which can be spread horizontally among other serotypes and acquire additional resistance determinants. Genes conferring antimicrobial resistance in *Salmonella* are often carried on integrons and plasmids and could be transmitted through conjugation. These are mobile DNA elements and play an important role in transmission and dissemination of antimicrobial

2013/652/EU Commission Decision sets an enhanced monitoring of antibiotic resistance (AMR) in bacteria from food and food-producing animals, which has been successfully implemented in all reporting and non-reporting member states. In accordance with the above legislation, the AMR monitoring started in 2014 and collected data referred on food and food-producing animals specifically targeted in different poultry populations and meat derived thereof. Two agents are responsible in performing the analyses of the data: EFSA and ECDC. The results are published in the first EU Summary Report on AMR [81] derived from 28 member states which reported data on AMR in zoonotic bacteria to the EFSA and 21 member states which submitted data to the ECDC. In the above report, the results showed that high proportions of human *Salmonella* isolates were resistant to tetracyclines (30.3%), sulphonamides (28.6%) and ampicillin (28.2%) and more than half (54.8%) of all isolates from humans were susceptible to the complete range of antimicrobial classes tested. A total of 8.8 and 1.1% of *Salmonella* isolates were resistant to ciprofloxacin to cefotaxime, respectively, which is thought to be an overall relatively low proportion of resistance to these clinically important antimicrobials. In the same report, resistance to third-generation cephalosporins was more common in *S. infantis* and *S. kentucky* with particularly high levels observed in Italy, most likely due to the circulation of a multiresistant and ESBL-producing (cefotaximase (CTX-M) type) clone of S. infantis. Also, an extremely high proportion (84.0%) of *S. kentucky* which showed high resistance to ciprofloxacin was mentioned. This is consistent with the dissemination of the ciprofloxacin-resistant *S. kentucky* ST198 strain in Europe and elsewhere since 2010 [84]. Overall, MDR in the EU was high (26.0%), with very high prevalence in some countries. It must be mentioned that some serovars exhibited very high to extremely high MDR. These were *S. kentucky* (74.6%), monophasic *S. typhimurium* 1,4,[5],12:i:- (69.4%) and *S. infantis* (61.9%). Another interesting observation derived from this study was the resistance to colistin which was commonly detected in *S. enteritidis* (67.5%, two member states)

resistance determinants among *Salmonella* strains [83].

226 Current Topics in Salmonella and Salmonellosis

and it is thought that could be due to intrinsic resistance in this serovar.

In another study [85], it was reported that over 80% of strains from both human and animal sources that were tested for their antimicrobial resistance, showed that resistance patterns were similar among strains from humans and animals: the commonest phenotype comprised resistance to ampicillin, sulphonamides, streptomycin, chloramphenicol, and tetracycline and Niki Mouttotou<sup>1</sup> , Shakeel Ahmad2 , Zahid Kamran3 , and Konstantinos C. Koutoulis4 \*

\*Address all correspondence to: kkoutoulis@vet.uth.gr

1 National Reference Laboratory of Salmonella and Antimicrobial Resistance, Ministry of Rural Development and Food, Chalkis, Greece

2 Al-Watania Poultry Institute of Technology, Al-Bukayriyah, Saudi Arabia

3 University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

4 Department of Poultry Diseases, Faculty of Veterinary Science, School of Health Sciences, University of Thessaly, Karditsa, Greece

## **References**


[14] De Jong B, Ekdahl K. Human salmonellosis in travellers is highly correlated to the prevalence of Salmonella in laying hen flocks. Eurosurveillance. 2006;**11:**E060706.1.

**References**

2010;**50:**882–889.

228 Current Topics in Salmonella and Salmonellosis

Risk Analysis; 2007

Bacteriol. 2004;**186:**5883–5898.

Microbiol. 2000;**38:**2465–2467.

EFSA J. 2015;**13(1):**3991.

Inc.; 2013; pp. 677–736. ISBN: 978-0-470-95899-5.

France; 2001

[1] Coburn B, Grassl GA, Finlay BB. Salmonella, the host and disease: a brief review.

[2] Majowicz S, Musto J, Scallan E, Angulo F, Kirk M, O'Brien S, Jones T, Fazil A, Hoekstra R. The global burden of nontyphoidal *Salmonella* gastroenteritis. Clin Infect Dis.

[3] EFSA. Report of the Task Force on Zoonoses Data Collection on the analysis of the baseline survey on the prevalence of *Salmonella* in turkey flocks, in the EU, 2006–2007. Part A:

[4] Vargas-Galindo Á. Probabilistic inversion in priority setting of food borne pathogens [MSc thesis]. Delft University of Technology: Department of Applied Mathematics and

[5] Popoff MY, Le Minor L. Antigenic formulas of the Salmonella serovars, 8th ed. WHO Collaborating Centre for Reference and Research on Salmonella, Institute Pasteur, Paris,

[6] Porwollik S, Boyd EF, Choy C, Cheng P, Florea L, Proctor E, McClelland M. Characterization of Salmonella enterica subspecies I genovars by use of microarrays. J

[7] Brenner F, Villar R, Angulo F, Tauxe R, Swaminathan B. *Salmonella* nomenclature. J Clin

[8] Jay LS, Davos D, Dundas M, Frankish E, Lightfoot D. Salmonella. In: Hocking AD (ed). Foodborne microorganisms of public health significance. 6th ed, Australian Institute of

[9] Gast RK. Salmonella infections. In: Swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair VL (eds). Diseases of Poultry. 13th ed, Ames, IA: John Wiley and Sons

[10] Allison MJ, Dalton HP, Escobar MR, Martin CJ. *Salmonella choleraesuis* infections in man: a report of 19 cases and a critical literature review. South Med J. 1969;**62:**593–596. [11] EFSA. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2013 Scientific Report of EFSA and ECDC.

[12] Castagna SMF, Schwartz P, Canal CW, Cardoso M. Presence of Salmonella sp. in the intestinal tract and tonsils/mandibular lymph nodes in pigs at slaughter. Arq Brasil Med

[13] Shimoni Z, Pitlik's K, Leibovici L. Nontyphoidal salmonella bacteremia: age-related differences in clinical presentation, bacteriology, and outcome. Clin Infect Dis. 1999;**28:**822–7.

Veter Zoo. 2004;**56(3):**300–306 DOI: 10.1590/S0102-09352004000300003

Food Science and Technology (NSW Branch), Sydney; 2003; pp. 207–266.

Immunol Cell Biol. 2007;**85:**112–118. DOI: 10.1038/sj.icb.7100007

*Salmonella* prevalence estimates. EFSA J. 2008;**134:**1–91.


[42] EFSA. Scientific opinion on microbiological risk assessment in feeding stuffs for foodproducing animals. Scientific opinion of the panel on biological hazards. EFSA J. 2008;**720:**1–84.

[28] Heaton JC, Jones K. Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review. J Appl Microbiol. 2008;**104(3):**613–626.

[29] Loewenstein M. An outbreak of salmonellosis propogated by person-to-person trans-

[30] Pether J, Scott R. *Salmonella* carriers: are they dangerous? A study to identify finger con-

[31] Dryden MS. Asymptomatic foodhandlers as the source of nosocomial salmonellosis. J

[32] Stein-Zamir C, Tallen-Gozani E, Abramson N, Shoob H, Yishai R, Agmon V, Reisfeld A, Valinsky L, Marva E. *Salmonella enterica* outbreak in a banqueting hall in Jerusalem: the

[33] Kendall ME, Crim S, Fullerton K, Han PV, Cronquist AB, Shiferaw B. Travel-associated enteric infections diagnosed after return to the United States, Foodborne Diseases Active Surveillance Network (FoodNet), 2004–2009. Clin Infect Dis. 2012;**54(Suppl 5):**S480–S487.

[34] Havelaar AH, Ivarsson S, Löfdahl M, Nauta MJ. Estimating the true incidence of campylobacteriosis and salmonellosis in the European Union, 2009. Epidemiol Infect.

[35] Hoffmann S, Maculloch B, Batz M. Economic burden of major foodborne illnesses acquired in the United States. In: Economic Research Service Economic Information Bulletin; Number 140, May 2015; pp.33; United States Department of Agriculture (USDA ). Available from: https://www.ers.usda.gov/webdocs/publications/eib140/52806\_eib140\_summary.pdf

[36] Commission Decision No. 2119/98/EC of the European Parliament and of the Council of 24 September 1998 setting up a network for the epidemiological surveillance and control

[37] Commission Decision No. 2000/96/EC of 22 December 1999 on the communicable diseases to be progressively covered by the community network under Decision No.

2119/98/EC of the European Parliament and of the Council. O J L. 2000;28:50–53.

[38] Rose N, Beaudeau F, Drouin P, Toux J, Rose V, Colin P. Risk factors for *Salmonella enterica* subsp. *enterica* contamination in French broiler-chicken flocks at the end of the rearing

[39] Marin C, Balasch S, Vega S, Lainez M. Sources of Salmonella contamination during

[40] Arsenault J, Letellier A, Quessy S, Normand V, Boulianne M. Prevalence and risk factors for Salmonella spp. and Campylobacter spp. caecal colonization in broiler chicken and

turkey flocks slaughtered in Quebec, Canada. Prevent Vet Med. 2007;**81:**250–264.

[41] Denagamage T, Jayarao B, Patterson P, Wallner-Pendleton E, Kariyawasam S. Risk factors associated with Salmonella in laying hen farms: systematic review of observational

broiler production in Eastern Spain. Prevent Vet Med. 2011;**98:**39–45.

studies. Avian Dis. 2015;**59(2):**291–302. DOI: 10.1637/10997-120214-Reg

of communicable diseases in the community. O J L. 1998;268:1–6.

period. Prevent Vet Med. 1999;**39:**265–277.

unseen hand of the epidemiological triangle? Israel Med Assoc J. 2009;**11:**94–97.

mission on an Indian reservation. Am J Epidemiol. 1974;**102:** 257–262.

Hosp Infect. 1994;**28:**195–208.

230 Current Topics in Salmonella and Salmonellosis

2013;**141(2):**293–302.

tamination with *Salmonella* by convalescent carriers. J Infect. 1982;**5:**81–88.


[69] EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J. 2014;**12(2):**3547 (312 pp.). DOI:10.2903/j.efsa.2013.3129

[56] King N, Wong T. Feasibility study: trace back of fresh poultry portions sold at retail. In:

[57] Dominguez SA, Schaffner DW. Survival of *Salmonella* in processed chicken products

[58] Gilbert S, Bayne G, Wong T, Lake R, Whyte R. Domestic food practices in New Zealand. 2005–2006 project report. In: ESR Client Report FW0640. Christchurch: ESR; 2006. [59] Gilbert S, Whyte R, Bayne G, Paulin S, Lake R, van der Logt P. Survey of domestic food

[60] McIntyre L, Bayne G, Gilbert S, Lake R. Domestic food practices in New Zealand. Freezer survey. In: ESR Client Report FW0735. Christchurch: ESR; 2007. Available from: http:// foodsafety.govt.nz/elibrary/industry/Domestic\_Food\_Practices-Baseline\_Information.pdf

[61] CAC. Report of the forty-second session of the Codex Committee on Food Hygiene, Kampala, Uganda, 29 November–3 December 2010. Codex Alimentarius Commission. Available from: https://ec.europa.eu/food/sites/food/files/safety/docs/codex\_ccfh\_42\_

[62] Anonymous. Outbreak of *Salmonella* Enteritidis 9a, Auckland, April-May 2005 (an inter-

[63] Hess IM, Neville LM, McCarthy R, Shadbolt CT, McAnulty JM. A Salmonella Typhimurium 197 outbreak linked to the consumption of lambs' liver in Sydney, NSW.

[64] Jansen A, Frank C, Stark K. Pork and pork products as a source for human salmonellosis in Germany. Berliner und Münchener tierärztliche Wochenschrift. 2007;**120(7-8):**340–346.

[65] Luzzi I, Galetta P, Massari M, Rizzo C, Dionisi AM, Filetici E, Cawthorne A, Tozzi A, Argentieri M, Bilei S, Busani L, Gnesivo C, Pendenza A, Piccoli A, Napoli P, Loffredo L, Trinito MO, Santarelli E, Ciofi degli Atti ML. An Easter outbreak of Salmonella Typhimurium DT 104A associated with traditional pork salami in Italy. Euro Surveill.

[66] Haeghebaert S, Duché L, Gilles C, Masini B, Dubreuil M, Minet JC, Bouvet P, Grimont F, Delarocque Astagneau E, Vaillant V. Minced beef and human salmonellosis: review of

[67] Little CL, Richardson JF, Owen RJ, de Pinna E, Threlfall EJ. Campylobacter and Salmonella in raw red meats in the United Kingdom: prevalence, characterization and

[68] EFSA. Panels on Biological Hazards (BIOHAZ), on Contaminants in the Food Chain (CONTAM) and on Animal Health and Welfare (AHAW), 2012. Scientific Opinion on the public health hazards to be covered by inspection of meat (poultry). EFSA J.

antimicrobial resistance pattern, 2003–2005. Food Microbiol. 2008;**25(3):**538–543.

2012;**10(6):**2741 (179 pp.). DOI:10.2903/j.efsa.2012.2741

the investigation of three outbreaks in France. Euro Surveill. 2001;**6(2):**21–26

handling practices in New Zealand. Int J Food Microbiol. 2007;**117:**306–311.

ESR Client Report FW10056. Christchurch: ESR; 2010.

during frozen storage. J Food Protect. 2009;**72:**2088–2092.

nal report provided by Public Health Services).

Epidemiol Infect. 2008;**136(4):**461–467.

agenda-item\_04.pdf

232 Current Topics in Salmonella and Salmonellosis

2007;**12(4):**11–12.


#### **Effects of Environment and Socioeconomics on** *Salmonella* **Infections Effects of Environment and Socioeconomics on**  *Salmonella* **Infections**

Hafiz Anwar Ahmad and Luma Akil Hafiz Anwar Ahmad and Luma Akil

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67501

#### **Abstract**

[81] EFSA. The European Union summary report on antimicrobial resistance in zoonotic and

[82] European Centre for Disease Prevention and Control (ECDC), European Food Safety Authority (EFSA), European Drugs Agency (EMEA), Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). Joint opinion on antimicrobial resistance (AMR) focused on zoonotic infections. EFSA J. 2009;**7(11):**1372. Available from: http:// www.ema.europa.eu/docs/en\_GB/document\_library/Other/2009/11/WC500015452.pdf

[83] Chen S,Zhao S, White DG, Schroeder CM, Lu R, Yang H, McDermott PF, Ayers S, Meng J. Characterization of multiple-antimicrobial-resistant *Salmonella* serovars isolated from retail meats. Appl Environ Microbiol. 2004;**70(1):**1–7. DOI:10.1128/AEM.70.1.1-7.2004

[84] Le Hello S, Bekhit A, Granier SA, Barua H, Beutlich J, Zając M, Münch S, Sintchenko V, Bouchrif B, Fashae K, Pinsard JL, Sontag L, Fabre L, Garnier M, Guibert V, Howard P, Hendriksen RS, Christensen JP, Biswas PK, Cloeckaert A, Rabsch W, Wasyl D, Doublet B, Weill FX. The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain. Front Microbiol. 2013;**4:**395. DOI:10.3389/

[85] Brisabois A, Cazin I, Breuil J, Collatz E. Surveillance of antibiotic resistance in Salmonella. Euro Surveill. 1997;2(3):pii=181. Available online: http://www.eurosurveillance.org/

[86] Voogd CE, van Leeuwen WJ, Guinée PA, Manten A, Valkenburg JJ. Incidence of resistance to ampicillin, chloramphenicol, kanamycin and tetracycline among Salmonella species isolated in the Netherlands in 1972, 1973 and 1974. Antonie van Leeuwenhoek.

[87] NARMS report for human isolates final report. National Center for Emerging and

DOI:10.1128%2FAEM.70.1.1-7.2004#pmc\_ext

fmicb.2013.00395

234 Current Topics in Salmonella and Salmonellosis

1977;**43(3–4):**269–281.

ViewArticle.aspx?ArticleId=181

Zoonotic Infectious Disease; 2013.

indicator bacteria from humans, animals and food. EFSA J. 2016;**14(2):**4380.

**Objectives:** *Salmonella* is a major public health concern particularly in areas of low socioeconomic status (SES) and high temperature. In this chapter, we examined several socioeconomic and environmental factors that may increase the spread of *Salmonella* in the southern states of the USA.

**Methods:** In our recent studies, relevant US-southern states data of foodborne illnesses, from 2002 to 2011, were collected and used in various analytical models. The associations among low socioeconomic status, climatic variables and *Salmonella* infections were determined using several software packages.

**Results:** Our studies showed a significant increase in *Salmonella* outbreaks in Mississippi during the observed periods with regional and district variations. Regression and neural network models revealed a moderate correlation between *Salmonella* infection rates and low socioeconomic factors. A seasonal trend was observed for *Salmonella* infections. In one of our study, an increase of 1°F (0.556°C) was shown to result in four new cases of *Salmonella* infection in Mississippi.

**Conclusions:** Geographic location besides socioeconomic status may contribute to the high rates of *Salmonella* outbreaks. There are consistent evidence that gastrointestinal infections with bacterial pathogens are positively correlated with ambient temperature. Warming trends in the USA may further increase rates of *Salmonella* infections.

**Keywords:** *Salmonella* infection, socioeconomic status, climate change, global warming

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

*Salmonella* is a serious foodborne pathogen with an estimated 94 million human cases of gastroenteritis and 155,000 deaths around the world each year [1]. It causes around 1.4 million human cases with 15,000 hospitalizations and more than 400 deaths in the USA annually [2].

