**2.3 Vaccine trials against** *Streptococcus uberis*

*S. uberis* is ubiquitous in the cow's environment accounting for a significant number of mastitis cases. It is found on-farm in water, soil, plant material, bedding, flies, hay, and feces [141]. As such, *S. uberis* is remarkably adaptable, affecting lactating and dry cows, heifers, and multiparous cows, causing clinical or subclinical mastitis, and even being responsible for persistent colonization without an elevation in the somatic cell count [142, 143]. It has been described as an environmental pathogen [108, 144–146] with potential as a contagious pathogen [142, 143, 147]. *S. uberis* has ability to persist within the mammary gland which lead to chronic mastitis that is difficult to treat [148]. Coliform bacteria are a major cause of clinical mastitis [149, 150]. A vaccine that prevents *S. uberis* mastitis is not available, control measures are limited to the implementation of good management practices. Recently vaccine efficacy trial with extract of biofilm-forming strains of *S. uberis* (UBAC®) (Hipra, Amir, Spain), was reported to reduce clinical severity [84]. It is not clear what kind of adative immunity is induced by UBAC® *S. uberis* vaccine [84] and it only conferred partial reduction in clinical severity of mastitis. Multiple intramammary vaccinations of dairy cows with killed *S. uberis* cells resulted in the complete protection from experimental infection with the homologous strain [103]. Similarly, subcutaneous vaccination of dairy cows with live *S. uberis* followed by intramammary booster vaccination with *S. uberis* cell surface extract protected against challenge with the homologous strain but was less effective against a heterologous strain [106]. Vaccination with *S. uberis* glyceraldehyde phosphate dehydrogenase C (GapC) protein induced immune responses that confer a significant reduction in inflammation post-challenge [107, 151]. The pauA is a plasminogen activator and also binds active protease plasmin [152]. It has been postulated that acquisition of plasmin may promote invasion [153]. Vaccination of dairy cows with PauA induced increased antibody titers that conferred reduction in clinical severity [154]. However, mutation of pauA did not alter ability to grow in milk or to infect lactating bovine mammary glands. It appears that the ability to activate plasminogen through PauA does not play a major role in pathogenesis of *S. uberis* to either grow in milk or infect bovine mammary gland [155].

*S. uberis* expresses several surface associated proteins such as *S. uberis* adhesion molecule (SUAM) and extracellular matrix binding proteins, which allow it to adhere to and internalize into mammary epithelial cells, successfully inducing IMI [156–158]. The *S. uberis* adhesion molecule (SUAM) plays a central role in the adherence of *S. uberis* to mammary epithelial cells [159–162]. Vaccination of dairy cows with SUAM induced strong immune resposes in vaccinated cows [163].

**195**

*Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major…*

The immune serum from SUAM vaccinated cows prevented *S. uberis* adhesion and invasion into mammary epithelial cells *in vitro* [163]. In vivo infusion of mammary quarters of dairy cows with *S. uberis* pre-incubated with immuneserum from SUAM vaccinated cows reduced clinical severity [164]. The SUAM gene deletion mutant strain is less pathogenic to mammary epithelial cells [165] and to dairy cows [159]. Controlled experimental efficacy studies using SUAM as vaccine antigen to control *S. uberis* mastitis showed that SUAM is immunogenic but the induced immunity was not protective. Following experimental IMI challenge with *S. uberis*, clinical signs emerged at about 48 h, along with increased levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8 in milk at 60 h post-infection [166]. Adaptive immune response cytokines such as IFN-γ promotes a cell-mediated immune response by enhancing functions such as macrophage bacterial killing, antigen presentation, cytotoxic T cell activation, and increased IgG2 levels. The IL-4 expression is associated with the antibodymediated response, which is generally linked to parasite resistance, allergic reactions, and increased levels of IgG1 [167, 168]. This partial protection by the SUSP vaccine can be improved with dose optimization, appropriate adjuvant, route of

In conclusion, it is clear that Bacterin vaccines have some protective effect against homologous strains, and single surface protein is not effective. Therefore; use of multiple surface proteins may induce better immunity that prevents clinical

Coliform bacteria are a major cause of clinical mastitis [149, 150]. Coliforms include the genera *Escherichia, Klebsiella*, and *Enterobacter* [169]. Eighty to ninety percent of coliform intramammary infection (IMI) develop clinical mastitis, and 10% will be severe and could lead to death [150]. *E. coli* usually infects the mammary glands during the dry period and progresses to inflammation and clinical mastitis during the early lactation with local and sometimes severe systemic clinical

