**2. Methodology**

The environmental investigation of *Salmonella* spp. was carried out in three poultry houses of a commercial farm located in the city of Monte Carmelo, Minas Gerais, Brazil, destined for the confinement of poultry of the species G. gallus, suitable for meat production. The evaluated poultry houses had a history of *Salmonella* Gallinarum isolation in a previously housed flock and, because of that, were previously submitted to the sanitary emptying technique for 149 days, after the sanitary management of washing and disinfection. The determination of the sample amount was performed as recommended by the Normative Instruction No. 78 of November 3, 2003 [12], with some modifications. With the aid of 80 sterile swabs randomly numbered from 1 to 80, zigzag drags were made over the floor, feeders, feed boxes, fans, misters, drinking fountains, meshes, and roof structure. Concomitantly to the collections, the samples were placed in individual sterile flasks, which were placed in refrigerated Styrofoam boxes. The sample collection procedures were performed aseptically, using personal protection equipment to avoid contamination. After collection, the samples were transported under refrigeration to the Microbiology Laboratory from the Lutheran University of Brazil, where they were stored at a temperature between 2 and 8°C until bacteriological processing.

To perform the diagnosis through bacterial isolation, the collected samples were submitted to isolation in broths and culture means highly selective for *Salmonella* spp., following the recommendations of Ordinance No. 126 of the Ministry of Agriculture, Livestock and Food Supply (MAPA) of November 3, 1995 [13], and also according to Silva's description [14]. Initially, the stage of the bacteriological

analyses consisted of the infusion of the swabs samples in a non-selective enrichment broth. The swabs were inoculated into 20 mL of BHI broth, followed by incubation at the temperature of 35 to 37°C for 18 to 24 hours. At the end of the incubation in BHI broth, 2 mL of each sample in non-selective broth was inoculated in 20 mL of Tetrathionate broth and in 20 mL of Rappaport Vassiliadis broth, where they were incubated at a temperature of 42 to 43°C for 18 to 24 hours. For isolation, MacConkey and Brilliant Green agars were used. With the aid of a flamed platinum loop, samples in selective enrichment broths were streaked until depleted onto two plates, one containing Bright Green agar and the other containing MacConkey agar, followed by incubation at a temperature of 35 to 37°C for 24 hours. At the end of the plate incubation, the reading of the plates proceeded to observe the growth of colonies. On the MacConkey agar, colorless colonies were considered suggestive of *Salmonella* spp. while those that appeared rosy on the Brilliant Green agar were considered suggestive of bacteria belonging to the *Salmonella* genus. In addition to the research, an epidemiological inquiry was prepared according to Bannow [15], with some modifications, to survey the sanitary history of the farm and the sanitary conditions of washing and disinfection management. The epidemiological investigation was applied to the farm owner responsible for the production process, in form of a questionnaire.

## **3. Results and discussion**

In the bacteriological isolation on plates, out of 80 samples of drag swabs submitted to diagnosis by plating, 52 samples showed growth of colonies with phenotypic characteristics suggestive of *Salmonella* spp. On plates containing MacConkey agar, there was a growth of colorless, plain, and circular colonies. Whereas on the Bright Green agar, the growth of isolated rosy, plain, and circular colonies occurred in the totality of positive samples. Therefore, when comparing the colonies obtained with the guidelines of Ordinance No. 126 of November 3, 1995 [13], 65% of the samples of drag swabs showed a growth suggestive of *Salmonella* spp. in the development of bacteriological diagnosis.

It is important to consider that the quantity of samples of drag swabs used in this study was defined based on Normative Instruction no. 78 from November 3, 2003 [12], with some modifications. The legislation in question recommends the use of 100 samples of swabs; however, only 80 were submitted to bacterial isolation. The modification does not seem to have influenced the bacteriological diagnosis results.

The isolation of *Salmonella* spp. from the poultry environment is representative of the risk of disease incidence that may result in financial losses related to industrial poultry production in Brazil, besides exerting an impact on the collective health of animals and humans [16]. In Brazil, these factors led the agency responsible for poultry health to establish the National Poultry Health Program and Ordinance 193 [17], which establishes standards for the prevention and control of *Salmonella* spp. in poultry and poultry products for human consumption [13]. Besides, Normative Instruction No. 50 of September 24, 2013, states that salmonellosis caused by *Salmonella* Enteritidis, *Salmonella* Gallinarum, *Salmonella* Pullorum, and *Salmonella* Typhimurium, when laboratorially confirmed, must be compulsorily reported to the official animal health service [18].