Contaminated eggs and poultry meat are common source of human salmonellosis. Wide range of domestic and wild animals, such as poultry and swine, can act as reservoirs for *Salmonella*. Institutions such as schools and nursing homes have often been linked to *Salmonella* outbreaks with devastating effects [3].

*Salmonella* rates in USA fluctuate considerably by geographic regions, with particularly higher rates in the Mid-Atlantic and New England States. This variation may be partly attributed to reporting differences. Salmonellosis rates between geographically and socio-economically similar to USA have been documented with as much as 200% differences between neighbouring states [1]. Southern USA, due to its socioeconomic status (SES), climatic changes and agricultural practices, is more vulnerable to increased outbreaks of foodborne illnesses compared to other parts of the country.

Emergence or resurgence of numerous infectious diseases is strongly influenced by environmental factors, such as climate or land use change [4]. Climate, weather, topology, hydrology and other geographical characteristics of the crop-growing site may influence the magnitude and frequency of transfer of pathogenic microorganisms from environmental sources [5].

## **2. Geographical variation and socioeconomic status effects**

Socioeconomic status (SES) is an important predictor of diseases. SES is frequently measured based on individual and community-level education, income, wealth, employment and family background when compared with other individuals or groups. Low SES is generally associated with greater morbidity and mortality of diseases [6]. Socioeconomic and demographic indicators can be used to predict the individuals and communities that are at an increased risk of acquiring infections. Generally, low socioeconomic status is an important predictor of several poor health outcomes including chronic diseases, mental illnesses and mortality.

In our previous study [7], we examined the extent of *Salmonella* infections in Mississippi and compared it with other southern states and with two referenced northern states of the USA (**Figure 1**) to determine the infections' correlation with socioeconomic status. Several analytical modelling approaches including geographical information system (GIS) and neural network (NN) were employed. Laboratory confirmed data of *Salmonella* cases, from 2002 to 2011, were collected for Mississippi, Alabama, Tennessee, Louisiana, Montana and Michigan. Southern states including Alabama, Tennessee and Louisiana were selected as neighbouring states of Mississippi, while Montana and MI were selected as reference states based on their geographical and climatic conditions. Monthly *Salmonella* outbreak cases were grouped by year and districts. Data sources for this study included the US Centers for Disease Control and

**Figure 1.** *Salmonella* Rates in Selected US States. MS-Mississippi; LA-Louisiana; TN-Tennessee; MI-Michigan; MT-Montana; AL-Alabama.

Prevention (CDC) and the respective States Department of health and Epidemiology [8–11]. Data were adjusted to 100,000 of population [12]. In addition to *Salmonella* infections, data for Mississippi, socioeconomic variables for its various counties, categorised by public health districts, for the year 2010–2011, were also retrieved [13]. The selected variables included poverty, uninsured, unemployment and primary care providers' rates.

Results of the study showed mostly positive correlation between low socioeconomic variables and increased rates of *Salmonella* infections; however, poverty rates were negatively correlated with *Salmonella* outbreaks.

Results of this study also revealed *Salmonella* rates in Mississippi to be twice than the average US *Salmonella* rates (36 cases/100,000 vs. 16.42 cases/100,000, respectively) [14]. Substantial regional differences in the incidence of *Salmonella* infections have also been reported previously [15]. A significant variation was observed in *Salmonella* outbreaks among the Mississippi districts through GIS mapping, regression analysis and NN models.

#### **2.1. Poverty, education and unemployment**

**1. Introduction**

236 Current Topics in Salmonella and Salmonellosis

with devastating effects [3].

to other parts of the country.

*Salmonella* is a serious foodborne pathogen with an estimated 94 million human cases of gastroenteritis and 155,000 deaths around the world each year [1]. It causes around 1.4 million human cases with 15,000 hospitalizations and more than 400 deaths in the USA annually [2]. Contaminated eggs and poultry meat are common source of human salmonellosis. Wide range of domestic and wild animals, such as poultry and swine, can act as reservoirs for *Salmonella*. Institutions such as schools and nursing homes have often been linked to *Salmonella* outbreaks

*Salmonella* rates in USA fluctuate considerably by geographic regions, with particularly higher rates in the Mid-Atlantic and New England States. This variation may be partly attributed to reporting differences. Salmonellosis rates between geographically and socio-economically similar to USA have been documented with as much as 200% differences between neighbouring states [1]. Southern USA, due to its socioeconomic status (SES), climatic changes and agricultural practices, is more vulnerable to increased outbreaks of foodborne illnesses compared

Emergence or resurgence of numerous infectious diseases is strongly influenced by environmental factors, such as climate or land use change [4]. Climate, weather, topology, hydrology and other geographical characteristics of the crop-growing site may influence the magnitude and frequency of transfer of pathogenic microorganisms from environmental sources [5].

Socioeconomic status (SES) is an important predictor of diseases. SES is frequently measured based on individual and community-level education, income, wealth, employment and family background when compared with other individuals or groups. Low SES is generally associated with greater morbidity and mortality of diseases [6]. Socioeconomic and demographic indicators can be used to predict the individuals and communities that are at an increased risk of acquiring infections. Generally, low socioeconomic status is an important predictor of several poor health outcomes including chronic diseases, mental illnesses and mortality.

In our previous study [7], we examined the extent of *Salmonella* infections in Mississippi and compared it with other southern states and with two referenced northern states of the USA (**Figure 1**) to determine the infections' correlation with socioeconomic status. Several analytical modelling approaches including geographical information system (GIS) and neural network (NN) were employed. Laboratory confirmed data of *Salmonella* cases, from 2002 to 2011, were collected for Mississippi, Alabama, Tennessee, Louisiana, Montana and Michigan. Southern states including Alabama, Tennessee and Louisiana were selected as neighbouring states of Mississippi, while Montana and MI were selected as reference states based on their geographical and climatic conditions. Monthly *Salmonella* outbreak cases were grouped by year and districts. Data sources for this study included the US Centers for Disease Control and

**2. Geographical variation and socioeconomic status effects**

Underreporting of enteric infections is a critical issue in disease surveillance systems. Generally, patients with severe symptoms tend to visit the doctor and are subsequently notified to health authorities. As of 2011, almost 23% of Mississippi populations are living under poverty with average per-capita income of \$32,000, although rural per-capita income lagged at \$29,550, according to the USDA Economic Research Service. There are 96 hospitals in Mississippi, 163 Rural Health Clinics, and 21 Federally Qualified Health Canters that provide services at 170 sites in the state. An average of 19% of Mississippi residents lacks health insurance [12, 13].

The west-central region of Mississippi showed higher rates of *Salmonella* infections and lower poverty rates (36%), when compared to the Delta region of high poverty. However, more medical facilities are available in west-central region, resulting in higher identification and reporting of diseases. In 2011, 20% of the populations in west-central region were college graduate, with 10% unemployment rate, while only 14% of populations in Delta region were college graduate and 13% were unemployed. Lower rates of shigellosis and salmonellosis in communities with high rates of unemployment were identified. It was speculated that the reduced access to health care due to lack of employment may lead to under-detection of a disease among the unemployed individuals [16].

Geographical variations in poverty rates were also observed in different districts of the state (**Figure 2**). In the Delta region of Mississippi, the poverty rate was 44.2%. The lowest *Salmonella*

**Figure 2.** Geographical variations in *Salmonella* rates and socioeconomic factors in Mississippi.

rates were observed in this region as well. With high rates of poverty, many individuals cannot afford to seek medical care, which suggested underreporting of the disease.

reporting of diseases. In 2011, 20% of the populations in west-central region were college graduate, with 10% unemployment rate, while only 14% of populations in Delta region were college graduate and 13% were unemployed. Lower rates of shigellosis and salmonellosis in communities with high rates of unemployment were identified. It was speculated that the reduced access to health care due to lack of employment may lead to under-detection of a

Geographical variations in poverty rates were also observed in different districts of the state (**Figure 2**). In the Delta region of Mississippi, the poverty rate was 44.2%. The lowest *Salmonella*

**Figure 2.** Geographical variations in *Salmonella* rates and socioeconomic factors in Mississippi.

disease among the unemployed individuals [16].

238 Current Topics in Salmonella and Salmonellosis

The northern region of the state including northeast, northwest, Tombigbee and Delta district had the highest rates of unemployment. An average of 42% increase in unemployment rate was observed in the region in 2011. Primary care provider rate was the lowest in the northwest and east-central regions of Mississippi. An average of 17% decrease in primary care provider rates was observed in these regions. On the other hand, highest rates of primary care providers were found in west-central and southeast regions of the state, with 2% increase from 2010 to 2011.

Our results are different from reported individual level epidemiologic studies that had found higher levels of foodborne infections among low education and low-income groups. Studies suggested that high socioeconomic status (HSES) groups may be overrepresented in incidence statistics. It is possible that lower socioeconomic status (LSES) groups tend not to have health insurance or do not seek medical care when needed due to financial constraints. Access to health care may be an important influence on rates of reported bacterial infections. In an economy without universal health care coverage, tendency to seek care for GI infection has been associated with having health insurance [17, 18]. However, the Affordable Care Act (ACA) is expected to expand insurance coverage to millions of people in the USA. As a result, rates of reported cases of diseases and infections are expected to increase. In future projects, we will try to understand the impact of Affordable Care Act of 2010 on diseases reporting, especially among minority and LSES groups.

It is quite possible that various SES groups have different exposures because of dietary differences, or differences in food safety behaviours [8]. Behavioural studies have revealed that high SES groups are more likely to eat undercooked foods, such as raw oysters and rare beef [9, 12]; while low SES groups are less likely to have adequately cool refrigerators [4].

Other studies had similarly utilised GIS to examine the relationships between area-based socioeconomic measures and incidence of salmonellosis [18, 19]. The results showed higher incidences of salmonellosis in groups with high education compared to the less educated groups suggesting the role of education in health-seeking behaviour and the predisposition for *Salmonella* infections at the population level [19].

Neural network modelling was shown to be a useful tool in this study to predict the correlation between socioeconomic factors and *Salmonella* outbreaks. A moderate correlation between actual and network predicted output was observed at 41%, a reasonable level considering the biological system. Artificial neural networks (ANNs) are non-linear mapping structures comparable to human brain. They have been shown to be universal and highly flexible function to approximate any data. ANNs were developed initially to model biological functions [20–23]. Neural network melding has been used previously for prediction of T-Cell epitopes [24], prediction of cancer using gene expression profiling [25], temperature prediction [26], diabetes prediction [21], poultry growth modelling [27], egg price forecasting [28], in addition to predicting the relation between obesity and high blood pressure [23].

In the USA, Mississippi ranked 50th among all the states for health care, according to the Commonwealth Fund, a non-profit foundation working to advance performance of the health care system. For the past 3 years, obese populations were accounted for more than 30% of Mississippi's residents and 22.8 % of the state's children. On top of obesity, Mississippi had the highest rates in the nation for high blood pressure, diabetes and adult inactivity [24].

Social and economic conditions underpin poverty and can directly or indirectly affect health status and health outcomes. Major epidemics emerge and chronic conditions cluster persist wherever poverty is widespread [5].

## **3. Effects of climatic variables**

#### **3.1. Temperature**

Diseases associated with climate change are estimated to comprise 4.6% of all environmental risks and hazards. Climate change, in the year 2000, contributed to about 2.4% of all diarrhoea outbreaks in the world, 6% of malaria outbreaks in certain developing countries and 7% of the episodes of dengue fever in some industrial countries. In total, the estimates showed that climate change related mortalities were 0.3%, whereas the related burden of disease was 0.4% [29].

Global average temperature, from 1906 to 2005, has warmed by 0.74°C; and since 1961, sea level has risen on average by 2 mm per year. On the other hand, Arctic sea ice has declined by 7.4% per decade while snow cover and glaciers have diminished in both hemispheres [4]. The climate change rate is faster now than in any other period during the last 1000 years. According to the United Nations Intergovernmental Panel on Climate Change, average global temperatures will increase between 1.8 and 4.0°C in next 90 years along with sea level rise of 18–59 cm [30, 31].

Changes in expected weather patterns can lead to the transfer of microbial contaminants to leafy vegetables and herbs. Dry periods can cause dust storms that settle dust particles on leafy vegetables. The rate of microbial growth was shown to increase with rise in temperature. It influences the population of insects and pests found in and around farms that transfer human pathogens to leafy vegetables as well. Relative humidity has been shown to have an effect on survival of human pathogens [32]. Climate change scenarios predict a change distribution of infectious diseases with warming temperature and changes in outbreaks associated with weather extremes, such as flooding and droughts.

Several infectious agents, vector organisms, non-human reservoir species, and rate of pathogen replication are sensitive to climatic conditions. Both *Salmonella* and *Vibrio cholera*, for example, proliferate more rapidly at higher temperatures, *Salmonella* in animal gut and food, cholera in water. In regions where low temperature, low rainfall, or absence of vector habitat restrict transmission of vector-borne disease, climatic changes could tip the ecological balance and trigger epidemics [31]. Furthermore, strong linear associations have been noted between temperature and notifications of Salmonellosis in European countries and Australia [31]. The USA is likely to experience increases in extreme heat, extreme cold, hurricanes, floods, wildfires, droughts, tornadoes and severe storms [33]. The health impacts of global climate change are expected to be widespread, geographically myriad and profoundly influenced by pre-existing social and economic disparities [34].

In the USA, Mississippi ranked 50th among all the states for health care, according to the Commonwealth Fund, a non-profit foundation working to advance performance of the health care system. For the past 3 years, obese populations were accounted for more than 30% of Mississippi's residents and 22.8 % of the state's children. On top of obesity, Mississippi had the highest rates in the nation for high blood pressure, diabetes and adult inactivity [24].

Social and economic conditions underpin poverty and can directly or indirectly affect health status and health outcomes. Major epidemics emerge and chronic conditions cluster persist

Diseases associated with climate change are estimated to comprise 4.6% of all environmental risks and hazards. Climate change, in the year 2000, contributed to about 2.4% of all diarrhoea outbreaks in the world, 6% of malaria outbreaks in certain developing countries and 7% of the episodes of dengue fever in some industrial countries. In total, the estimates showed that climate change related mortalities were 0.3%, whereas the related burden of disease was 0.4% [29]. Global average temperature, from 1906 to 2005, has warmed by 0.74°C; and since 1961, sea level has risen on average by 2 mm per year. On the other hand, Arctic sea ice has declined by 7.4% per decade while snow cover and glaciers have diminished in both hemispheres [4]. The climate change rate is faster now than in any other period during the last 1000 years. According to the United Nations Intergovernmental Panel on Climate Change, average global temperatures will increase between 1.8 and 4.0°C in next 90 years along with sea level rise of

Changes in expected weather patterns can lead to the transfer of microbial contaminants to leafy vegetables and herbs. Dry periods can cause dust storms that settle dust particles on leafy vegetables. The rate of microbial growth was shown to increase with rise in temperature. It influences the population of insects and pests found in and around farms that transfer human pathogens to leafy vegetables as well. Relative humidity has been shown to have an effect on survival of human pathogens [32]. Climate change scenarios predict a change distribution of infectious diseases with warming temperature and changes in outbreaks associated

Several infectious agents, vector organisms, non-human reservoir species, and rate of pathogen replication are sensitive to climatic conditions. Both *Salmonella* and *Vibrio cholera*, for example, proliferate more rapidly at higher temperatures, *Salmonella* in animal gut and food, cholera in water. In regions where low temperature, low rainfall, or absence of vector habitat restrict transmission of vector-borne disease, climatic changes could tip the ecological balance and trigger epidemics [31]. Furthermore, strong linear associations have been noted between temperature and notifications of Salmonellosis in European countries and Australia [31]. The USA is likely to experience increases in extreme heat, extreme cold, hurricanes, floods,

with weather extremes, such as flooding and droughts.

wherever poverty is widespread [5].

240 Current Topics in Salmonella and Salmonellosis

**3. Effects of climatic variables**

**3.1. Temperature**

18–59 cm [30, 31].

The southern states, including Mississippi's climate, has been fluctuating with extreme patterns. The average temperatures in Mississippi have varied significantly over the past century, with an average of 1°F increase, since the late 1960s. Extreme rainfall events, primarily thunderstorms, have increased as well. While rainfall totals have changed little, seasonal trends are apparent, summers have become slightly drier and winters slightly wetter [33]. On an average, 29 tornadoes are reported annually in Mississippi; the highest number was in 2008 with 109 tornadoes. In addition, during the past decade, Mississippi had experienced multiple hits by hurricanes including the devastating Katrina in 2005 [33].

Global warming and the climate change have contributed to the spread of several foodborne pathogens [5, 30]. In our previous research, we determined the extent of *Salmonella* infections in Mississippi along with its correlation to climate variations [35]. Monthly data of *Salmonella* outbreaks from 2001 to 2011 were obtained from Mississippi State Department of Health and Department of Epidemiology. In addition, meteorological data, including average air temperatures, minimum and maximum, and total precipitation for the selected station across the state were collected from the Southeast Regional Climate Center, available at: http://www. sercc.com/climateinfo/monthly\_seasonal.html.

Analysis of variance was performed to determine the seasonal change in *Salmonella* outbreaks during the study period. Time series analysis, including the Mann-Kendall test and a Seasonal trend test, was applied to quantify the relationships between the temperature and the number of notified cases of *Salmonella*.

Our results indicated an increase in temperature is positively correlated with *Salmonella* infections. A seasonal trend was also observed in this study with the highest outbreaks during the summer to early fall (**Figure 3**).

The positive relationship between temperature and *Salmonella* infections, observed in our study, using regression and NN models, was similar to recent findings from Australia, Europe, North America and Asia with similar trends [36–38]. Endemic regions for *Salmonella* outbreaks include developing countries in South Central and South East Asia; and many parts of Africa, Middle East and Latin America. In the same study, we found that an increase of 1°F (0.556°C) in Mississippi was shown to result in four new cases of *Salmonella* (**Figure 4**). Studies also found that weekly counts of enteric bacterial disease cases generally increased with weekly temperature after adjusting for seasonal and long-term trends [37, 39]. Another study [40] had suggested that a potential 1°C rise in mean weekly maximum temperature may be associated with an 8.8% increase in the weekly number of cases, and a 1°C rise in mean weekly minimum temperature may lead to a 5.8% increase in the weekly number of cases.