Iron is an essential nutrient for the growth of coliforms [170]. However, free iron is limited in the bovine milk because most iron is bound to citrate and to a lesser extent to lactoferrin, transferrin, xanthine oxidase, and some caseins [171] and maintained at concentrations below levels required to support coliform growth [172]. To overcome this limited iron source, coliforms express multiple iron transport systems [173], which include synthesis of siderophores (e.g., enterobactin, aerobactin, ferrichrome) that bind iron with high affinity [174], the expression of iron-regulated outer membrane proteins (IROMP) that binds to ferric siderophore complexes to transport into bacterial cell and enzymes to utlize the chelated iron [173]. The siderophores are too large (600 to 1200 Da) to pass through the porin channels of the bacterial outer membrane [175, 176]. Therefore, the siderophores require specific IROMP to enable their passage across the bacterial outer membrane into the periplasm [177, 178]. The enterobactin is a siderophore with the highest affinity for iron, and it is produced by most pathogenic *E. coli* and *Klebsiella* spp. [179–181]. The aerobactin is another siderophore that was detected in only 12% of *E. coli* isolated from mastitis cases [182]. Enterobactin is the primary siderophore of *Escherichia coli* and many other Gram-negative bacteria [183]. Coliform bacteria also developed the ability to take up iron directly from naturally occurring organic iron-binding acids, including citrate [173, 184]. The citrate iron uptake system requires ferric dicitrate for induction [184]. More than 0.1 mM citrate is required for the induction of this system under iron-restricted conditions [184]. The ferric

*DOI: http://dx.doi.org/10.5772/intechopen.94227*

injection, and timing of vaccination.

**2.4 Vaccine trials against** *E. coli* **mastitis**

disease and production losses.

manifestations.

*Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major… DOI: http://dx.doi.org/10.5772/intechopen.94227*

The immune serum from SUAM vaccinated cows prevented *S. uberis* adhesion and invasion into mammary epithelial cells *in vitro* [163]. In vivo infusion of mammary quarters of dairy cows with *S. uberis* pre-incubated with immuneserum from SUAM vaccinated cows reduced clinical severity [164]. The SUAM gene deletion mutant strain is less pathogenic to mammary epithelial cells [165] and to dairy cows [159]. Controlled experimental efficacy studies using SUAM as vaccine antigen to control *S. uberis* mastitis showed that SUAM is immunogenic but the induced immunity was not protective. Following experimental IMI challenge with *S. uberis*, clinical signs emerged at about 48 h, along with increased levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8 in milk at 60 h post-infection [166]. Adaptive immune response cytokines such as IFN-γ promotes a cell-mediated immune response by enhancing functions such as macrophage bacterial killing, antigen presentation, cytotoxic T cell activation, and increased IgG2 levels. The IL-4 expression is associated with the antibodymediated response, which is generally linked to parasite resistance, allergic reactions, and increased levels of IgG1 [167, 168]. This partial protection by the SUSP vaccine can be improved with dose optimization, appropriate adjuvant, route of injection, and timing of vaccination.

In conclusion, it is clear that Bacterin vaccines have some protective effect against homologous strains, and single surface protein is not effective. Therefore; use of multiple surface proteins may induce better immunity that prevents clinical disease and production losses.

#### **2.4 Vaccine trials against** *E. coli* **mastitis**

Coliform bacteria are a major cause of clinical mastitis [149, 150]. Coliforms include the genera *Escherichia, Klebsiella*, and *Enterobacter* [169]. Eighty to ninety percent of coliform intramammary infection (IMI) develop clinical mastitis, and 10% will be severe and could lead to death [150]. *E. coli* usually infects the mammary glands during the dry period and progresses to inflammation and clinical mastitis during the early lactation with local and sometimes severe systemic clinical manifestations.

Iron is an essential nutrient for the growth of coliforms [170]. However, free iron is limited in the bovine milk because most iron is bound to citrate and to a lesser extent to lactoferrin, transferrin, xanthine oxidase, and some caseins [171] and maintained at concentrations below levels required to support coliform growth [172]. To overcome this limited iron source, coliforms express multiple iron transport systems [173], which include synthesis of siderophores (e.g., enterobactin, aerobactin, ferrichrome) that bind iron with high affinity [174], the expression of iron-regulated outer membrane proteins (IROMP) that binds to ferric siderophore complexes to transport into bacterial cell and enzymes to utlize the chelated iron [173]. The siderophores are too large (600 to 1200 Da) to pass through the porin channels of the bacterial outer membrane [175, 176]. Therefore, the siderophores require specific IROMP to enable their passage across the bacterial outer membrane into the periplasm [177, 178]. The enterobactin is a siderophore with the highest affinity for iron, and it is produced by most pathogenic *E. coli* and *Klebsiella* spp. [179–181]. The aerobactin is another siderophore that was detected in only 12% of *E. coli* isolated from mastitis cases [182]. Enterobactin is the primary siderophore of *Escherichia coli* and many other Gram-negative bacteria [183]. Coliform bacteria also developed the ability to take up iron directly from naturally occurring organic iron-binding acids, including citrate [173, 184]. The citrate iron uptake system requires ferric dicitrate for induction [184]. More than 0.1 mM citrate is required for the induction of this system under iron-restricted conditions [184]. The ferric

*Animal Reproduction in Veterinary Medicine*

incidence of mastitis in dairy cows.