From a sanitary point of view, Payment and Riley [19] recommend that the longer the fallow period, the better the effectiveness of disinfection protocols. They also point out that the processes of cleaning and disinfection of poultry houses

### *Effect of Fallowing on the Viability of* Salmonella *spp. in Poultry Facilities DOI: http://dx.doi.org/10.5772/intechopen.110291*

associated with fallowing between flocks have shown to be extremely efficient in reducing the environmental persistence of *Salmonella* spp. Thus, when evaluating the epidemiological inquiry conducted in this research, it is important to stress that the fallow period applied and analyzed in this study was 149 days, and although it was longer than 4 months, it was still not enough to eliminate the pathogen from the environment.

Naturally, *Salmonella* species are eliminated in large numbers from the gastrointestinal tract of the infected birds and can remain in the fecal material for long periods, contaminating soil and water [20]. In particular, *Salmonella* spp. persist for more than 28 months in dry feces and dust. The fallow period evaluated in the present research was 149 days, and it is important to consider the notes by Gast [21], who states that the survival time of *Salmonella* spp. in chicken feces can be 9 days in the environment and that in the soil it can remain for up to 280 days.

According to Jaenisch [9], fallowing is a period that extends from the process of cleaning and disinfection of poultry facilities until the housing of the subsequent flock. The authors also state that the fallow period should be applied in a complementary way to the procedures for cleaning and disinfection of the facilities in order to enhance them, being determinant to achieve success in disinfection processes, especially the technical protocols related to the elimination of *Salmonella* spp. from poultry production farms. Furthermore, it becomes important to point out that the investigation carried out, through the application of an epidemiological inquiry and bacterial plating, revealed that the fallowing technique applied to three poultry farms with a history of *Salmonella* Gallinarum isolation was not effective, according to the bacteriological results.

The results obtained from the epidemiological inquiry presented important information related to the *Salmonella* Gallinarum transmission chain in broiler flocks. Based on the anamnesis performed with the owner of the poultry unit, the outbreak of Avian Typhoid was diagnosed on April sixteenth of the year 2014 and affected a flock of 33,000 birds housed in the same poultry houses where the drag swabs were collected in this research. As also described by Silva [14], the flock diagnosed with *Salmonella* Gallinarum was affected by 80% mortality and 60% morbidity, whose birds showed only clinical signs of inappetence and prostration on the bedding. At the time, all flocks housed on the farm were also affected and diagnosed with *Salmonella* Gallinarum. The flock diagnosed with *Salmonella* Gallinarum, as well as all other flocks diagnosed, was sacrificed and incinerated, depopulating all farm facilities. After the flocks were disposed of, the bedding from all the poultry houses underwent disinfection by fermentation or windrowing before being sent to the landfill in ditches. Similarly, the remaining feed was sent to the landfill along with the bedding.

It becomes relevant to point out that in the poultry houses with birds primodiagnosed with *Salmonella* Gallinarum, where the drag swabs were performed for the present study, a fallowing of 149 days counted from the end of the disinfection of the facilities was applied. According to Salle and Silva [22], the washing and disinfection of poultry houses and facilities are necessary for an effective control of pathogenic microorganisms and cannot be done randomly or irrationally, but with a scientific basis of knowledge. Thus, sanitation and disinfection measures were adopted in all poultry houses after the disposal of bedding and feed. For this purpose, the houses were swept and mechanically scraped to remove the remains of organic matter, such as remains of bedding, feces, and encrusted feed. Consequently, the facilities and equipment were thoroughly washed with high-pressure water, starting from the upper and ending with the lower portions of the facilities. The procedure was

repeated using water and detergent, and the action was waited for 30 minutes, followed by a third high-pressure wash. After the washing, the facilities and equipment were disinfected only once with Farmasept® Plus, whose chemical base consists of glutaraldehyde and benzalkonium chloride, at a dilution of 1.0 milliliter to 1.0 liter of water. After drying, hydrated lime was applied to the floor in the proportion of 500 g/m<sup>2</sup> of the premises. Workers' clothing and technical recording instruments were incinerated.