The US-southern states climate is generally warm and wet, with mild and humid winters. The average annual temperatures in the region have increased by about 2°F since 1970, and the average annual temperatures in the region are projected to increase by 4 to 9°F by 2080 [41].

**Figure 3.** Seasonal trend in *Salmonella* cases.

Climate change and extreme events may increase the spread of foodborne diseases in this region, particularly in the disadvantaged states, such as Mississippi.

Increased growth *of Salmonella* at higher temperatures leads to higher concentration of *Salmonella* in the food supply, particularly during the warmer months. Poor cooking practices are also more common during these summer months (picnics, barbecues, etc.). Temperature

**Figure 4.** Correlation between *Salmonella* outbreaks and increase in temperature.

may affect the transmission of *Salmonella* infections via several causal pathways, such as direct effects on bacterial proliferation and indirect effects on eating habits during hot days. The optimum temperature for the growth of *Salmonellae* is between 35 and 37°C. The growth is greatly decreased at less than 15°C. Ambient temperature influences the development of *Salmonella* at various stages in the food chain, including bacterial loads on raw food production, transport and inappropriate storage [37, 42].

Studies showed that an increase in the ambient temperature correlated positively with an increase in human *Salmonella* with a delay of approximately five weeks. *Salmonella* has the ability to multiply itself within 20 min at ambient temperatures; this growth rate however, is increased at 30°C or higher temperature. Additionally, cross-contamination and undercooking of meat during barbecuing is more likely to occur during summer months [43].

There is consistent evidence that gastrointestinal infection with bacterial pathogens is positively correlated with ambient temperature, as warmer temperatures enable rapid replications of pathogens. *Salmonella* notifications peak in summer and the rate of notifications has been shown to be positively and linearly correlated with the mean temperature of the previous month or week [44, 45]. Some of this increase in summer months may be due to changed eating behaviours (more 'eating out' while on holidays and attending outdoor functions such as barbecues). Ambient temperatures contribute directly to pathogen multiplication in foods and thus the likelihood of infection. Furthermore, it was noted that enteric diseases in temperate latitudes have a seasonal pattern, with the highest incidence of illnesses during the summer months. A study of foodborne illnesses in the UK found a relationship between the incidence of disease and the temperature in the month preceding the illness [46]. It is believed that the survival and growth of certain enteric pathogens are, within limits, positively correlated with ambient temperature [39].

Climate change and extreme events may increase the spread of foodborne diseases in this

Increased growth *of Salmonella* at higher temperatures leads to higher concentration of *Salmonella* in the food supply, particularly during the warmer months. Poor cooking practices are also more common during these summer months (picnics, barbecues, etc.). Temperature

region, particularly in the disadvantaged states, such as Mississippi.

**Figure 4.** Correlation between *Salmonella* outbreaks and increase in temperature.

**Figure 3.** Seasonal trend in *Salmonella* cases.

242 Current Topics in Salmonella and Salmonellosis

Rates of *Salmonella* are expected to increase in the future as climate change causes ambient temperatures to rise above the average, contributing to around 1000 extra cases annually. This links to an annual difference of approximately 1200 lost workdays and \$120,000 in the cost of health care and surveillance by 2050 [44]. By considering a suite of future climate scenarios, the UN-Intergovernmental Panel on Climate Change projected global surface temperature increases between 1.1 and 6.4°C over the next century. Studies had shown that the main health risks caused by climate change include health impacts of weather disasters, health impacts of temperature extremes, including heat waves, mosquito-borne infectious diseases, foodborne infectious diseases (including those due to *Salmonella, Campylobacter* and many other microbes), water-borne infectious diseases and other health risks from poor water quality. In addition, diminished food availability, costs/affordability, nutritional consequences, and increases in urban air pollution are also reported as health risks from climate change [44, 47, 48].

Higher ambient temperatures are main concerns on farm and during food processing and should be considered as an early warning for increased numbers of foodborne infections with 4–6-week lag time. Heightened surveillance during such times may act as a mitigation and enhance the preventive measures. Proper hygiene during slaughter, processing, wholesale and retail sale should be carefully implemented and monitored for further safeguards. More importantly, active consumer education through mass media and other sources regarding the potential danger of consuming contaminated food with *Salmonella* or *Campylobacter*, especially during the summer months should be properly disseminated to mitigate the increased infection rates of *Salmonella*.

#### **3.2. Precipitation effect**

In our previous study [35], no correlation between monthly average precipitation rate and *Salmonella* was observed. A better association with *Salmonella* outbreaks was noted in studies using daily or weekly rates of precipitation. Other studies, however, indicated that maximum and minimum temperatures, relative humidity and rainfall were positively correlated with the number of cases of *Salmonella* with a lag time of 2–8 weeks. It was reported that rainfall, especially heavy rainfall events, may affect the frequency and level of contamination of drinking water, and consequently enteric infection. A strong association between drinking water quality, precipitation and gastroenteritis was reported [49].

A study by Jiang *et al*. [50] suggested that extreme temperature and precipitation events are associated with salmonellosis. It was shown that the frequency and intensity of such extreme events are increasing and will continue to do so in the coming decades as a result of changing climate [50]. The influence of precipitation on salmonellosis is not always immediate but most often delayed by 2–4 weeks [51]. Precipitation likely increases salmonellosis incidence shortly after a rainfall event by increasing pathogen loads in household rainwater tanks through runoff. The delayed effect of rainfall on salmonellosis is also likely to be through increased pathogen loads in surface water which is then used to irrigate or process fresh produce that later may be consumed raw [51].

Climatic changes impact the emergence or re-emergence of infectious disease agents. There are some general principles of pathogen emergence, which are associated with changes in ecology and agriculture, technology and industry, globalization, human behaviour and demographics, epidemiological surveillance and microbial adaptation [52, 53]. It is important to recognise that pathogen emergence usually occurs as a consequence of a combination of two or more specific factors [54].

## **4. Modelling approaches**

#### **4.1. Regression analysis**

Multiple regression analysis were carried out to test the relationship between *Salmonella* rates and socioeconomic factors, including poverty, uninsured, unemployment and primary care providers' rates. Socioeconomic factors were used as classification variables and *Salmonella* infection rate as a response variable. Regression analysis was also performed for climate factors, where temperature and precipitation were used as independent (classification) variables and *Salmonella* outbreaks as dependent (response) variable.

#### **4.2. Neural network modelling of** *Salmonella* **and temperature**

More importantly, active consumer education through mass media and other sources regarding the potential danger of consuming contaminated food with *Salmonella* or *Campylobacter*, especially during the summer months should be properly disseminated to mitigate the

In our previous study [35], no correlation between monthly average precipitation rate and *Salmonella* was observed. A better association with *Salmonella* outbreaks was noted in studies using daily or weekly rates of precipitation. Other studies, however, indicated that maximum and minimum temperatures, relative humidity and rainfall were positively correlated with the number of cases of *Salmonella* with a lag time of 2–8 weeks. It was reported that rainfall, especially heavy rainfall events, may affect the frequency and level of contamination of drinking water, and consequently enteric infection. A strong association between drinking water

A study by Jiang *et al*. [50] suggested that extreme temperature and precipitation events are associated with salmonellosis. It was shown that the frequency and intensity of such extreme events are increasing and will continue to do so in the coming decades as a result of changing climate [50]. The influence of precipitation on salmonellosis is not always immediate but most often delayed by 2–4 weeks [51]. Precipitation likely increases salmonellosis incidence shortly after a rainfall event by increasing pathogen loads in household rainwater tanks through runoff. The delayed effect of rainfall on salmonellosis is also likely to be through increased pathogen loads in surface water which is then used to irrigate or process fresh produce that later

Climatic changes impact the emergence or re-emergence of infectious disease agents. There are some general principles of pathogen emergence, which are associated with changes in ecology and agriculture, technology and industry, globalization, human behaviour and demographics, epidemiological surveillance and microbial adaptation [52, 53]. It is important to recognise that pathogen emergence usually occurs as a consequence of a combination of

Multiple regression analysis were carried out to test the relationship between *Salmonella* rates and socioeconomic factors, including poverty, uninsured, unemployment and primary care providers' rates. Socioeconomic factors were used as classification variables and *Salmonella* infection rate as a response variable. Regression analysis was also performed for climate factors, where temperature and precipitation were used as independent (classification) variables

increased infection rates of *Salmonella*.

244 Current Topics in Salmonella and Salmonellosis

quality, precipitation and gastroenteritis was reported [49].

and *Salmonella* outbreaks as dependent (response) variable.

**3.2. Precipitation effect**

may be consumed raw [51].

two or more specific factors [54].

**4. Modelling approaches**

**4.1. Regression analysis**

Neural network models for temperature effects on *Salmonella* outbreaks were developed using @RISK and NeuroShell2 software packages. NeuroShell2 is a program that mimics the human brain's ability to classify patterns or to make predictions or decisions based upon past experience. The network is exposed to the problem being predicted or classified, and the software will 'learn' the patterns from training data and will make its own classifications, predictions or decisions when presented with new data. NN models are particularly useful when there are implicit interactions and complex relationships in the data.

Over the last few years, artificial neural networks, as nonlinear modelling techniques, had been proposed for use in predictive microbiology [55–61]. In our study, two neural network models, General Regression Neural Network (GRNN) model and Polynomial Net model, were used to predict the effects of temperature on *Salmonella* outbreaks in Mississippi. Several architectures of neural network models were developed to establish the best fitting models. Both of the reported models showed a significant correlation between temperature and *Salmonella* outbreak. GRNN model and Monte Carlo simulation for predicting survival and growth of *Salmonella* on raw chicken skin, as a function of serotype, temperature and time, were used in previous studies as well [62].

Monthly data for temperature and *Salmonella* data in Mississippi from 2001 to 2011 were used to build these models. Temperature was used as an input while *Salmonella* outbreaks as output variables (**Figure 5**). A General Regression Neural Network Model and Polynomial Net Models were selected from the software design architecture. Twenty per cent of the data were extracted for testing, and 80% were used for training the NN models. A test data file was applied to previously saved trained NN models and outputs were generated. Results were exported to Excel, and graphs were created to show the association between actual data and the predicted model.

*Salmonella* outbreaks and socioeconomic data for Mississippi districts were used for NN models. Mean and Standard deviation were calculated for each variable, including *Salmonella*, poverty, uninsured and unemployment and primary care providers' rates. Those means and SD were subsequently used to generate data with 500 iterations using @RISK in Risk Normal distribution. The simulated data were then used as training examples for the NN models, while the original data were used for testing with NeuroShell2 software.

Advanced NNs were selected and the simulated data files were imported. The network was built by defining input variables as poverty, uninsured, unemployment and primary care providers' rates, while *Salmonella* outbreaks as output. A General Regression Neural Network (GRNN) model was selected from the software design architecture. This model was trained with the simulated data. The test file containing the original data was imported to the system and applied to previously saved trained NN models. Results were exported to Excel where graphs were created to show the association between actual data and the predicted model.

**Figure 5.** Neural network models for *Salmonella* and temperature correlation.

#### **4.3. GIS mapping**

A GIS incorporates hardware, software and data for capturing, managing, analysing and displaying all forms of geographically referenced information.

Study area for GIS map: Mississippi (32.9906° N, 89.5261° W) is located in the southern USA. It is bordered by TN on the north, Gulf of Mexico on the south, AL on the east and Arkansas and LA on the west. It covers a total area of 47,689 square miles. GIS allows for the integration and analysis of geographic data, such as coordinates and area perimeters, and tabular data (i.e., attributes of geographic data points). Data are imported into mapping software in layers, where each layer represents a different visual component of the map. Shape files are layers which provide visual output of coordinates and area perimeters on the map.

Mississippi counties' data were grouped by public health districts. Background map was obtained from ESRI ArcGIS online resources. Maps' layers for *Salmonella*, unemployment and primary care providers' rates were created for the years 2010 and 2011, to visually analyse areas with higher disease rates and socioeconomic status (**Figure 2**).

## **5. Conclusions**

Human foodborne illnesses are significant public health concerns. Socioeconomic status and climate changes contribute to the increased rates of *Salmonella*. A significant correlation between increase in outbreaks of *Salmonella* and the lower socioeconomic status was observed in several studies. Understanding the geographical and economic relation with infectious diseases will help to determine effective methods to reduce outbreaks within these communities. Climate changes in the USA are likely to increase the severity, frequency, timing and duration of extreme weather events, which consecutively will increase the associated health risks. The transmission of *Salmonella* to humans is a complex ecological process; warmer temperatures in combination with differences in eating pattern, may contribute to enteric infections.

Modelling approaches, such as neural network were shown to be a useful tool to model and predict outbreaks. Neural network models accounting for non-linearity may predict better association than regression models. Geographical information system mapping was also shown to be a very useful instrument to map and visualise the areas and districts of highest *Salmonella* outbreaks in addition to socioeconomic status.

Regression and neural network models were used to determine the correlation between increase in temperature and increase in *Salmonella* outbreaks. Considering the seasonal variation, neural network models turned out to be better predictor models.

## **Acknowledgements**

The reported research work is funded by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number G12MD007581. The content is solely the responsibility of the authors and does not represent in any form or shape the official views of the National Institutes of Health.

## **Author details**

**4.3. GIS mapping**

246 Current Topics in Salmonella and Salmonellosis

A GIS incorporates hardware, software and data for capturing, managing, analysing and dis-

Study area for GIS map: Mississippi (32.9906° N, 89.5261° W) is located in the southern USA. It is bordered by TN on the north, Gulf of Mexico on the south, AL on the east and Arkansas and LA on the west. It covers a total area of 47,689 square miles. GIS allows for the integration and analysis of geographic data, such as coordinates and area perimeters, and tabular data (i.e., attributes of geographic data points). Data are imported into mapping software in layers, where each layer represents a different visual component of the map. Shape files are layers

which provide visual output of coordinates and area perimeters on the map.

playing all forms of geographically referenced information.

**Figure 5.** Neural network models for *Salmonella* and temperature correlation.

Hafiz Anwar Ahmad\* and Luma Akil

\*Address all correspondence to: hafiz.a.ahmad@jsums.edu

1 Department of Biology-Environmental Science, Jackson State University, Jackson, MS, USA

2 Department of Behavioral and Environmental Health, Jackson State University, Jackson, MS, USA

## **References**


[14] Centers for Disease Control and Prevention. Food Safety. Retrieved 15 March 2014, from http://www.cdc.gov/foodsafety/ Last Accessed 8/15/2016

**References**

248 Current Topics in Salmonella and Salmonellosis

[1] Hoelzer K., Isabel A., Switt M. and Wiedmann M. "Animal Contact as a Source of Human

[2] Callaway T.R., Edrington T.S, Anderson R.C., Byrd J.A. and Nisbet D.J. "Gastrointestinal Microbial Ecology and the Safety of Our Food Supply as Related to Salmonella". J Anim

[3] Brichta-Harhay D.M., Arthur T.M., Bosilevac J.M., Kalchayanand N., Shackelford S.D., Wheeler T.L. and Koohmaraie M. "Diversity of Multidrug-Resistant *Salmonella enterica* Strains Associated with Cattle at Harvest in the United States". Appl Environ Microbiol

[4] Mills, J. N., Gage, K. L., & Khan, A. S. (2010). Potential influence of climate change on vector-borne and zoonotic diseases: a review and proposed research plan. *Environmental* 

[5] World Health Organization. Food Safety. (2009) Available at: http://www.who.int/food-

[6] Newman K.L., Leon J.S., Rebolledo P.A. and Scallan E. "The Impact of Socioeconomic Status on Foodborne Illness in High-Income Countries: A Systematic Review". Epidemiol

[7] Akil L. and Ahmad H.A. "Salmonella Infections Modelling in Mississippi Using Neural Network and Geographical Information System (GIS)". BMJ Open 6 no. 3 (2016): e009255.

[8] TN Department of Health. Communicable Disease Interactive Data. Available at: http:// health.state.tn.us/ceds/WebAim/WEBAim\_criteria.aspx Last Accessed 8/15/2016

[9] State of Louisiana Department of Health and Hospitals. Infectious Diseases Reports. Available at: http://new.dhh.louisiana.gov/index.cfm/newsroom/archives/64 Last Accessed 8/15/2016

[10] Montana Department of Public Health and Human Services Surveillance. Available at: http:// www.dphhs.mt.gov/publichealth/cdepi/surveillance/index.shtml Last Accessed 8/15/2016

[11] Division of Communicable Disease, Bureau of Epidemiology, Michigan Department of Community Health. Reportable Infectious Diseases in Michigan 2002–2011 Summaries. Available at: http://www.michigan.gov/documents/mdch/2005\_CD\_Epi\_Profile\_177917\_7. pdf http://www.michigan.gov/documents/mdch/2009\_CDEpiProfile\_337815\_7.pdf http:// www.michigan.gov/documents/mdch/2011\_CDEpiProfile\_400563\_7.pdf Last Accessed

[12] United States Census Bureau. Available at: www.Census.gov Last Accessed 8/15/2016

site/\_static/31,0,211.html Last Accessed 8/15/2016

[13] MSDH, County Health Ranking Mississippi. Available at: http://msdh.ms.gov/msdh-

Non-typhoidal Salmonellosis". Vet Res 42 (2011): 34.

Sci 86 no. 14 Suppl (2008): E163–E172.

77 no. 5 (2011): 1783–1796.

safety/en/last access 2009

8/15/2016

*health perspectives, 118*(11), 1507.