**2.3 Vaccine trials against** *Streptococcus uberis*

of most mastitis-causing bacteria to attach and internalize into mammary epithelial cells. Furthermore, evaluation of mastitis vaccines is complicated by the absence of uniform challenge study models, and lack of uniform route(s) of vaccination, time of vaccination, adjuvants, and challenge dose. There is an increasing need for development of better vaccines that overcome these problems. Most mastitis vaccines are killed whole bacterial cells (Bacterin) vaccines [75, 77, 80, 88, 89, 91–95, 97–99] that are difficult to improve because of difficulty to specifically identify an immunogenic component that induced partial or some protective effect. In this regard, some of the current efforts to use a mixture of purified surface proteins as vaccine antigens [100] to induce immunity than killed whole bacterial cells (Bacterin) is encouraging. A better understanding of natural and acquired immunological defenses of the mammary gland coupled with detailed knowledge of the pathogenesis of each mammary pathogen should lead to the development of improved methods of reducing the

*S. uberis* is ubiquitous in the cow's environment accounting for a significant

number of mastitis cases. It is found on-farm in water, soil, plant material, bedding, flies, hay, and feces [141]. As such, *S. uberis* is remarkably adaptable, affecting lactating and dry cows, heifers, and multiparous cows, causing clinical or subclinical mastitis, and even being responsible for persistent colonization without an elevation in the somatic cell count [142, 143]. It has been described as an environmental pathogen [108, 144–146] with potential as a contagious pathogen [142, 143, 147]. *S. uberis* has ability to persist within the mammary gland which lead to chronic mastitis that is difficult to treat [148]. Coliform bacteria are a major cause of clinical mastitis [149, 150]. A vaccine that prevents *S. uberis* mastitis is not available, control measures are limited to the implementation of good management practices. Recently vaccine efficacy trial with extract of biofilm-forming strains of *S. uberis* (UBAC®) (Hipra, Amir, Spain), was reported to reduce clinical severity [84]. It is not clear what kind of adative immunity is induced by UBAC® *S. uberis* vaccine [84] and it only conferred partial reduction in clinical severity of mastitis. Multiple intramammary vaccinations of dairy cows with killed *S. uberis* cells resulted in the complete protection from experimental infection with the homologous strain [103]. Similarly, subcutaneous vaccination of dairy cows with live *S. uberis* followed by intramammary booster vaccination with *S. uberis* cell surface extract protected against challenge with the homologous strain but was less effective against a heterologous strain [106]. Vaccination with *S. uberis* glyceraldehyde phosphate dehydrogenase C (GapC) protein induced immune responses that confer a significant reduction in inflammation post-challenge [107, 151]. The pauA is a plasminogen activator and also binds active protease plasmin [152]. It has been postulated that acquisition of plasmin may promote invasion [153]. Vaccination of dairy cows with PauA induced increased antibody titers that conferred reduction in clinical severity [154]. However, mutation of pauA did not alter ability to grow in milk or to infect lactating bovine mammary glands. It appears that the ability to activate plasminogen through PauA does not play a major role in pathogenesis of

*S. uberis* to either grow in milk or infect bovine mammary gland [155].

*S. uberis* expresses several surface associated proteins such as *S. uberis* adhesion molecule (SUAM) and extracellular matrix binding proteins, which allow it to adhere to and internalize into mammary epithelial cells, successfully inducing IMI [156–158]. The *S. uberis* adhesion molecule (SUAM) plays a central role in the adherence of *S. uberis* to mammary epithelial cells [159–162]. Vaccination of dairy cows with SUAM induced strong immune resposes in vaccinated cows [163].

**194**

citrate transport system is the major iron acquisition system utilized by *E. coli* [173] to grow in the mammary gland. The mammary gland is an iron-restricted environment, and bovine milk contains approximately 7 mM citrate [185] which is ideal for induction of ferric citrate transport sytem.