In this way, it is important to choose disinfectants that are ideal and compatible with the needs, taking into account the type of microorganism that one intends to control, the place and the object to be disinfected. For the disinfection of the poultry houses analyzed in this study, Farmasept® Plus was the disinfectant used in the recommended dosage in the datasheet of the laboratory producer. Sesti [11] points out that the chemical bases of these disinfectants are indicated for the elimination of bacteria of the *Salmonella* genus. Consequently, the results of the bacteriological exams suggest that the fallowing process applied in association with the hygiene and disinfection process, up to the moment of the bacteriological analyses, was not effective in eliminating *Salmonella* Gallinarum from the installations and equipment, despite configuring itself as a prophylaxis technique to enhance the cleaning and disinfection processes applied in the environment.

Bannow [15] points out that the epidemiological investigation by inquiry in poultry production units is essential, for this practice has the purpose of performing an epidemiological triage pertinent to salmonellosis that affects poultry production and that can also affect public health. Through the epidemiological inquiry tool, it was observed that the birds affected by *Salmonella* Gallinarum did not present clinical signs suggesting infection by this pathogen. According to Oliveira [23], in general, birds affected by salmonellosis have mortality above the standard for the strain, presenting diarrhea, ruffled feathers, fallen wings, and dyspnea, in addition to depression and anorexia. However, Paiva [24] points out that infection by *Salmonella* spp. can develop asymptomatically and sources of infection can become lifelong carriers. Also, by means of an epidemiological inquiry carried out on a farm suitable for broiler production, they found that a batch of infected birds did not present clinical signs suggestive of infection by *Salmonella* spp. [14]. Furthermore, according to Miranda [25] and Silva [14], the absence of *Salmonella* spp. isolation in plating may be associated with the high sanitary control that is applied to national poultry farms, as evidenced by the epidemiological inquiry in a poultry production unit studied.

The clinical manifestations of Avian Typhoid are usually observed in the adult stage of the host [23]. At this age, the birds show somnolence, with prostration, anorexia, diarrhea with yellow to greenish coloration, and a drop in egg laying, evolving to death in a few days. However, Bannow [15] describes the clinical occurrence of Avian Typhoid with isolation of *Salmonella* Gallinarum in two-week-old birds. In this way, it is important to consider that 35-day-old birds presented scientific epidemiological evidence that supports the research of *Salmonella* Gallinarum in young birds, even if they do not present clinical disease [26]. In this study, the epidemiological investigation also revealed that the researched farm is concerned with the health of its birds, which can be verified by noting that the farm uses a single-age housing system, controls the flow of vehicles and visitors, requires a bath for access to the facilities, has technical protocols for cleaning and disinfection of the aviaries, adopts fallowing between flocks, and uses good quality feed and chlorinated water for bird consumption. Moreover, it is possible to observe that the farm was built in a region with low poultry density.

### *Effect of Fallowing on the Viability of* Salmonella *spp. in Poultry Facilities DOI: http://dx.doi.org/10.5772/intechopen.110291*

According to Fernandes [7], the lack of hygiene involving the environment and facilities favors the approach of synanthropic animals, such as flies, birds, vultures, and rodents, which may contribute to dispersing *Salmonella* Gallinarum throughout poultry farms. The authors also recommend the use of bacterial control methods, such as composting and dug and impermeable tanks for manure fermentation, to prevent the spread of *Salmonella* spp. They also highlight that the means of transporting poultry, manure, and eggs are configured as efficient ways to disperse the bacteria, especially when the vehicles enter production units without proper prior hygiene and disinfection. Furthermore, workers who move around poultry farms can act as a means of transmission of the agent of Avian Typhoid. They also orient that the creation of poultry flocks with multiple ages should also be avoided in a productive process. Moreover, according to Gast [21], vertical transmission should be an imminent and constant concern to avoid the introduction of *Salmonella* spp. in farms. Studies also point out that any negligence in the biosecurity program can lead to intrauterine transmission to the progeny. Therefore, infection-free flocks depend on specific and efficient prophylaxis measures to control, prevent, and eradicate *Salmonella* spp. through sanitary programs, which reflect directly on the health of the birds [27, 28].

Studies carried out by Silva [14] for the control of *Salmonella* spp. indicated that fallowing, applied as a prophylactic measure in poultry facilities with a history of isolation of *Salmonella* Gallinarum, configures itself as an effective technique for the elimination of this pathogen. In addition, they highlighted that the epidemiological inquiry was an efficient and recommended tool to investigate the epidemiological relations of the etiologic agent with the host and the poultry production environment. In counterpart to the present research, Silva [14] evaluated the effect of fallowing for a much superior period than the one considered in this work, which allows us to infer that longitudinal bacteriological studies are extremely important for the employment of epidemiology in poultry production.