Infect 143 no. 12 (2015): 2473–2485.


[43] Milazzo A., et al. "The Effect of Temperature on Different Salmonella Serotypes During Warm Seasons in a Mediterranean Climate City, Adelaide, Australia." Epidemiol Infect 144 no. 6 (2016): 1231–1240.

[28] Ahmad H.A. and Mariano M. "Comparison of Forecasting Methodologies Using Egg

[29] Kendrovski, Vladimir, and Dragan Gjorgjev. *Climate Change: Implication for Food-Borne Diseases (Salmonella and Food Poisoning Among Humans in R. Macedonia)*. INTECH Open

[30] Patz J.A., Olson S.H., Uejio C.K. and Gibbs H.K. "Disease Emergence from Global

[31] McMichael A.J., Woodruff R.E. and Hales S. "Climate Change and Human Health:

[32] Hunter PR. Climate change and waterborne and vector-borne disease. Journal of applied

[33] National Oceanic and Atmospheric Administration: U.S. Climate Extremes Index. Available at: http://www.ncdc.noaa.gov/extremes/cei/retrieved Last Accessed 4/20/2012

[34] Sheffield P.E. and Landrigan P.J. "Global Climate Change and Children's Health: Threats and Strategies for Prevention". Environ Health Perspect 119 no. 3 (2011): 291–298.

[35] Akil L., Reddy R.S. and Ahmad H.A. "Effects of Climate Change on Salmonella Infections". Foodborne Pathog Dis 11 no. 12 (2014): 974–980. doi:10.1089/fpd.2014.1802.

[36] D'Souza R.M. "Climatic Factors Association with Hospitalization and Emergency Room

[37] Zhang Ying, Bi Peng and Hiller Janet E. "Climate Variations and Salmonella Infection in Australian Subtropical and Tropical Regions". Sci Total Environ 408 (2010): 524–530.

[38] Taylor E., Kastner J. and Renter D. "Challenges Involved in the Salmonella Saintpaul Outbreak and Lessons Learned". (2009). Retrieved from http://krex.k-state.edu

[39] Fleury Manon, Charron Dominique F., Holt John D., Allen O. Brian and Maarouf Abdel R. "A Time Series Analysis of the Relationship of Ambient Temperature and Common Bacterial Enteric Infections in Two Canadian Provinces." Int J Biometeorol 50 no. 6

[40] Zhang G., Brown E.W. and González-Escalona N. "Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in

[41] Karl T.R., Melillo J.M. and Peterson T.C. (Eds.). USGCRP. Global Climate Change Impacts in the United States. United States Global Change Research Program. Cambridge

[42] Junejaa Vijay K., Melendresb Martin Valenzuela, Huanga Lihan, Gumudavellic Vinod, Subbiahc Jeyamkondan and Thippareddi Harshavardhan. "Modeling the Effect of Temperature on Growth of Salmonella in Chicken". Food Microbiol

Climate and Land Use Change". Med Clin N Am 92 (2008): 1473–1491.

Price as a Test Case." Poultry Sci 85 no. 4 (2006): 798–807.

Present and Future Risks." Lancet 367 (2006): 859–869.

Presentations of Diarrhea". Epidemiology 16 (2005): S60.

Produce." Appl Environ Microbiol 77 no. 18 (2011): 6495–6501.

microbiology. 2003 May 1; 94(s1):37-46.

Access Publisher, 2012.

250 Current Topics in Salmonella and Salmonellosis

(2006): 385–391.

24 (2007): 328–335.

University Press, New York (2009).


#### **Application of Ionizing Radiation for Control of** *Salmonella* **in Food Application of Ionizing Radiation for Control of**  *Salmonella* **in Food**

Małgorzata E. Szczawińska Małgorzata E. Szczawińska

[56] Ibarra, Juan G., and Yang Tao. "Estimation of internal temperature in chicken meat by means of mid-infrared imaging and neural networks." In *Photonics East (ISAM, VVDC,* 

[57] Valero A., Hervás C., García-Gimeno R.M. and Zurera G. "Product Unit Neural Network Models for Predicting the Growth Limits of Listeria Monocytogene." Food Microbiol 24

[58] McKee K.T., Shields T.M., Jenkins P.R., Zenilman J.M. and Glass G.E. "Application of a Geographic Information System to the Tracking and Control of an Outbreak of

[59] Hervás C., Martínez J.A., Zurera G., García R.M. and Martínez J.A. "Optimization of Computational Neural Network for Its Application in the Prediction of Microbial

[60] Khanzadi S., Gharibzadeh S., Raoufy M.R., Razavilar V., Khaksar R. and Radmehr B. "Application of Artificial Neural Networks to Predict Clostridium Botulinum Growth as a Function of Zataria Multiflora Essential Oil, Ph, Nacl and Temperature." J Food Saf 30

[61] Jacoboni I., Martelli P.L., Fariselli P., De Pinto V. and Casadio R. "Prediction of the Transmembrane Regions of β-Barrel Membrane Proteins with a Neural Network-Based

[62] Oscar T.P. "A Quantitative Risk Assessment Model for Salmonella and Whole Chickens."

*IEMB)*, pp. 24-31. International Society for Optics and Photonics, 1999.

no. 5 (2007): 452–464.

252 Current Topics in Salmonella and Salmonellosis

no. 2 (2010): 490–505.

Shigellosis." Clin Infect Dis 31 (2000): 728–733.

Predictor." Protein Sci 10 no. 4 (2001): 779–787.

Int J Food Microbiol 93 (2004): 231–247.

Growth in Food." Food Sci Technol Int 7 (2001): 159.

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67408

#### **Abstract**

Ionizing radiation (gamma rays from the radionuclides cobalt-60 or cesium-137, e-beams) is an effective, nonthermal method to reduce or eliminate food-borne pathogens, including *Salmonella* spp. both in raw and in cooked meats, poultry, fish, and shellfish. Irradiation treatment, applied as the final processing step, seems to be particularly promising in the case of packed food products, including ready-to-eat food. Final packaged food products can be contaminated from post-lethality exposure, that is, after heat treatment and before packaging. The application of ionizing radiation after packaging can eliminate or considerably reduce both saprophytic and pathogenic microflora in final products. It is of particular importance in the case of ready-to-eat food which is not subjected to heat treatment before consumption. According to hurdle concept technology, the combination of existing and novel preservation methods can ensure safety of food by applying all treatments as mild as possible. Irradiation treatment can be combined with the use of natural antibacterial compounds, such as extracts of spices and herbs, or various packaging systems. Doses of ionizing radiation required for the inactivation of *Salmonella* spp. in fish and seafood are lower than those used for meats and poultry.

**Keywords:** *Salmonella*, radiation, meat, poultry, fish

## **1. Introduction**

In the last years, the great consumer interest in "natural" or "fresh" foods, nonprocessed or only minimally processed, has caused an increasing interest in nonthermal preservation methods, that is, ionizing radiation, ultraviolet radiation, high-pressure processing (HPP), pulsed electric field (PEF), high-pressure carbon dioxide (HPCD), the use of natural antibacterial compounds, such as extracts of spices and herbs, or the application of various packaging

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

systems. However, at the same time consumer demand for ensuring food safety has to be met. Those two ideas are very often tough to reconcile in practice.

A great number of studies have shown that ionizing radiation improve the safety of various foods of animal as well as plant origin. Food irradiation is a process which can be used to inactivate both food-borne pathogens and microorganisms causing spoilage of food, thus extending storage of foods such as red meats, poultry, fish, and so on. It can also extend the storage of vegetables by prevention of sprouting (potatoes, onions, and garlic) or fruits by the delay of ripening. At present time, this technology may be used not only to raw foods but also as post-lethality treatment. The product may be exposed to the post-lethality processing environment into which the product is routed after having been subjected to an initial lethality treatment. The foodstuffs may be exposed to the environment in the area of establishment as a result of, for example, slicing, peeling, and re-bagging, or other procedures. Hotdog products are examples of readyto-eat (RTE) meat and poultry products that receive a lethality treatment to eliminate pathogens (core temperatures of +70° to +72°C must be reached due to cooking) and they are subsequently exposed to the environment during peeling, slicing, and repackaging operations. Then, the technology of irradiation, used as an intervention step, can be applied to the final product or sealed package of product in order to reduce or eliminate the level of pathogens resulting from contamination from post-lethality exposure. Thus, for example, vacuum-packaged ready-to-eat (RTE) meat products may be subjected to irradiation to reduce or eliminate dangerous food-borne pathogens such as *Salmonella* spp. and *Listeria monocytogenes* in a final food product. According to hurdle concept technology, the combination of existing and novel preservation methods can ensure safety of food by applying all treatments as mild as possible [1, 2].

A good example of such combination of preservation methods (low-dose irradiation and modified atmosphere packaging (MAP)) is the work of Chouliara et al. [3] who investigated the combined effect of gamma irradiation (2 and 4 kGy) and modified atmosphere (MA) packaging (30% CO2 /70% N2 and 70% CO2 /30% N<sup>2</sup> ) on shelf-life extension of fresh chicken meat stored under refrigeration. The authors noted the reduction of the number of various groups of bacteria (from 1 to 5 logs), including Enterobacteriaceae family. Sensory evaluation showed that the combination of irradiation at 4 kGy and MAP (70% CO2 /30% N<sup>2</sup> ) resulted in the highest shelf-life extension by 12 days compared to the air-packaged samples. A study of Grant and Patterson [4] is another good example of hurdle concept technology: mild heating combined with low-dose irradiation. In this study, thermal treatment (70, 65, or 60°C) was applied alone, directly post 0.8 kGy irradiation or post irradiation combined with refrigerated storage on inactivation of *L. monocytogenes* and *Salmonella typhimurium* inoculated into beef and gravy. The researchers observed heat sensitization of *S. typhimurium* at 60°C, but not at either 65 or 70°C like in the case of *L. monocytogenes*. In another study [5], the influence of heating and low-dose irradiation *S. typhimurium* in MDCM (mechanically deboned chicken meat) was examined. The researchers noted that salmonellae irradiated with 0.9 kGy were more heat sensitive; this effect was maintained during 6 weeks of refrigerated storage.

Those readers who want to deepen their knowledge of the subject can find an extensive description of microbiological issues associated with all muscle foods, their specific spoilage, safety issues, and their control for meat, poultry, and seafood in the work provided by Sofos et al. [6].

Thermal treatment is a very effective method for eliminating *Salmonella* spp. in foods. This organism is rather sensitive to pasteurization temperatures used in meat processing. Properly conducted heat treatment in industrial food processing should cause complete inactivation of these bacteria in meat and meat products; however, recontamination of ready-to-eat meat products with *Salmonella* spp. after cooking, as well as subsequent storage at abuse temperatures at food establishments or at a consumer's home, can cause a significant risk to human health. Szczawińska et al. [7] inoculated commercial, smoked, cured, and cooked ham with *Salmonella* enteritidis and stored the samples at abused temperature (15°C). Lag time for *S.*  enteritidis was at that temperature only 139.08 h, that is, less than 6 days [7]. Usually, the length of time for storage of such product recommended by the food manufacturer is much longer than the time mentioned above. It means that a consumer can contract food-borne salmonellosis during the recommended length of storage time for such ready-to-eat meat product if it was recontaminated with *Salmonella*. Thus, due to beneficial effects of ionizing radiation treatment of final packaged food product (RTE), we can expect that *Salmonella* (and other vegetative bacterial pathogens which show similar radiation resistance, e.g., *L. monocytogenes*) will be significantly reduced or eliminated.

## **2. The use of ionizing radiation**

systems. However, at the same time consumer demand for ensuring food safety has to be met.

A great number of studies have shown that ionizing radiation improve the safety of various foods of animal as well as plant origin. Food irradiation is a process which can be used to inactivate both food-borne pathogens and microorganisms causing spoilage of food, thus extending storage of foods such as red meats, poultry, fish, and so on. It can also extend the storage of vegetables by prevention of sprouting (potatoes, onions, and garlic) or fruits by the delay of ripening. At present time, this technology may be used not only to raw foods but also as post-lethality treatment. The product may be exposed to the post-lethality processing environment into which the product is routed after having been subjected to an initial lethality treatment. The foodstuffs may be exposed to the environment in the area of establishment as a result of, for example, slicing, peeling, and re-bagging, or other procedures. Hotdog products are examples of readyto-eat (RTE) meat and poultry products that receive a lethality treatment to eliminate pathogens (core temperatures of +70° to +72°C must be reached due to cooking) and they are subsequently exposed to the environment during peeling, slicing, and repackaging operations. Then, the technology of irradiation, used as an intervention step, can be applied to the final product or sealed package of product in order to reduce or eliminate the level of pathogens resulting from contamination from post-lethality exposure. Thus, for example, vacuum-packaged ready-to-eat (RTE) meat products may be subjected to irradiation to reduce or eliminate dangerous food-borne pathogens such as *Salmonella* spp. and *Listeria monocytogenes* in a final food product. According to hurdle concept technology, the combination of existing and novel preservation methods can

Those two ideas are very often tough to reconcile in practice.

254 Current Topics in Salmonella and Salmonellosis

ensure safety of food by applying all treatments as mild as possible [1, 2].

/70% N2 and 70% CO2

that the combination of irradiation at 4 kGy and MAP (70% CO2

sensitive; this effect was maintained during 6 weeks of refrigerated storage.

aging (30% CO2

A good example of such combination of preservation methods (low-dose irradiation and modified atmosphere packaging (MAP)) is the work of Chouliara et al. [3] who investigated the combined effect of gamma irradiation (2 and 4 kGy) and modified atmosphere (MA) pack-

stored under refrigeration. The authors noted the reduction of the number of various groups of bacteria (from 1 to 5 logs), including Enterobacteriaceae family. Sensory evaluation showed

est shelf-life extension by 12 days compared to the air-packaged samples. A study of Grant and Patterson [4] is another good example of hurdle concept technology: mild heating combined with low-dose irradiation. In this study, thermal treatment (70, 65, or 60°C) was applied alone, directly post 0.8 kGy irradiation or post irradiation combined with refrigerated storage on inactivation of *L. monocytogenes* and *Salmonella typhimurium* inoculated into beef and gravy. The researchers observed heat sensitization of *S. typhimurium* at 60°C, but not at either 65 or 70°C like in the case of *L. monocytogenes*. In another study [5], the influence of heating and low-dose irradiation *S. typhimurium* in MDCM (mechanically deboned chicken meat) was examined. The researchers noted that salmonellae irradiated with 0.9 kGy were more heat

Those readers who want to deepen their knowledge of the subject can find an extensive description of microbiological issues associated with all muscle foods, their specific spoilage, safety issues, and their control for meat, poultry, and seafood in the work provided by Sofos et al. [6].

) on shelf-life extension of fresh chicken meat

) resulted in the high-

/30% N<sup>2</sup>

/30% N<sup>2</sup>

According to the Codex General Standard for Irradiated Foods [8], the following sources of ionizing radiation can be used:


Compared to γ-rays, e-beams are characterized by a low penetrative capacity; therefore, e-beam irradiation is particularly useful for products which can be processed in thin layers or surface-contaminated products.

The dose of radiation received is commonly measured in grays. One gray is a derived unit of ionizing radiation. It is defined as the absorption of one joule of radiation energy in a mass of one kilogram (1 Gy = 1 J/kg). The gray has superseded the older unit—the rad (1 Gy = 100 rad). The **gray** (symbol: **Gy**) is used as a measure of absorbed dose.

According to several objectives for food (fresh or processed meats, poultry, and seafood) irradiation, the following terms are used [9]:


**(c) Radappertization** is based on a similar concept ("botulinum cook") like in canning industry. It should ensure complete elimination of spore formers in foods, thus significant shelflife extension (years) and botulism food safety; doses range 30–40 kGy. The term was established to honor Nicolas Appert who invented the method of preserving food from spoilage by placing it in hermetically sealed containers and then sterilized by heat treatment.

In this review, special attention will be paid to **radicidation**. In case of this technology, one of the most important pathogens, *Salmonella* spp., public health problem, has been the main target for control, particularly in meat and poultry products (i.e., for example, see Ref. [10]). The most recent European Food Safety Authority (EFSA) summary report has informed us that the total number of food-borne outbreaks in Europe was 5251, including water-borne outbreaks [11]. *Salmonella* caused 20.0% of all reported food-borne outbreaks in European Union (EU) and it was the second most frequent cause of outbreaks; the largest number of reported food-borne outbreaks was caused by viruses (20.4% of all outbreaks) [11]. High level of noncompliance was noted for poultry meat [11]. Monitoring activities and control programs for *Salmonella* in fresh broiler meat are based on sampling at the slaughterhouse and/or at processing or cutting plants and at retail. In 2014, *Salmonella* was found in 0.6% of the 2263 units of RTE broiler meat products tested at retail or at processing (0.4% of single samples and 1.7% of batches) [11].

As in previous years, the two most commonly reported *Salmonella* serovars in 2014 were *S. enteritidis* and *S. typhimurium*, representing 44.4% and 17.4%, respectively, of all reported serovars in confirmed human cases [11]. Generally, there was no major change as regards *Salmonella*contaminated foodstuffs compared with previous years. *Salmonella* was most frequently detected in fresh turkey meat (3.5%), fresh broiler (2.2%), pig (0.5%), and bovine meat (0.1%) [11]. It should be emphasized that according to the European legislation on microbiological criteria for foodstuffs [12] *Salmonella* spp. is currently included both in food safety as well as food hygiene criteria.