Ferric enterobactin receptor, FepA, is an 81 kDa iron regulated outer membrane protein (IROMP), that binds to ferric enterobactin complex to transoport iron into the bacterial cell [186, 187]. Vaccination of dairy cows with FepA elicited an increased immunological response in serum and milk [188]. Bovine IgG directed against FepA inhibited the growth of coliform bacteria by interfering with the binding of the ferric enterobactin complex [189]. Ferric citrate receptor, FecA, is an 80.5-kDa IROMP that is responsible for the binding of ferric dicitrate [190] and transport into the bacterial cell. The FecA, is conserved among coliforms isolated from cases of naturally occurring mastitis [191]. The iron-regulated outer membrane proteins, FepA and FecA are ideal vaccine candidates because they are surface exposed, antigenic, and conserved among isolates from IMI.

Immunization of dairy cows with FepA induced significantly higher serum and whey anti-FepA IgG titers than in *E. coli* J5 vaccinates [188]. Results of *in vitro* growth inhibition studies demonstrated that antibody specific for blocking ferric enterobactin-binding site (anti-FepA) inhibited the growth of *E. coli* in vitro [192]. Cows immunized with FecA did have increased antibody titers in serum and mammary secretions compared with *E. coli* J5 immunization and unimmunized control cows [193, 194]. Antibody purified from colostrum inhibited the growth of *E. coli* when cultured in synthetic media modified to induce FecA expression [193]. Despite their antigenicity, the use of either FepA or FecA alone were not sufficient to prevent mastitis. The FecA and FepA are antigenically distinct [191].

Intramammary infection with *E. coli* induced expression and release of proinflammatory cytokines such as TNF-alpha, IL-8, IL-6, and IL-1 [195, 196]. Recently it has been shown with mouse mastitis models that IL-17A and Th17 cells are instrumental in the defense against *E. coli* IMI [197, 198]. However, the role of IL-17 in bovine *E. coli* mastitis is not well defined. Results of a recent vaccine efficacy study against *E. coli* mastitis suggested that cell-mediated immune response has more protective effect than humoral response [199]. The cytokine signaling pathways that lead to efficient bacterial clearance is not clearly defined.

The four coliform vaccines which include 1) J-5 Bacterin® (Zoetis, Kalamazoo, MI) [82, 83], 2) Mastiguard®, 3) J Vac® (Merial-Boehringer Ingelheim vet medical, Inc., Duluth, GA) and 4) Endovac-bovi® (IMMVAC) (Endovac Animal Health, Columbia, MO). Of the four coliform vaccines, J-5 Bacterin® and Mastiguard® are believed to have the same component, which is J5 Bacterin. The J Vac® is a different bacterin-toxoid. The Endovac-Bovi® contains mutant *Salmonella typhimurium* bacterin toxoid. All coliform mastitis vaccine formulations use gram-negative core antigens to produce non-specific immunity directed against endotoxin (LPS) [119]. The efficacy of these vaccines has been demonstrated in both experimental challenge trials and field trials in commercial dairy herds [109–111]. The principle of these bacterins is based upon their ability to stimulate the production of antibodies directed against common core antigens that gram-negative bacteria share. These vaccines are considered efficacious even though the rate of intramammary infection is not significantly reduced in vaccinated animals because they significantly reduce the clinical effects of the infection. Experimental challenge studies have demonstrated that J5 vaccines are able to reduce bacterial counts in milk and result in fewer clinical symptoms [109]. Vaccinated cows may become infected with gramnegative mastitis pathogens at the same rate as control animals but have a lower rate of development of clinical mastitis [111], reduced the duration of IMI [110], reduced production, culling, and death losses [200, 201].

**197**

**Author details**

Oudessa Kerro Dego

Tennessee, Knoxville, TN, USA

\*Address all correspondence to: okerrode@utk.edu

provided the original work is properly cited.

*Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major…*

There is an increasing need for the development of effective vaccines against major bacterial bovine mastitis pathogens. A better understanding of the natural and acquired immunological defenses of the mammary gland coupled with detailed knowledge of the pathogenesis of each mammary pathogen should lead to the development of improved methods of reducing the incidence of mastitis in dairy cows (**Table 1**).

Department of Animal Science, Institute of Agriculture, The University of

© 2020 The Author(s). Licensee IntechOpen. 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,

*DOI: http://dx.doi.org/10.5772/intechopen.94227*

*Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major… DOI: http://dx.doi.org/10.5772/intechopen.94227*

There is an increasing need for the development of effective vaccines against major bacterial bovine mastitis pathogens. A better understanding of the natural and acquired immunological defenses of the mammary gland coupled with detailed knowledge of the pathogenesis of each mammary pathogen should lead to the development of improved methods of reducing the incidence of mastitis in dairy cows (**Table 1**).