The main reason for the use of food irradiation is the ability of ionizing radiation to inactivate populations of microorganisms including pathogenic bacteria, parasites, and viruses. Depending on irradiation dose, food-borne pathogens can be injured or killed due to DNA damage. Radiation sensitivity depends on many factors such as species of microorganisms, age of cells, and their number. It is also affected by the environment (buffer solution, laboratory medium, or real food product). Thus, the effect of radiation on microorganisms is dependent on intrinsic and extrinsic factors which include temperature, water activity, pH, chemical composition, and structure of food and gaseous environment. Radiation resistance of bacteria is much higher at freezing temperatures than at chill temperatures; however, irradiation of frozen food offers much better results in some foods because it significantly reduces or eliminates some negative sensory changes caused by, for example, lipid oxidation. D10 values (D10 value is defined as decimal reduction dose or the dose of ionizing radiation required for a 90% inactivation of viable colony-forming unit (CFU) or by one logarithmic cycle) are higher in foods with a low water activity because the lack of water means that there are less OH radicals available to cause DNA damage. Hence, higher doses of ionizing radiation have to be used to ensure the elimination of pathogenic bacteria in dry foods such as spices [13].

Some authors observed different effects of meat irradiation depending on radiation source. Rajkowski et al. [14] discovered in their study that D10 values for *S. typhimurium* DT 104 irradiated in ground pork with gamma rays were 0.56–0.62 kGy, whereas D10 values for the same organism treated with e-beams ranged from 0.42 to 0.43 kGy.

**(c) Radappertization** is based on a similar concept ("botulinum cook") like in canning industry. It should ensure complete elimination of spore formers in foods, thus significant shelflife extension (years) and botulism food safety; doses range 30–40 kGy. The term was established to honor Nicolas Appert who invented the method of preserving food from spoilage by placing it in hermetically sealed containers and then sterilized by heat treatment.

In this review, special attention will be paid to **radicidation**. In case of this technology, one of the most important pathogens, *Salmonella* spp., public health problem, has been the main target for control, particularly in meat and poultry products (i.e., for example, see Ref. [10]). The most recent European Food Safety Authority (EFSA) summary report has informed us that the total number of food-borne outbreaks in Europe was 5251, including water-borne outbreaks [11]. *Salmonella* caused 20.0% of all reported food-borne outbreaks in European Union (EU) and it was the second most frequent cause of outbreaks; the largest number of reported food-borne outbreaks was caused by viruses (20.4% of all outbreaks) [11]. High level of noncompliance was noted for poultry meat [11]. Monitoring activities and control programs for *Salmonella* in fresh broiler meat are based on sampling at the slaughterhouse and/or at processing or cutting plants and at retail. In 2014, *Salmonella* was found in 0.6% of the 2263 units of RTE broiler meat products tested at retail or at processing (0.4% of single samples and 1.7% of batches) [11].

As in previous years, the two most commonly reported *Salmonella* serovars in 2014 were *S. enteritidis* and *S. typhimurium*, representing 44.4% and 17.4%, respectively, of all reported serovars in confirmed human cases [11]. Generally, there was no major change as regards *Salmonella*contaminated foodstuffs compared with previous years. *Salmonella* was most frequently detected in fresh turkey meat (3.5%), fresh broiler (2.2%), pig (0.5%), and bovine meat (0.1%) [11]. It should be emphasized that according to the European legislation on microbiological criteria for foodstuffs [12] *Salmonella* spp. is currently included both in food safety as well as food

The main reason for the use of food irradiation is the ability of ionizing radiation to inactivate populations of microorganisms including pathogenic bacteria, parasites, and viruses. Depending on irradiation dose, food-borne pathogens can be injured or killed due to DNA damage. Radiation sensitivity depends on many factors such as species of microorganisms, age of cells, and their number. It is also affected by the environment (buffer solution, laboratory medium, or real food product). Thus, the effect of radiation on microorganisms is dependent on intrinsic and extrinsic factors which include temperature, water activity, pH, chemical composition, and structure of food and gaseous environment. Radiation resistance of bacteria is much higher at freezing temperatures than at chill temperatures; however, irradiation of frozen food offers much better results in some foods because it significantly reduces or eliminates some negative sensory changes caused by, for example, lipid oxidation. D10 values (D10 value is defined as decimal reduction dose or the dose of ionizing radiation required for a 90% inactivation of viable colony-forming unit (CFU) or by one logarithmic cycle) are higher in foods with a low water activity because the lack of water means that there are less OH radicals available to cause DNA damage. Hence, higher doses of ionizing radiation have to be used to ensure the elimination of pathogenic bacteria in dry foods such as spices [13].

Some authors observed different effects of meat irradiation depending on radiation source. Rajkowski et al. [14] discovered in their study that D10 values for *S. typhimurium* DT 104

hygiene criteria.

256 Current Topics in Salmonella and Salmonellosis

However, Miyahara and Miyahara [15] concluded that both gamma rays and e-beams were similarly effective while irradiating ground beef patties inoculated with *S. enteritidis*.

The use of ionizing radiation as a means of reducing the risk to human health from foodborne pathogens, including *Salmonella* spp., is being extensively researched. It seems that the application of ionizing radiation to preserve food or eliminate pathogenic bacteria from food has been so intensively studied like not any other scientific field, because of consumer concerns, particularly associated with fear of nuclear energy and very often occurring confusion between terms, for example, radiation, radioactive contamination, or radioactivity. In general, consumer is rather reluctant to this technology due to well-known nuclear accidents (e.g., Chernobyl and Fukushima) believing that the process of food irradiation can make food radioactive, thus unsafe. Interestingly, there is much less consumer resistance to the highpressure-processing technology which is used to treat wide range of foods including those of animal origin, for example, RTE products. To date, health and safety authorities in over 60 countries worldwide, for example, the United States, France, Belgium, the Netherlands, Canada, Australia, and New Zealand, granted clearances for irradiation of more than 60 different foods [16]. Frog legs are the most often irradiated food items [17].

Currently, the International Atomic Energy Agency (IAEA) is responsible for updating and maintaining various irradiation databases as resources for researchers, government officials, and the general public. European Food Safety Authority [18] summarized and evaluated an opinion on the efficacy and microbiological safety of irradiation of food taking into consideration recommendations from the two panels: BIOHAZ (the EFSA Panel on Biological Hazards) and CEF (the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids).

EFSA emphasizes its standpoint that food irradiation should only be used in conjunction with an integrated food safety management program. With regard to efficacy and microbiological safety, the BIOHAZ Panel recommended that the application of food irradiation should be based on risk assessment and on the desired risk reduction rather than on predefined food classes/commodities and doses [18]). Concerning the safety assessment of irradiation of food, according to the BIOHAZ Panel, there are no microbiological risks for the consumer linked to the use of food irradiation and its consequences on the food microflora. EFSA's experts conclude that the irradiation dose needed to inactivate food-borne pathogens depends on the targeted pathogen, on the reduction required, and on the physical state of the food, **regardless of the food classes** as previously proposed [18].

#### **2.1. Reducing** *Salmonella* **spp. in red meats and poultry**

Vegetative food-borne bacteria, such as *Salmonella* spp. and *L. monocytogenes*, are moderately sensitive to ionizing radiation. The medium-dose irradiation processes reduce their populations by several logs. As previously mentioned, various factors influence radiation sensitivity of bacterial cells. The presence of proteins can exert a protective effect on microorganisms subjected to radiation treatment. Maxcy and Tiwari [19] studied the effect of fat content in beef on radioresistance of *S. enteritidis*. They found D10 value higher in beef with lower level of fat (0.70 kGy) compared to lower D10 value obtained for salmonellae irradiated in beef with higher content of fat (0.49 kGy). Assuming that the low fat level in the meat is correlated with a higher protein content and because the proteins have the properties of free radicals scavenging, it can be suggested that the higher content of protein in meat protects more the bacteria against the damaging effects of radiation treatment.

There have been frequently voiced concerns that the reduction of the competitive microflora by radiation treatment could facilitate growth of pathogens contaminating the food after irradiation or that food pathogens which survived irradiation can grow better than the indigenous, competitive microflora. Dickson and Olson [20] studied the first problem; ground beef was irradiated at 0, 2, or 4 kGy, thus reducing the number of saprophytic microorganisms which cause food spoilage, and then inoculated with a mixture of four serotypes of salmonellae. The meat was stored at 4°C, temperature proper for storage, and at two abused temperatures 15 and 25°C. Bacterial growth was monitored during storage. The authors observed that there was no significant difference in lag-phase duration or generation time, irrespective of the dose to which the ground beef had previously been exposed. This suggests that, although irradiation eliminates a significant portion of the spoilage microflora in ground beef, the absence of this microflora provides no competitive advantage to the growth of salmonellae in ground beef [20]. Szczawińska [21] studied the effect of irradiation on the survival rate of non-sporing bacteria (*Staphylococcus aureus, S. typhimurium, Escherichia coli, Pseudomonas fluorescens*) during conventional methods of meat preservation (heating, chilling, freezing, salting, curing, and smoking). On the basis of the conducted experiments, it can be concluded that irradiated bacteria stored under conditions preventing their growth die faster compared to unirradiated bacteria or their survival rate is almost identical like unirradiated ones; those organisms which are stored under conditions that allow their growth show a worse adaptability to the environment and begin to grow after a certain delay [21]. In another work, Szczawińska et al. [22] studied the growth of salmonellae in mechanically deboned chicken meat (MDCM), which was irradiated at 0, 1.25, and 2.5 kGy and inoculated with *S. dublin, S. enteritidis*, and *S. typhimurium*. Subsequently, the inoculated MDCM was stored at 5, 10, or 20°C and bacterial numbers were determined over storage time. The results of the study suggested that there was no greater risk from the same number of *Salmonella* cells contaminating irradiated MDCM compared to unirradiated one. In the same study, irradiated indigenous microflora had doserelated increased lag phases and decreased rates of multiplication compared with that of the indigenous microflora in the unirradiated control [22]. Kim and Thayer [23] discovered that the gamma-injured *S. typhimurium* cells on mechanically deboned chicken meat were much more sensitive to heat than the nonirradiated cells, which implies that any cells surviving the irradiation process were unlikely to survive cooking. This increased sensitivity of the salmonellae to gamma radiation was retained during refrigerated storage of the irradiated chicken. Kim and Thayer [23] explained the mechanism of the heat sensitivity of *S. typhimurium* subjected to ionizing radiation. The results proved that combined effects of irradiation and heating were always beneficial in regard to food safety due to synergistic (when heating is applied after irradiation) or additive (when heating is applied before irradiation) effects depending on the order of both treatments. Therefore, on the basis of these studies it can be concluded that any microorganisms which survive irradiation are more sensitive to intrinsic or extrinsic factors, such as temperature, water activity, pH, and so on, compared to unirradiated organisms.

Irradiation of fresh meat up to an overall average dose of 2 kGy was proposed by the SCF in 1986 [24]. Implication of meat in food-borne salmonellosis still remains a concern, particularly in the countries or regions where traditional dishes are served and consumed as raw and cold. In the Netherlands, Belgium, such meat product is "filet américain" composed of raw beef meat, and often raw egg. Kampelmacher [25] reported that a dose of only 1 kGy decreased *Salmonella* number in such a product by two log cycles. Rajkowski et al. [14] examined the effect of e-beam and gamma rays irradiation on the mixture of *S. typhimurium* DT104 strains inoculated into three ground pork products containing various fat contents and obtained D10 values for salmonellae from 0.42 to 0.62 kGy. The data prove that the content of fat had no effect on radiation resistance of salmonellae. The D10 values are similar to the values reported by Szczawińska [26] for *S. typhimurium* strains inoculated into poultry meat. Clavero et al. [27] subjected raw ground beef patties inoculated with mixture of serovars of *S. dublin, S. typhimurium*, and *S. enteritidis* to gamma irradiation (60°C) treatment. The influence of two levels of fat (8–14% (low fat) and 27–28% (high fat)) and temperature (frozen (−17 to −15°C) and refrigerated (3–5°C)) on the inactivation of pathogens by irradiation was investigated. D10 values for salmonellae in beef patties ranged from 0.618 to 0.800 kGy. The authors discovered that temperature did not have a significant effect when salmonellae were irradiated in high-fat ground beef.

of fat (0.70 kGy) compared to lower D10 value obtained for salmonellae irradiated in beef with higher content of fat (0.49 kGy). Assuming that the low fat level in the meat is correlated with a higher protein content and because the proteins have the properties of free radicals scavenging, it can be suggested that the higher content of protein in meat protects more the

There have been frequently voiced concerns that the reduction of the competitive microflora by radiation treatment could facilitate growth of pathogens contaminating the food after irradiation or that food pathogens which survived irradiation can grow better than the indigenous, competitive microflora. Dickson and Olson [20] studied the first problem; ground beef was irradiated at 0, 2, or 4 kGy, thus reducing the number of saprophytic microorganisms which cause food spoilage, and then inoculated with a mixture of four serotypes of salmonellae. The meat was stored at 4°C, temperature proper for storage, and at two abused temperatures 15 and 25°C. Bacterial growth was monitored during storage. The authors observed that there was no significant difference in lag-phase duration or generation time, irrespective of the dose to which the ground beef had previously been exposed. This suggests that, although irradiation eliminates a significant portion of the spoilage microflora in ground beef, the absence of this microflora provides no competitive advantage to the growth of salmonellae in ground beef [20]. Szczawińska [21] studied the effect of irradiation on the survival rate of non-sporing bacteria (*Staphylococcus aureus, S. typhimurium, Escherichia coli, Pseudomonas fluorescens*) during conventional methods of meat preservation (heating, chilling, freezing, salting, curing, and smoking). On the basis of the conducted experiments, it can be concluded that irradiated bacteria stored under conditions preventing their growth die faster compared to unirradiated bacteria or their survival rate is almost identical like unirradiated ones; those organisms which are stored under conditions that allow their growth show a worse adaptability to the environment and begin to grow after a certain delay [21]. In another work, Szczawińska et al. [22] studied the growth of salmonellae in mechanically deboned chicken meat (MDCM), which was irradiated at 0, 1.25, and 2.5 kGy and inoculated with *S. dublin, S. enteritidis*, and *S. typhimurium*. Subsequently, the inoculated MDCM was stored at 5, 10, or 20°C and bacterial numbers were determined over storage time. The results of the study suggested that there was no greater risk from the same number of *Salmonella* cells contaminating irradiated MDCM compared to unirradiated one. In the same study, irradiated indigenous microflora had doserelated increased lag phases and decreased rates of multiplication compared with that of the indigenous microflora in the unirradiated control [22]. Kim and Thayer [23] discovered that the gamma-injured *S. typhimurium* cells on mechanically deboned chicken meat were much more sensitive to heat than the nonirradiated cells, which implies that any cells surviving the irradiation process were unlikely to survive cooking. This increased sensitivity of the salmonellae to gamma radiation was retained during refrigerated storage of the irradiated chicken. Kim and Thayer [23] explained the mechanism of the heat sensitivity of *S. typhimurium* subjected to ionizing radiation. The results proved that combined effects of irradiation and heating were always beneficial in regard to food safety due to synergistic (when heating is applied after irradiation) or additive (when heating is applied before irradiation) effects depending on the order of both treatments. Therefore, on the basis of these studies it can be concluded that any microorganisms which survive irradiation are more sensitive to intrinsic or extrinsic factors, such as temperature, water activity, pH, and so on, compared to unirradiated organisms.

bacteria against the damaging effects of radiation treatment.

258 Current Topics in Salmonella and Salmonellosis

D10 values for *Salmonella* spp. have been reported [28] to range from 0.38 to 0.77 kGy at 2°C in mechanically deboned chicken; sensitivity of *Salmonella* spp. to ionizing radiation has been found to be highly dependent on serovars. Similarly, the D10 values were reported by Szczawińska [26] for *S. typhimurium* strains inoculated into poultry meat, whereas a D10 value of 0.57 kGy has been observed for the pathogen in ground beef treated at 18–20°C [29].

In another work by Thayer et al. [30], *Musculus longissimus dorsi* from beef, pork, and lamb and turkey breast and leg meats were inoculated with *Salmonella* spp., and the gamma radiation resistance of the pathogens was determined at 5°C under identical conditions. The authors concluded that the *D*-value for a mixture of *Salmonella* spp. was significantly lower on pork than on beef, lamb, turkey breast, and turkey leg meats; however, all *D*-values were within expected ranges. Thayer et al. [31] studied the survival of salmonellae in vacuum-canned, commercial MDCM. The MDCM was challenged with *S. enteritidis* (ca 10<sup>4</sup> CFU/g of meat) followed by irradiation to 0, 1.5, and 3.0 kGy and storage at 5°C for 0, 2, and 4 weeks. The researchers reported that the number of salmonellae in unirradiated MDCM decreased about one log cycle after 1 month of storage; however, in meat irradiated with 3.0 kGy dose the presence of this pathogen was not detected at the very beginning of storage. Thayer and Boyd also found that *S. typhimurium* was more resistant to gamma radiation when vacuum packaged than when air was present during irradiation [32]. The final equations predict a reduction in the number of surviving *Salmonella* in mechanically deboned chicken meat. If MDCM is irradiated at −20°C with a dose of 1.50 kGy in air then the expected reduction of this pathogen is 2.53 and 2.12 logs in vacuum. After 3.0 kGy dose, at −20°C in air the level of bacteria will be lower by 4.78 and 4.29 logs in vacuum [32].

Bacteria are more resistant when irradiated at frozen temperatures compared to chill or ambient temperatures; Szczawińska [26] reported that the mean D10 value for 13 *Salmonella* strains irradiated in chicken meat using gamma rays at 4°C amounted to 0.575 kGy, whereas for samples irradiated in a frozen state (at −18°C) the mean D10 value amounted to 0.687 kGy. Gamma-irradiated broiler halves packed in polyethylene pouches with the dose of 2.5 kGy should ensure *Salmonella* reduction adequate to eliminate naturally occurring contamination. In frozen poultry meat, similar effects can be expected after a dose of 3.5 kGy [26]. In the same work, Szczawińska [26] discovered that the packaging material exerted a very strong effect on radiation resistance of *S. typhimurium*. Two strains of *S. typhimurium* were irradiated in ground chicken meat at temperatures +4 and −18°C. D10 values obtained for salmonellae irradiated at +4 and packed in PE pouches were 0.194 and 0.210 kGy, whereas D10 values obtained for salmonellae packed in PA/PE laminate pouches at the same temperature were 0.424 and 0.533 kGy. D10 values obtained for salmonellae irradiated at -18°C and packed in PE pouches were 0.412 and 0.633 kGy, whereas D10 values obtained for salmonellae packed in PA/PE laminate pouches at the same temperature were 0.538 and 0.721 kGy. Thus, the contribution of food-packaging material and packaging system is a very important issue in this technology. Irradiation was also combined with curing salts. The combined effects of 1-kGy irradiation dose and curing salts (NaNO2 and NaCl) on the survival of *S. typhimurium, S. agona, and S. cholerasuis* in pork meat were studied by Szczawiński et al. [33]. Salmonellae were inoculated in ground *M. longissimus dorsi*, and irradiated at 1 kGy dose. The three experimental groups were designed. The meat was treated with 100 mg NaNO2 , 200 mg NaNO2 , and 200 mg NaNO2 plus 3% NaCl. Meat samples were stored at 0–2°C for 3 weeks or at 20°C for 7 days. The authors reported that irradiation at 1 kGy dose reduced *Salmonella* number by 1.2–2 logs and that an additive effect of curing salts and irradiation was observed at low temperature of storage, and that synergistic effect of irradiation and curing salts was observed at temperature abuse [33].

Poultry, as already mentioned, as regards the radicidation, has been recognized as one of the best candidates for irradiation aiming a reduction or elimination of food-borne pathogenic bacteria such as *Salmonella* spp. and *Campylobacter* spp. Irradiation of poultry up to an overall average dose of 7 kGy was proposed by the Scientific Committee on Food [24] with the purpose to improve microbiological safety.

*Salmonella* caused 38.18%, the highest number of outbreaks and human cases among all causative agents according to data of EFSA for 2014 [34]. Raw poultry meat and poultry products are vehicles of those two food-borne pathogenic bacteria. In the EU, in 2013 [34], *Salmonella* was detected in 3.5% of the broiler meat. At retail, the overall proportion of *Salmonella*-positive samples was 7.5%, higher than at slaughterhouse (4.9%) and at the processing plant (2.6%) level [34]. Since December 2011, a *Salmonella* criterion for *S. enteritidis* and *S. typhimurium* in raw poultry entered into force [35].

In 2013, EFSA [34] reported that *Salmonella* was found in 0.3% of the 4776 samples of RTE broiler meat products tested at retail or at processing (0.1% of single samples and 1.9% of batches). Of the 2100 tested units of RTE products from turkey meat, only 0.1% in total were found to be *Salmonella*-positive [34].

Kudra et al. [36] studied the survival of *S. typhimurium* subjected to irradiation combined with high-CO2 + CO MAP in chicken meat product. The authors did not find significant difference between D10 values for bacteria irradiated in vacuum (0.55 kGy) or in high-CO2 + CO MAP (0.54 kGy). The dose of 1.5 kGy decreased the number of salmonellae by three logs. *Salmonella* presence was detected in both packaging systems during cold storage. During storage of this meat product at temperature abuse (25°C), *Salmonella* was able to grow in both packaging systems. The authors concluded that low-dose irradiation is a suitable method for destruction of this pathogen; however, packaging system did not exert significant influence on *Salmonella* number during storage at low temperature. The authors concluded that if the initial contamination of these pathogens is high, cross-contamination of ready-to-eat food at temperature abuse of the product is likely to continue to be a food safety concern regardless of irradiation treatment doses or packaging treatments.

Gamma-irradiated broiler halves packed in polyethylene pouches with the dose of 2.5 kGy should ensure *Salmonella* reduction adequate to eliminate naturally occurring contamination. In frozen poultry meat, similar effects can be expected after a dose of 3.5 kGy [26]. In the same work, Szczawińska [26] discovered that the packaging material exerted a very strong effect on radiation resistance of *S. typhimurium*. Two strains of *S. typhimurium* were irradiated in ground chicken meat at temperatures +4 and −18°C. D10 values obtained for salmonellae irradiated at +4 and packed in PE pouches were 0.194 and 0.210 kGy, whereas D10 values obtained for salmonellae packed in PA/PE laminate pouches at the same temperature were 0.424 and 0.533 kGy. D10 values obtained for salmonellae irradiated at -18°C and packed in PE pouches were 0.412 and 0.633 kGy, whereas D10 values obtained for salmonellae packed in PA/PE laminate pouches at the same temperature were 0.538 and 0.721 kGy. Thus, the contribution of food-packaging material and packaging system is a very important issue in this technology. Irradiation was also combined with curing salts. The combined effects of 1-kGy irradiation

*erasuis* in pork meat were studied by Szczawiński et al. [33]. Salmonellae were inoculated in ground *M. longissimus dorsi*, and irradiated at 1 kGy dose. The three experimental groups were

3% NaCl. Meat samples were stored at 0–2°C for 3 weeks or at 20°C for 7 days. The authors reported that irradiation at 1 kGy dose reduced *Salmonella* number by 1.2–2 logs and that an additive effect of curing salts and irradiation was observed at low temperature of storage, and that synergistic effect of irradiation and curing salts was observed at temperature abuse [33]. Poultry, as already mentioned, as regards the radicidation, has been recognized as one of the best candidates for irradiation aiming a reduction or elimination of food-borne pathogenic bacteria such as *Salmonella* spp. and *Campylobacter* spp. Irradiation of poultry up to an overall average dose of 7 kGy was proposed by the Scientific Committee on Food [24] with the pur-

*Salmonella* caused 38.18%, the highest number of outbreaks and human cases among all causative agents according to data of EFSA for 2014 [34]. Raw poultry meat and poultry products are vehicles of those two food-borne pathogenic bacteria. In the EU, in 2013 [34], *Salmonella* was detected in 3.5% of the broiler meat. At retail, the overall proportion of *Salmonella*-positive samples was 7.5%, higher than at slaughterhouse (4.9%) and at the processing plant (2.6%) level [34]. Since December 2011, a *Salmonella* criterion for *S. enteritidis* and *S. typhimurium* in

In 2013, EFSA [34] reported that *Salmonella* was found in 0.3% of the 4776 samples of RTE broiler meat products tested at retail or at processing (0.1% of single samples and 1.9% of batches). Of the 2100 tested units of RTE products from turkey meat, only 0.1% in total were

Kudra et al. [36] studied the survival of *S. typhimurium* subjected to irradiation combined with high-CO2 + CO MAP in chicken meat product. The authors did not find significant difference between D10 values for bacteria irradiated in vacuum (0.55 kGy) or in high-CO2 + CO MAP (0.54 kGy). The dose of 1.5 kGy decreased the number of salmonellae by three logs. *Salmonella* presence was detected in both packaging systems during cold storage. During storage of this meat product at temperature abuse (25°C), *Salmonella* was able to grow in both packaging

and NaCl) on the survival of *S. typhimurium, S. agona, and S. chol-*

, 200 mg NaNO2

, and 200 mg NaNO2

plus

dose and curing salts (NaNO2

260 Current Topics in Salmonella and Salmonellosis

designed. The meat was treated with 100 mg NaNO2

pose to improve microbiological safety.

raw poultry entered into force [35].

found to be *Salmonella*-positive [34].

Szczawińska [26] reported that the mean D10 value for 13 *Salmonella* strains irradiated in chicken meat using gamma rays at 4°C amounted to 0.575 kGy, whereas for samples irradiated in a frozen state (at −18° C) the mean D10 value amounted to 0.687 kGy. Gamma-irradiated broiler halves packed in polyethylene pouches with the dose of 2.5 kGy should ensure *Salmonella* reduction adequate to eliminate naturally occurring contamination; in frozen poultry meat, similar effects can be expected after a dose of 3.5 kGy [26].

Nassar et al. [37] evaluated the survival of *Salmonella virchow* inoculated into raw chicken carcasses as a result of radiation treatment (dose range of 2–7 kGy) or disinfection with three chemical substances. The presence of salmonellae in chicken meat was not detected after 7 kGy dose; however, after chemical disinfection this pathogen was still present.

On the basis of the various published data, it seems that the dose up to 7 kGy for frozen poultry and about 3.5 kGy for unfrozen meat can be recommended to reduce the most radioresistant vegetative pathogenic bacteria by five logs [18].

Thayer et al. [38] compared gamma radiation resistance of a mixture of salmonellae (*S. dublin, S. enteritidis, S. newport, S. senftenberg, and S. typhimurium*) in the so-called "exotic" meats such as ground bison, ostrich, alligator, and caiman meats at 5°C. The type of meat did not significantly alter the radiation resistance of salmonellae, and the *D*-value of 0.53 ± 0.02 kGy for *Salmonella* spp. was obtained. In the conclusions, authors emphasized that the efficacy of the radiation treatment in elimination of *Salmonella* spp. in exotic meats and non-exotic meats (e.g., poultry) is similar, thus similar control measures can be applied to ensure exotic meat safety. When considering cooked chilled and other ready-to-eat poultry meat products, the food-borne pathogens of higher concern are represented by *L. monocytogenes* and *Salmonella* spp. Hence, stricter microbiological criteria for poultry meat products intended to be eaten cooked, as amended by the Commission Regulation (EU) No 365/2010, which enhance food safety, must be respected by EU members [39]. Another (EC) regulation [40], which lays down general rules for food business operators on the hygiene of foodstuffs, requires food business operators to comply with microbiological criteria for foodstuffs. Regulation (EC) No 853/2005 [41], which sets specific hygiene rules for foods of animal origin, also requires that food business operators ensure compliance with microbiological criteria.

Radiation sensitivity of *L. monocytogenes* was determined by many authors (i.e., for example, see Ref. [42]). Reported D10 values for *L. monocytogenes* in cooked turkey nuggets were about 0.70 kGy, making *L. monocytogenes* generally more radiation-resistant than *Campylobacter* and *Salmonella*. Taking into consideration similar radiation sensitivity of *L. monocytogenes* and *Salmonella* spp., it can be assumed that doses of ionizing radiation effective for the inactivation of *L. monocytogenes* will be sufficient to inactivate salmonellae.

#### **2.2. Reducing of** *Salmonella* **spp. in ready-to-eat foods**

Ready-to-eat foods deserve special interest. Very often, they contain not only cooked poultry or other meats and cooked seafoods but also raw meats which are consumed without heat treatment (e.g., "filet américain" composed of raw beef meat). Thus, those complex RTE foods may represent an individual specific hazard to consumers since they are often composed of a mixture of several types of ingredients. RTE foods vary by country and may include, for example, dried meat (beef jerky), uncooked and fermented minced meat products (salami), cooked offal or minced meat products (chicken liver pâté or luncheon sausage), and cooked whole meat products (ham) [43]. Gormley et al. [44] conducted a wide study on microbiological quality of ready-to-eat specialty meats (2359 samples of continental sausages, cured/fermented, and dried meats) and reported that 0.4% were unacceptable due to the presence of *Salmonella* spp. or *L. monocytogenes* (>10(2) CFU/g). These unacceptable meats were all prepacked prior to supply to retail premises indicating that contamination with bacterial pathogens occurred earlier in the production chain; the authors emphasize how important it is to prevent food contamination before final packaging and to control conditions of storage.

Song et al. [45] investigated the efficacy of radiation treatment and fumaric acid on the reduction of *L. monocytogenes* and *S. typhimurium* inoculated into sliced ham. The authors noted the decrease of number of listeriae and salmonellae by 2.42 and 3.78 logs, respectively, after irradiation of this ready-to-eat product while the decrease of only one log for both organisms was found after acid treatment.

The US Food and Drug Administration (FDA) is currently evaluating a petition to allow irradiation of RTE meats in the United States including deli turkey, ham, pastrami, beef bologna, bacon bits, and pepperoni. The basis for the petition is data reported by Sommers and Mackay [46].

The authors observed in their study that irradiation of food-borne pathogenic bacteria with a dose of 3.75 kGy on ready-to-eat meats caused reduction of bacteria comparable to that obtained due to pasteurization, that is, minimum of five logs.

Sommers and Boyd [47] discovered that doses in the range of 2–4 kGy eliminate *Salmonella* spp. in many RTE foods.

The ability of ionizing radiation to inactivate *E. coli* O157:H7, *Salmonella, L. monocytogenes*, and *S. aureus* inoculated onto a frankfurter on a roll product containing the antimicrobials sodium diacetate and potassium lactate in the presence of an MA (100% N2 , 50% N<sup>2</sup> plus 50% CO2 , or 100% CO2 ) was investigated. The authors reported that the radiation resistance (D10 values) for *Salmonella* in frankfurter on a roll product was from 0.61 to 0.71 kGy. MA had no effect on the radiation resistance of the pathogens. During a 2-week storage period under mild temperature abuse (10°C), both salmonellae and other pathogens were not able to proliferate on the frankfurter on a roll product, regardless of the MA used. Although the pathogens were unable to proliferate on the frankfurter on a roll product during the storage period, the application of a postpackaging intervention step was needed to actually inactivate the food-borne pathogens. The authors concluded that, when applied as a terminal intervention as part of a HACCP plan, food irradiation could reduce the risk of food-borne pathogens on complex ready-to-eat foods such as sandwiches. It seems that intervention technologies including ionizing radiation, antimicrobials, and modified atmospheres (MAs) can be used to inhibit the growth of or inactivate food-borne pathogens on complex ready-to-eat foods such as sandwiches. Cárcel et al. [48] in their study elaborated mathematical model for the most efficient elimination of *Salmonella* spp. from two poultry products taking into consideration shelf life and sensory attributes. It was concluded that in the case of hamburgers, the optimum calculated dose was 2.04 kGy, which guaranteed the safety of the product and provided the best combination of sensory and instrumental attributes. As regards the steaks, the optimum assessed dose was 1.11 kGy, significantly lower than for hamburgers.

According to the data of a research project of a joint Food and Agriculture Organization/ International Academy of Engineering (FAO/IAE), the application of ionizing radiation combined with other methods used for food preservation offers improved safety of many various prepared meals and longer shelf life [49].

Kang et al. [50] studied the efficacy of radiation treatment combined with leek (*Allium tuberosum* R.) extract on the survival of several food-borne pathogens inoculated in pork jerky. The authors used doses in the range of 0.5–4 kGy. The *D*10-value for *S. typhimurium* irradiated with leek extract was 0.32 kGy and without this extract 0.39 kGy. The results prove that this combination strengthens both microbiological safety and shelf life of this meat.

#### **2.3. Reducing** *Salmonella* **spp. in fish and shellfish**

**2.2. Reducing of** *Salmonella* **spp. in ready-to-eat foods**

262 Current Topics in Salmonella and Salmonellosis

conditions of storage.

Mackay [46].

100% CO2

spp. in many RTE foods.

was found after acid treatment.

Ready-to-eat foods deserve special interest. Very often, they contain not only cooked poultry or other meats and cooked seafoods but also raw meats which are consumed without heat treatment (e.g., "filet américain" composed of raw beef meat). Thus, those complex RTE foods may represent an individual specific hazard to consumers since they are often composed of a mixture of several types of ingredients. RTE foods vary by country and may include, for example, dried meat (beef jerky), uncooked and fermented minced meat products (salami), cooked offal or minced meat products (chicken liver pâté or luncheon sausage), and cooked whole meat products (ham) [43]. Gormley et al. [44] conducted a wide study on microbiological quality of ready-to-eat specialty meats (2359 samples of continental sausages, cured/fermented, and dried meats) and reported that 0.4% were unacceptable due to the presence of *Salmonella* spp. or *L. monocytogenes* (>10(2) CFU/g). These unacceptable meats were all prepacked prior to supply to retail premises indicating that contamination with bacterial pathogens occurred earlier in the production chain; the authors emphasize how important it is to prevent food contamination before final packaging and to control

Song et al. [45] investigated the efficacy of radiation treatment and fumaric acid on the reduction of *L. monocytogenes* and *S. typhimurium* inoculated into sliced ham. The authors noted the decrease of number of listeriae and salmonellae by 2.42 and 3.78 logs, respectively, after irradiation of this ready-to-eat product while the decrease of only one log for both organisms

The US Food and Drug Administration (FDA) is currently evaluating a petition to allow irradiation of RTE meats in the United States including deli turkey, ham, pastrami, beef bologna, bacon bits, and pepperoni. The basis for the petition is data reported by Sommers and

The authors observed in their study that irradiation of food-borne pathogenic bacteria with a dose of 3.75 kGy on ready-to-eat meats caused reduction of bacteria comparable to that

Sommers and Boyd [47] discovered that doses in the range of 2–4 kGy eliminate *Salmonella*

The ability of ionizing radiation to inactivate *E. coli* O157:H7, *Salmonella, L. monocytogenes*, and *S. aureus* inoculated onto a frankfurter on a roll product containing the antimicrobials sodium

for *Salmonella* in frankfurter on a roll product was from 0.61 to 0.71 kGy. MA had no effect on the radiation resistance of the pathogens. During a 2-week storage period under mild temperature abuse (10°C), both salmonellae and other pathogens were not able to proliferate on the frankfurter on a roll product, regardless of the MA used. Although the pathogens were unable to proliferate on the frankfurter on a roll product during the storage period, the application of a postpackaging intervention step was needed to actually inactivate the food-borne pathogens. The authors concluded that, when applied as a terminal intervention as part of a HACCP

) was investigated. The authors reported that the radiation resistance (D10 values)

, 50% N<sup>2</sup>

plus 50% CO2

, or

obtained due to pasteurization, that is, minimum of five logs.

diacetate and potassium lactate in the presence of an MA (100% N2

Raw fish and shellfish can be contaminated with pathogenic bacteria such as *Salmonella, Shigella, Vibrio parahaemolyticus, vulnificus, Vibrio cholerae, S. aureus*, and viruses.

According to the data delivered by the United States Department of Agriculture (USDA) [51], *Salmonella* was found in 21% of 153 aquaculture catfish collected from aquaculture ponds and retail markets. The U.S. Food and Drug Administration data from 1998 to 2004 on examination of seafood import refusal identified *Salmonella* contamination to be the most frequent violation in catfish (41.91% of violation categories). Hatha and Laksmanaperumalsamy [52] found *Salmonella* spp. in 14–25% of fish belonging to 18 families. On the basis of the data presented in the literature, along with outbreak data and FDA import refusal data, it can be concluded that the highest microbial hazard associated with catfish consumption is *Salmonella* spp. Raw finfish might contain *V. parahaemolyticus, Salmonella* spp., or *L. monocytogenes* [53]. According to the report of USDA, nontyphoidal *Salmonella* spp. in raw and RTE catfish are considered as higher priority microbial hazards [51]. In terms of risk assessment related to catfish consumption, USDA estimated that the mean reduction of *Salmonella* per catfish serving caused by frying is about two logs, and caused by baking is about three logs [51]. Thus, it seems that such reduction of *Salmonella* spp. number, taking into consideration that naturally contaminated foods contain usually low levels of salmonellae, may significantly lower the risk of food-borne disease due to consumption of contaminated catfish. These findings could partially explain the differences between a significant contamination of raw finfish by pathogenic bacteria and relatively small number of outbreaks in which etiologic agent is *Salmonella* spp.

The monthly data on import refusal published in the USA prove that 1/10 of the refused products are seafood products and that second in terms of rejection reason is the detection of *Salmonella* spp. [54]. Risk analysis conducted in New Zealand by Reed [55] for fillet meat of *Pangasius* spp. fish from Vietnam considered that contamination of fillets with water not of a suitable purity could result in the presence of exotic strains of *Corynebacterium diphtheriae, E. coli, Salmonella* spp., *V. cholerae*, and *Cryptosporidia* spp., which is a risk to human health. Shabarinath et al. [56] studied the prevalence of *Salmonella* in seafood samples by conventional culture and by a DNA-based molecular technique, polymerase chain reaction (PCR). Using PCR, which was considered to be better method, they isolated *Salmonella* spp. from over 50% of seafood samples collected from the southwest coast of India; 14 of 19 isolates belonged to serovar *Salmonella enterica* Weltevreden.

The FAO experts in their report, after thorough evaluation of *Salmonella* spp. problem related to seafood, concluded that good hygienic practices during aquaculture production and biosecurity measures can minimize but not eliminate *Salmonella* in products of aquaculture [57].

Among various seafood, shrimp as the largest single seafood commodity in value terms (at around 15% of the total value of internationally traded fishery products in 2012) mainly produced in developing countries such as South and East Asia and Latin America deserves special attention [58] particularly that the consumption of this commodity consumption has been trending upward.

Norhana et al. [59] in their comprehensive review paper on prevalence, persistence, and control of *Salmonella* and *Listeria* in shrimp and shrimp products indicated that the continued reporting of the presence of these bacteria in fresh and frozen shrimps, and even in the lightly preserved and ready-to-eat products, shows that the existing hygienic practices in fishery industry are insufficient to eliminate these pathogens which have been isolated from shrimps and shrimp products on a regular basis since the 1980s. Shrimp is frequently imported from tropical and subtropical areas and reports indicate that the product does not always meet the microbiological standards set for EU-producing countries or USA, because of either contaminated production sites or unhygienic processing conditions.

*Salmonella* bacteria are associated with pond water, sediment, and shrimp throughout the culture cycle, including the pre-stocking period, farming phase, and harvest. Untreated chicken manure used to fertilize ponds and droppings from aquatic birds are significant sources of *Salmonella*. The survival rate of the microorganism is enhanced by nutrients, manure, and feed present in the pond system and by the favorable interaction of various biological and physical factors [60]. Shrimps are usually eaten fully cooked. The major health hazards with these products are contamination during or after processing.

Pinu et al. [61] evaluated the microbiological condition of the frozen shrimps found in the local markets and departmental chain shops of Dhaka city. Pathogenic bacterial load was found greater in the samples of departmental shops rather than that of local markets. The researchers found *Salmonella* spp., *Vibrio* spp., and *Shigella* spp. in shrimps' samples and discovered that the samples collected from local markets and departmental shops were heavily contaminated and were of special concern for human consumption.

Asai et al. [62] reported that the examination of 353 samples of 29 types of seafood revealed that *S. enterica* serotype Weltevreden was isolated from two of 47 black tiger prawn samples. The contamination levels of *Salmonella* were in a range of <30–40 most probable number per 100 g. Asai concluded that these results indicate the possibility that shrimp and prawns contribute to food-borne infections.

The monthly data on import refusal published in the USA prove that 1/10 of the refused products are seafood products and that second in terms of rejection reason is the detection of *Salmonella* spp. [54]. Risk analysis conducted in New Zealand by Reed [55] for fillet meat of *Pangasius* spp. fish from Vietnam considered that contamination of fillets with water not of a suitable purity could result in the presence of exotic strains of *Corynebacterium diphtheriae, E. coli, Salmonella* spp., *V. cholerae*, and *Cryptosporidia* spp., which is a risk to human health. Shabarinath et al. [56] studied the prevalence of *Salmonella* in seafood samples by conventional culture and by a DNA-based molecular technique, polymerase chain reaction (PCR). Using PCR, which was considered to be better method, they isolated *Salmonella* spp. from over 50% of seafood samples collected from the southwest coast of India; 14 of 19 isolates

The FAO experts in their report, after thorough evaluation of *Salmonella* spp. problem related to seafood, concluded that good hygienic practices during aquaculture production and biosecurity measures can minimize but not eliminate *Salmonella* in products of aquaculture [57]. Among various seafood, shrimp as the largest single seafood commodity in value terms (at around 15% of the total value of internationally traded fishery products in 2012) mainly produced in developing countries such as South and East Asia and Latin America deserves special attention [58] particularly that the consumption of this commodity consumption has been

Norhana et al. [59] in their comprehensive review paper on prevalence, persistence, and control of *Salmonella* and *Listeria* in shrimp and shrimp products indicated that the continued reporting of the presence of these bacteria in fresh and frozen shrimps, and even in the lightly preserved and ready-to-eat products, shows that the existing hygienic practices in fishery industry are insufficient to eliminate these pathogens which have been isolated from shrimps and shrimp products on a regular basis since the 1980s. Shrimp is frequently imported from tropical and subtropical areas and reports indicate that the product does not always meet the microbiological standards set for EU-producing countries or USA, because of either contami-

*Salmonella* bacteria are associated with pond water, sediment, and shrimp throughout the culture cycle, including the pre-stocking period, farming phase, and harvest. Untreated chicken manure used to fertilize ponds and droppings from aquatic birds are significant sources of *Salmonella*. The survival rate of the microorganism is enhanced by nutrients, manure, and feed present in the pond system and by the favorable interaction of various biological and physical factors [60]. Shrimps are usually eaten fully cooked. The major health hazards with these

Pinu et al. [61] evaluated the microbiological condition of the frozen shrimps found in the local markets and departmental chain shops of Dhaka city. Pathogenic bacterial load was found greater in the samples of departmental shops rather than that of local markets. The researchers found *Salmonella* spp., *Vibrio* spp., and *Shigella* spp. in shrimps' samples and discovered that the samples collected from local markets and departmental shops were heavily

belonged to serovar *Salmonella enterica* Weltevreden.

264 Current Topics in Salmonella and Salmonellosis

nated production sites or unhygienic processing conditions.

products are contamination during or after processing.

contaminated and were of special concern for human consumption.

trending upward.

In recent years, safety risks are associated to the consumption of raw or subjected to mild heat treatment fish and shellfish; molluscan shellfish (oysters, clams, mussels, and scallops) are often consumed whole and raw. Huss et al. [54] and Olgunoglu [63] in his comprehensive review on *Salmonella* in fish and fishery products show that the pathogens of concern in this seafood include both bacteria (e.g., *Vibrio* spp., *Salmonella* spp., *L. monocytogenes, Shigella* spp., *C. jejuni*), viruses (e.g., hepatitis A virus and norovirus), and parasites. Molluscan shellfish feed on phytoplankton and zooplankton. They are passive feeders that filter and concentrate pathogens present in harvest area. Their environment, particularly near-shore harvest water, is contaminated from sewage, which may contain pathogens from both human and animal fecal sources (e.g., *V. cholerae* O1 and O139, *Salmonella* spp.). Also, poor sanitary practices on the harvest vessel, poor aquacultural practices, and transportation can cause contamination of fishery products.

As observed in previous years, the food category with the highest level of non-compliance at processing was RTE fishery products (4.7% of single samples and 10.8% of batches), mainly in smoked fish [34].

Distribution of strong-evidence outbreaks by food vehicle in the EU in 2014 indicated that crustaceans, shellfish, molluscs, and products thereof were responsible for 8.1% of outbreaks with strong evidence (data from 592 outbreaks with strong evidence) [34]. Taking the above-mentioned data into consideration, health authorities in many countries including European Community emphasized that the increasing trend in raw fish consumption (sushi, sashimi, salmon, etc.) has been identified as a risk to human health. Oysters and mussels can cause food-borne illness. Consumer can contract food-borne salmonellosis due to consumption of raw oysters.

It is generally known that the best method of controlling pathogens is to use a postharvest treatment. Some treatments, such as thermal treatment, ionizing radiation, and high hydrostatic pressure processing, reduce the number of pathogenic microorganisms (bacteria and viruses) while the long-term freezing most widely used method of food preservation is mainly effective in controlling parasites.

Brands et al. [64] reported that *Salmonella* was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained *Salmonella*. Isolation tended to be bay specific. The vast majority (78/101) of *Salmonella* isolates from oysters were *S. enterica* serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Bakr et al. [65] showed that out of the 150 seafood samples examined, collected from 11 localities in Alexandria, Egypt, Salmonella was isolated from 10% of samples (shrimp, oyster, and mussel). In 1986, the Scientific Committee for Foods [24] recommended that fish and shellfish could be irradiated at doses up to 3 kGy. In the United States, FDA has approved the use of ionizing radiation for the control of *V. parahaemolyticus* and *V. vulnificus* and other food-borne pathogens in fresh or frozen molluscan shellfish. Irradiation of fresh and frozen molluscan shellfish may not exceed an absorbed dose of 5.5 kGy [53]. Also, FDA proposes radiation treatment for the control of food-borne bacteria in crustaceans with a dose of 6.0 kGy. The D10 values cited in the published literature for several *Salmonella* serotypes in grass prawns and shrimp homogenate ranged from 0.30 to 0.59 kGy. Thus, irradiation of crustaceans at a maximum absorbed dose of 6.0 kGy would be effective at controlling pertinent pathogens. The petitioner requested a maximum absorbed dose of 6.0 kGy to achieve a six-log reduction of *L. monocytogenes*. It can be expected that this dose should also eliminate majority of non-sporing pathogenic bacteria including *Salmonella*. Irradiation of fish and shellfish is intended, similarly like in the case of other foods to extend shelf life, reduce pathogen load, and inactivate parasites. Irradiation has been applied to fresh, frozen, as well as dried fish, fish products, and shellfish [18]. As for other foods, pathogenic bacteria are more resistant to irradiation in frozen state compared to chilled one. Most studies indicate that irradiation at doses recommended by the SCF (3 kGy) should yield two to five logs reduction of pathogenic, nonspore-forming bacteria for the majority of fish and fish products. Sommers and Rajkowski [66] determined the radiation D10 values for *Salmonella* inoculated onto seafood samples (scallops, lobster meat, blue crab, swordfish, octopus, and squid). The samples were frozen and irradiated in the frozen state (−20°C); D10 values for *Salmonella* ranged from 0.47 to 0.70 kGy. By contrast, the radiation D10 value for *Salmonella* suspended on frozen pork was 1.18 kGy. They concluded that radiation dose needed to inactivate these food-borne pathogens on frozen seafood is significantly lower than that for frozen meat or frozen vegetables. *Salmonella* spp. and other primary pathogens of concern can also be introduced after pasteurization. Some fishery products are cooked before they are packaged; therefore, they are at risk for recontamination between cooking and packaging (e.g., vacuum packaging, modified atmosphere packaging). Kamat and Thomas [67] evaluated the effect of fat content in fish on radiation sensitivity of *L. monocytogenes, Bacillus cereus, S. typhimurium*, and *Yersinia enterocolitica*. The radiation response of all those pathogens was examined in sardine with high fat and golden anchovy with low fat. The results clearly suggest that regardless of the level of lipid in fish, the application of a 3 kGy dose at refrigeration temperature would effectively decontaminate approximately 10<sup>5</sup> CFU g−1 of all the organisms tested, except spores of *B. cereus*. The authors concluded that the studies revealed a lack of influence of lipid levels in fish on radiation resistance of four food-borne bacterial pathogens.

Jakabi et al. [68] studied the survival of *S. enteritidis* and *S. infantis* inoculated into oysters and sensory properties as the result of irradiation with doses in the dose range of 0.5–3.0 kGy. The number of those both *Salmonella* populations decreased after a 3.0 kGy dose by five to six logs. The authors also discovered that oysters irradiated with the highest dose were still alive and concluded that a dose of 3.0 kGy could be considered effective in inactivating *Salmonella* in oysters without changing their odor, flavor, or appearance.

The SCF [24] recommended that shrimps could be irradiated at doses of 5 kGy which is considered to be an effective decontamination method. Ito et al. [69] reported that the dose of gamma irradiation necessary to reduce both *S. typhimurium* and *L. monocytogenes* in frozen shrimps at a level of below 10−4 per gram was about 3.5 kGy. Sinanoglou et al. [70] irradiated using a cobalt-60 gamma source frozen molluscs (squid, octopuses, and cuttlefish) and crustaceans (shrimp) with different doses. The authors noted the substantial decrease of mesophiles number in shrimp irradiated with the dose of 2.5 kGy, whereas after the dose of 4.7 kGy the presence of those bacteria in squid was not detected. Shrimp is considered separately from fish and shellfish given that certain pathogens (i.e., *L. monocytogenes*) require doses about 3 kGy for several log10 reduction. Sommers et al. [71] evaluated the effect of cryogenic freezing (−82°C, 3 min), and gamma irradiation on the survival of mixture of *Salmonella* spp. (*S. schwarzengrund, S. bahrenfeld, S. weltevreden*, and *S. panama* isolated from seafood, including shrimp), on raw frozen shrimp. D10 values for salmonellae irradiated in shrimp were about 0.56 kGy. The authors observed the decrease of *Salmonella* spp. number after cryogenic freezing and irradiation with a dose of 2.25 kGy by over five logs and that this effect persisted during 3 months storage at −20°C. The authors conclude that radiation treatment combined with cryogenic freezing offers big benefits in regard to frozen shrimp.

Nerkar and Bandekar [72] studied radiation resistance of *S. typhimurium* and *S. enteritidis* inoculated at 1 × 10<sup>8</sup> cells/ml in shrimp homogenate and they determined that the D10 value was in the range from 0.30 to 0.40 kGy. Finally, they concluded that a dose of 4.0 kGy could be used to completely eliminate *Salmonella* in frozen prepackaged shrimp.

Luo et al. [73] studied radioresistance of non-spore-forming and spore-forming pathogenic microorganisms inoculated into shelf-stable foods, semi-dried pork, and fish which have been vacuum-packaged. The water activity (aw) of semi-dried food products ranged between 0.930 and 0.940 for pork, and 0.852 and 0.895 for fish. The authors observed that *S. enteritidis* was eliminated at a dose of 2.5 kGy in semi-dried fish, and the minimum irradiation dose required to inactivate this pathogen in pork was 5 kGy.

#### **2.4. Reducing** *Salmonella* **spp. in frog's legs**

food-borne pathogens in fresh or frozen molluscan shellfish. Irradiation of fresh and frozen molluscan shellfish may not exceed an absorbed dose of 5.5 kGy [53]. Also, FDA proposes radiation treatment for the control of food-borne bacteria in crustaceans with a dose of 6.0 kGy. The D10 values cited in the published literature for several *Salmonella* serotypes in grass prawns and shrimp homogenate ranged from 0.30 to 0.59 kGy. Thus, irradiation of crustaceans at a maximum absorbed dose of 6.0 kGy would be effective at controlling pertinent pathogens. The petitioner requested a maximum absorbed dose of 6.0 kGy to achieve a six-log reduction of *L. monocytogenes*. It can be expected that this dose should also eliminate majority of non-sporing pathogenic bacteria including *Salmonella*. Irradiation of fish and shellfish is intended, similarly like in the case of other foods to extend shelf life, reduce pathogen load, and inactivate parasites. Irradiation has been applied to fresh, frozen, as well as dried fish, fish products, and shellfish [18]. As for other foods, pathogenic bacteria are more resistant to irradiation in frozen state compared to chilled one. Most studies indicate that irradiation at doses recommended by the SCF (3 kGy) should yield two to five logs reduction of pathogenic, nonspore-forming bacteria for the majority of fish and fish products. Sommers and Rajkowski [66] determined the radiation D10 values for *Salmonella* inoculated onto seafood samples (scallops, lobster meat, blue crab, swordfish, octopus, and squid). The samples were frozen and irradiated in the frozen state (−20°C); D10 values for *Salmonella* ranged from 0.47 to 0.70 kGy. By contrast, the radiation D10 value for *Salmonella* suspended on frozen pork was 1.18 kGy. They concluded that radiation dose needed to inactivate these food-borne pathogens on frozen seafood is significantly lower than that for frozen meat or frozen vegetables. *Salmonella* spp. and other primary pathogens of concern can also be introduced after pasteurization. Some fishery products are cooked before they are packaged; therefore, they are at risk for recontamination between cooking and packaging (e.g., vacuum packaging, modified atmosphere packaging). Kamat and Thomas [67] evaluated the effect of fat content in fish on radiation sensitivity of *L. monocytogenes, Bacillus cereus, S. typhimurium*, and *Yersinia enterocolitica*. The radiation response of all those pathogens was examined in sardine with high fat and golden anchovy with low fat. The results clearly suggest that regardless of the level of lipid in fish, the application of a 3 kGy dose at refrigeration temperature would effectively decontaminate

CFU g−1 of all the organisms tested, except spores of *B. cereus*. The authors

concluded that the studies revealed a lack of influence of lipid levels in fish on radiation resis-

Jakabi et al. [68] studied the survival of *S. enteritidis* and *S. infantis* inoculated into oysters and sensory properties as the result of irradiation with doses in the dose range of 0.5–3.0 kGy. The number of those both *Salmonella* populations decreased after a 3.0 kGy dose by five to six logs. The authors also discovered that oysters irradiated with the highest dose were still alive and concluded that a dose of 3.0 kGy could be considered effective in inactivating *Salmonella* in

The SCF [24] recommended that shrimps could be irradiated at doses of 5 kGy which is considered to be an effective decontamination method. Ito et al. [69] reported that the dose of gamma irradiation necessary to reduce both *S. typhimurium* and *L. monocytogenes* in frozen shrimps at a level of below 10−4 per gram was about 3.5 kGy. Sinanoglou et al. [70] irradiated using a cobalt-60 gamma source frozen molluscs (squid, octopuses, and cuttlefish) and crustaceans (shrimp) with different doses. The authors noted the substantial decrease of mesophiles

approximately 10<sup>5</sup>

266 Current Topics in Salmonella and Salmonellosis

tance of four food-borne bacterial pathogens.

oysters without changing their odor, flavor, or appearance.

The skin of frogs and their internal organs are often contaminated with *Salmonella* spp. and other pathogens, such as *E. coli* and *S. aureus*. Although frog's legs are cooked before consumption, there is a risk for cross-contamination.

The highest radiation dose for frog's legs suggested by the Scientific Committee for Foods is 5 kGy [18]. The most important hazard arises from contamination with *Salmonella* and other fecal pathogens occurring in frog's legs at the time of deep-freezing. *E. coli* and *S. aureus* have been also found in frog's legs. Tambunan's [74] studies showed that irradiating frog legs artificially contaminated with *Salmonella* up to 10<sup>6</sup> /g before freezing a dose of 3 kGy and above resulted in no detection of the bacteria. If irradiation was carried out after freezing, a dose of 4 kGy and above has to be used. The latter procedure appears to be more feasible commercially than the former one. It was concluded that a combination of chlorination, freezing, and irradiation with a dose ranging from 3 to 6 kGy should provide sufficient conditions for the elimination of *Salmonella* in the product.

## **3. Concluding remarks**

Ionizing radiation in industry can be used to reduce the level of *Salmonella* spp. in both raw and cooked meats, poultry, and seafood. This intervention technology can be regarded as a Critical Control Point in the HACCP plan. Irradiation treatment, applied as the final processing step, seems to be of particular importance in the case of packed food products, including ready-toeat food. In the USA, FDA [75] proposes radiation treatment with the maximum dose of 4.5 kGy for a variety of raw meats and meat products for the improvement of microbial safety and for shelf-life extension.

The data from literature prove that the *D*-values for *L. monocytogenes* are similar to those reported for *Salmonella* spp. irradiated under similar conditions. Thus, *Salmonella* spp. in meats, poultry, and fish and shellfish including ready-to-eat foods may be controlled by the same dose required for *L. monocytogenes*.

It should be noted, however, that dose range used for radicidation (2.5–10 kGy) is not sufficient to sterilize foods. Thus, all additional control measures (e.g., an unbroken cold chain, appropriate handling of raw meat, and procedures for cleaning disinfection and waste disposal, etc.) should maintain or even increase the beneficial effects of radiation treatment.

Referring to irradiation facilities, electron beams are much more useful for packs of relatively thin cooked, sliced meats, and other ready-to-eat products while gamma radiation is more suited for treating whole carcasses [76].

## **Author details**

Małgorzata E. Szczawińska

Address all correspondence to: malgorzata\_szczawinska@sggw.pl

Warsaw University of Life Sciences – SGGW, Faculty of Veterinary Medicine, Department of Food Hygiene and Public Health Protection, Warsaw, Poland

## **References**


[6] Sofos J., Flick G., Nychas G., O'Bryan C., Ricke S., Crandall P. Meat, Poultry, and Seafood, p 111-167. In: Doyle M, Buchanan R (ed), Food Microbiology. ASM Press, Washington, DC. 4th ed. 2013. DOI: 10.1128/9781555818463.ch6.

seems to be of particular importance in the case of packed food products, including ready-toeat food. In the USA, FDA [75] proposes radiation treatment with the maximum dose of 4.5 kGy for a variety of raw meats and meat products for the improvement of microbial safety

The data from literature prove that the *D*-values for *L. monocytogenes* are similar to those reported for *Salmonella* spp. irradiated under similar conditions. Thus, *Salmonella* spp. in meats, poultry, and fish and shellfish including ready-to-eat foods may be controlled by the

It should be noted, however, that dose range used for radicidation (2.5–10 kGy) is not sufficient to sterilize foods. Thus, all additional control measures (e.g., an unbroken cold chain, appropriate handling of raw meat, and procedures for cleaning disinfection and waste disposal, etc.) should maintain or even increase the beneficial effects of radiation treatment.

Referring to irradiation facilities, electron beams are much more useful for packs of relatively thin cooked, sliced meats, and other ready-to-eat products while gamma radiation is more

Warsaw University of Life Sciences – SGGW, Faculty of Veterinary Medicine, Department of

[1] Leistner L., Gorris L.G.M.: Food preservation by hurdle technology. Trends in Food

[2] Farkas J.: Combination of irradiation with mild heat treatment. Food Control. 1990;1:

[3] Chouliara E., Badeka A., Savvaidis I., Kontaminas MG.: Combined effect of irradiation and modified atmosphere packaging on shelf-life extension of chicken breast meat: microbiological, chemical and sensory changes. European Food Research and

[4] Grant I.R., Patterson M.F.: Combined effect of gamma radiation and heating on the destruction of *Listeria monocytogenes* and *Salmonella typhimurium* in cook-chill roast beef and gravy. International Journal of Food Microbiology. 1995;27:117–128. DOI:

[5] Thayer D.W., Songprasertchai S., Boyd G.: Effects of heat and ionizing radiation on *Salmonella typhimurium* in mechanically deboned chicken meat. J. Food Prot. 1991;54:

Technology. 2008;226:877–888. DOI: 10.1007/s00217-007-0610-3.

and for shelf-life extension.

268 Current Topics in Salmonella and Salmonellosis

same dose required for *L. monocytogenes*.

suited for treating whole carcasses [76].

Address all correspondence to: malgorzata\_szczawinska@sggw.pl

Food Hygiene and Public Health Protection, Warsaw, Poland

Science and Technology. 1995;6:41–46.

10.1016/0168-1605(94)00156-Z.

**Author details**

**References**

223–229.

718–724.

Małgorzata E. Szczawińska


[33] Szczawiński J., Szczawińska M., Szulc M.: Combined effects of irradiation and curing salts (NaNO2 and NaCl) upon the behaviour of salmonellae in meat. Archiv fur Lebensmittelhygiene. 1985;36:(3):55–58.

[20] Dickson J.S., Olson D.G.: Growth rates of *Salmonella* and *Escherichia coli* O157:H7 in irra-

[21] Szczawińska M., Szczawiński J., Szulc M.: The survival of irradiated bacteria during conventional methods of meat preservation. Archiv fur Lebensmittelhygiene 1983;34:53–58.

[22] Szczawińska M.E., Thayer D.W., Phillips J.G.: Fate of unirradiated *Salmonella* in irradiated mechanically deboned chicken meat. International Journal of Food Microbiology.

[23] Kim A.Y., Thayer D.W.: Mechanism by which gamma irradiation increases the sensitivity of *Salmonella typhimurium* ATCC 14028 to heat. Applied and Environmental

[24] SCF (Scientific Committee on Food). Food - Science and Techniques. Reports of the

[25] Kampelmacher E.H.: Irradiation of food: a new technology for preserving and ensuring

[26] Szczawińska M.: Prospects for elimination of salmonellae from poultry by irradiation in Poland. Food Irradiation in the Middle East and Europe. Proceedings of the Final Coordination Meeting of an FAO/IAEA Coordinated Research Programme organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture and held in

Cadarache, France, 8–12 March 1993. IAEA-TECDOC-754, pp. 77–96, July 1994.

[27] Clavero M.R., Monk J.D., Beuchat L.R., Doyle M.P., Brackett R.E.: Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation. Applied and Environmental Microbiology. 1994;60:2069–2075.

[28] Thayer D.W., Boyd G., Muller W.S., Lipson C.A., Hayne W.C., Baer S.H. Radiation resis-

[29] Tarkowski J.A., Beumer R.R., Kampelmacher E.H.: Low gamma irradiation of raw meat.

[30] Thayer D.W., Boyd G., Fox I. B., Ir., Lakritz L., Hampson I.W.: Variations in radiation sensitivity of foodborne pathogens associated with the suspending meat. I. Food Science.

[31] Thayer D.W., Boyd G., Huhtanen C.N.: Effects of ionizing-radiation and anaerobic refrigerated storage on indigenous microflora, *Salmonella*, and *Clostridium botulinum* type-A and type-B in vacuum-canned, mechanically deboned chicken meat. Journal of

[32] Thayer D.W., Boyd G.: Survival of *Salmonella typhimurium* ATCC 14028 on the surface of chicken legs or in mechanically deboned chicken meat gamma irradiated in air or vacuum at temperatures of -20 to +20 degree. Poultry Science. 1991:70:1026–1033.

tance of *Salmonella*. Journal of Industrial Microbiology. 1990;5:383–390.

International Journal of Food Microbiology. 1984;1:25–31.

diated beef. Journal of Food Protection 2001;64:1828–1831.

Scientific Committee for Food (Eighteenth Series). 1986.

the hygiene of foods. Fleischwirtschaft 1984;64:322–327.

1991;14:313–324.

270 Current Topics in Salmonella and Salmonellosis

1995a;60:63–67.

Food Protection. 1995;58:752–757.

Microbiology. 1996; 62:1759**–**1763.


[56] Shabarinath S., Kumar H.S., Khushiramani R., Karunasagar I.: Detection and characterization of *Salmonella* associated with tropical seafood. International Journal of Food Microbiology. 2007;114:227–233. DOI: 10.1016/j.ijfoodmicro.2006.09.012.

[44] Gormley F.J., Little C.L., Grant K.A., de Pinna E., McLauchlin J.: The microbiological safety of ready-to-eat specialty meats from markets and specialty food shops: a UK wide study with a focus on *Salmonella* and *Listeria monocytogenes*. Food Microbiology.

[45] Song H.J., Lee J.H., Song K.B.: Effects of irradiation and fumaric acid treatment on the inactivation of *Listeria monocytogenes* and *Salmonella typhimurium* inoculated on sliced ham. Radiation Physics and Chemistry. 2011. Doi:10.1016/j.radphyschem.2011.06.012.

[46] Sommers C.H., Mackay W.J.: Irradiation of Ready-To-Eat Meat Products. In: Institute of Food Technologist Series: Food Irradiation Research and Technology, 2nd ed. Fan X and Sommers CH., editors. 2012. DOI: 10.1002/9781118422557.ch11. This edition first published 2013 © 2013 Blackwell Publishing and the Institute of Food Technologists.

[47] Sommers C.H., Boyd G.: Radiation sensitivity and postirradiation growth of foodborne pathogens on a ready-to-eat frankfurter on a roll product in the presence of modified

[49] IAEA (International Atomic Energy Agency), Vienna, Irradiation to Ensure the Safety and Quality of Prepared Meals. STI/PUB/1365. 2009. ISBN 978-92-0-111108-1.

[50] Kang M., Kim H.-J., Jayasena D.D., Bae S.Y., Yong I.H., Lee M., Jo C.: Effects of combined treatments of electron-beam irradiation and addition of Leek (Allium tuberosum) extract on reduction of pathogens in pork jerky. Foodborne Pathogens and Disease.

[51] Bauer N., Cook V., Disney T., Ebel E., Guo C., Johnston J., La Barre D., Lee J., McCoy E., Morrison J., Schlosser W., Williams M.: DRAFT Risk Assessment of the Potential Human Health Effect of Applying Continuous Inspection to Catfish, Prepared by the Risk Assessment Division Office of Public Health Science Food Safety and Inspection

[52] Hatha AAM and Laksmanaperumalsamy P.: Prevalence of *Salmonella* in fish and crustaceans from markets in Coimbatore, South India. Food Microbiology.1997;14:111–116.

[53] FDA Guidance for the Industry: Fish and Fishery Products Hazards and Controls Guidance Department Of Health And Human Services Public Health Service Food and Drug Administration Center For Food Safety And Applied Nutrition Office Of Food

[54] Huss H.H., Ababouch L., Gram L.: Assessment and management of seafood safety and

[55] Reed C.: Import risk analysis: Frozen, skinless and boneless fillet meat of *Pangasius* spp. fish from Vietnam for human consumption MAF Biosecurity New Zealand, 2008.

Service United States Department of Agriculture. December, 2010.

quality. FAO Fisheries Technical Paper. No. 444. Rome, FAO. 2004.

atmosphere and antimicrobials. Journal of Food Protection. 2006;69:2436–2440. [48] Cárcel J.A., Benedito J., Cambero M.I., Cabeza M.C., Ordóñez J.A.: Modeling and optimization of the E-beam treatment of chicken steaks and hamburgers, considering food safety, shelf-life, and sensory quality. Food and Bioproducts Processing. 2015;96:133–

144. DOI: http://dx.doi.org/10.1016/j.fbp.2015.07.006

2012;9:1083–1087. DOI: 10.1089/fpd.2012.1249.

Safety. Fourth Edition – APRIL 2011.

2010;27:243–249. Doi: 10.1016/j.fm.2009.10.009. Epub 2009 Oct 21.

272 Current Topics in Salmonella and Salmonellosis


[69] Hitoshi I., Harun Or R., Naruemon S., Pitaya A., Pongpen R., Isao I.: Effect of gammairradiation on frozen shrimps for decontamination of pathogenic bacteria. Radiation

[70] Sinanoglou V.J., Batrinou A., Konteles S., Sflomos K.: Microbial population, physicochemical quality, and allergenicity of molluscs and shrimp treated with cobalt-60

[71] Sommers C.H., Rajkowski K.T., Sheen S., Samer C., Bender E.: The effect of cryogenic freezing followed by gamma radiation on the survival of *Salmonella* spp. on frozen shrimp. Journal of Food Processing & Technology2011;S8. http://dx.doi.org/10.4172/2157-

[72] Nerkar D.P., Bandekar J.R.: Elimination of *Salmonella* from frozen shrimp by gamma

[73] Luo X., Cui S., Li Y., Jiang T.: A Study On The Use Of Irradiation In Combination With Vacuum Packaging To Produce Non-Sterile Shelf-Stable Food, Semi-Dried Meat, Seafood And Vegetables. In: Radiation Processing For Safe, Shelf-Stable And Ready-To-Eat Food. Proceedings of a final Research Co-ordination Meeting held in Montreal, Canada, 10–14 July 2000. IAEA, VIENNA, 2003 IAEA-TECDOC-1337 ISBN 92–0–100703–5 ISSN 1011–

[74] Tambunan P.R.: Elimination of *Salmonella* in frog legs by irradiation. Final Report to the International Atomic Energy Agency on Contract No 2711/R1/RB. Central Research

[75] Federal Register: Irradiation in the Production, Processing and Handling of Food, A Rule by the Food and Drug Administration on 11/30/2012 Fed Regist. 2012 Nov

[76] Farkas J.: Irradiation of poultry meat. In: Food Safety Control in the Poultry Industry. G.

Physics and Chemistry 1993;42:279–282. DOI: 10.1016/0969-806X(93)90249-T.

gamma radiation. Journal of Food Protection. 2007;70:958–966.

radiation. Journal of Food Safety, 1990;10(3):175–180.

Institute for Fisheries, Indonesia. 1984.

7110.S8-001

274 Current Topics in Salmonella and Salmonellosis

4289, p.194-198.

30;77(231):71312–71316.

Mead. Cambridge, 2005:433–452.

## *Edited by Mihai Mares*

The genus Salmonella comprises an important number of bacterial species able to colonize and infect numerous animal species and humans. Although more than a hundred years passed since its discovery, Salmonella still represents a redoubtable and successful microorganism, difficult to deal with. Whether we discuss about typhoid fever or food poisoning, the public health and financial consequences are practically incalculable. The costs attributable to Salmonella contamination of meat, eggs, and vegetables are also very high worldwide. Antimicrobial resistance in Salmonella isolates is an emerging threat not only in humans, and special measures should be addressed to this global problem. The book Current Topics in Salmonella and Salmonellosis contains a series of reviews about all-important issues concerning these subjects. It comprises 14 chapters grouped in 4 sections emphasizing new insights into pathogenesis, bacterial detection and antibiotic resistance, infections in animals, risk factors, and control strategies. The new genomic data and the exhaustive presentation

of molecular pathogenesis bring novelty to the book and can help to improve our knowledge about Salmonella-induced diseases.

Current Topics in Salmonella and Salmonellosis

Current Topics in Salmonella

and Salmonellosis

*Edited by Mihai Mares*

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