**2. Prospects for effective vaccines against major bacterial mastitis pathogens**

Despite decades of research to develop effective vaccines against major bacterial bovine mastitis pathogens such as *Staphylococcus aureus*, *Streptococcus uberis,* and *E. coli*, the effective intramammary immune mechanism is still poorly understood, perpetuating reliance on antibiotic therapies to control mastitis in dairy cows. Dependence on antimicrobials is not sustainable because of their limited efficacy [46, 47] and increased risk of emergence of antimicrobial-resistant bacteria that pose serious public health threats [4, 72–74]. Neither of the two currently available commercial Bacterin vaccines against *S. aureus* (**Table 1**), Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the USA and Startvac® (Hipra, Girona, Spain) in Europe and other countries, confer protection from new intramammary infection under field trials as well as under controlled experimental challenge studies [75–81].

There are four commercial vaccines against *E. coli* mastitis which include 1) the Eviracor®J5 (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) (**Table 1**). The Endovac-bovi® is a cross-protective vaccine made of genetically engineered R/17 mutant strain of *Salmonella typhimurium* and the core somatic antigen mutant J-5 strain of *E. coli* combined with an immune-potentiating adjuvant (IMMUNEPlus®). Endovac-bovi significantly reduces diseases caused by Gramnegative bacteria producing various endotoxins and protects against *E. coli* mastitis and other endotoxin-mediated diseases caused by *E. coli*, *Salmonella*, *Pasteurella multocida*, and *Mannheimia hemolytica*. The UBAC® (Hipra, Amir, Spain) [84] is a recently developed vaccine against *S. uberis* mastitis with label claim of partial reduction in clinical severity of *S. uberis* mastitis.


**189**

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

autologous vaccine compared with Startvac®

encapsulated in biodegradable microspheres

Bacterin from two strains (α and α + β hemolytic) plus supernatants from non-hemolytic strain

MASTIVAC I Whole-cell lysate Improved udder health

Live pathogenic *S.* 

DNA primed and protein boosted

Bacterin + antibiotic

vaccine containing CP types 5, 8 and 336 with FIA or Alum adjuvants

therapy

Whole-cell lysate Whole-cell trivalent

*aureus*

**Vaccine Vaccine component Protective effect Reference**

Both vaccines decreased herd prevalence of *S. aureus* mastitis but no other differences in terms of improvement of udder

Induced antibodies that were more opsonic for neutrophils and inhibited adhesion to mammary epithelium.

Vaccinated cows had 70% protection from infection compared to less than 10% protection in control

in addition to specific protection against *S. aureus* infection

Induce activation of immune cells in mammary gland and

Induced cellular and humoral immune responses that provide partial protection against *S. aureus*

and cellular immune

and cellular immune

cases of chronic intramammary *S. aureus* infections

clinical severity and duration of clinical disease. None of the experimental Bacterins has significant effects

responses

responses

*S. aureus* intramammary infection cure rate increased

Elicited antibody responses specific to the 3 capsular polysaccharides

[78]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[88]

[80]

[89]

[98]

health

cows

blood

DNA Induced humoral

DNA Induced humoral

Bacterin Eliminated some

Bacterin Lysigin reduced the

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

**Experimental**

Whole-cell lysate from two strains

Live pathogenic *S. aureus* through IM

route

Fibronectin binding protein and clumping factor A

Protein A of *S. aureus* with the green fluorescent protein

Plasmid encoding bacterial antigen

Polyvalent *S. aureus*

Lysigin® with three-isolates based experimental Bacterin

Polyvalent *S. aureus*

Bacterin

β-gal

Bacterin

Bestvac® Vs Startvac herd-specific

Whole-cell lysate Bacterin

*Mastitis Pathogen*


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

*Animal Reproduction in Veterinary Medicine*

the antibiotic to eliminate the pathogen.

reduction in clinical severity of *S. uberis* mastitis.

*S. aureus* Lysigin® Bacterin: Somatic

Startvac® Bacterin: *E. coli* J5

**Commercial**

**pathogens**

lenge studies [75–81].

*Mastitis Pathogen*

mastitis, determining the susceptibility/resistance of the pathogen, and proper dose, duration, and frequency of treatment to ensure effective concentrations of

**2. Prospects for effective vaccines against major bacterial mastitis** 

Despite decades of research to develop effective vaccines against major bacterial bovine mastitis pathogens such as *Staphylococcus aureus*, *Streptococcus uberis,* and *E. coli*, the effective intramammary immune mechanism is still poorly understood, perpetuating reliance on antibiotic therapies to control mastitis in dairy cows. Dependence on antimicrobials is not sustainable because of their limited efficacy [46, 47] and increased risk of emergence of antimicrobial-resistant bacteria that pose serious public health threats [4, 72–74]. Neither of the two currently available commercial Bacterin vaccines against *S. aureus* (**Table 1**), Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the USA and Startvac® (Hipra, Girona, Spain) in Europe and other countries, confer protection from new intramammary infection under field trials as well as under controlled experimental chal-

There are four commercial vaccines against *E. coli* mastitis which include 1) the Eviracor®J5 (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) (**Table 1**). The Endovac-bovi® is a cross-protective vaccine made of genetically engineered R/17 mutant strain of *Salmonella typhimurium* and the core somatic antigen mutant J-5 strain of *E. coli* combined with an immune-potentiating adjuvant (IMMUNEPlus®). Endovac-bovi significantly reduces diseases caused by Gramnegative bacteria producing various endotoxins and protects against *E. coli* mastitis and other endotoxin-mediated diseases caused by *E. coli*, *Salmonella*, *Pasteurella multocida*, and *Mannheimia hemolytica*. The UBAC® (Hipra, Amir, Spain) [84] is a recently developed vaccine against *S. uberis* mastitis with label claim of partial

**Vaccine Vaccine component Protective effect Reference**

Reduced SCC, clinical mastitis, and chronic

concluded no such

Decreased duration of IMI, transmissibility of *S. aureus,* coliforms,

not associated with a decrease in mastitis

[85–87]

[80, 81, 88–90]

[77]

[75]

IMI

effect

and CNS

antigen containing phage types I, II, III, IV with different strains of *S. aureus*

**" "** Field-based studies

and *S. aureus* CP type 8 with SAAC

" " Use of the vaccine was

**188**


*SAAC: slime associated antigenic complex, SASP: Staphylococcus aureus surface proteins, SCSP: Staphylococcus chromogenes surface proteins, CP: Capsular polysaccharide, GapC: Glyceraldehyde-3-phosphate dehydrogenase C, pauA: plasminogen activator protein, FIA: Freund's incomplete adjuvant, Efb: fibrinogen-binding protein, LukM: leukocidin subunit M.*

**191**

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

Intramammary immunity can be induced locally in the mammary gland or systemically in the body and cross from the body into the mammary glands. Mammary gland pathogen that enters through teat opening interact with host innate defense system primarily with macrophages in the mammary gland. Macrophages recognize invading pathogens through its pattern recognition receptors (PRR) which binds to pathogen associated molecular patterns (PAMPs) and engulf and break down the foreign pathogen into small peptides and load on to MHC-II molecules move to the supramammary lymph nodes and display on its surface to the T cells. Naïve T cells bind with peptide on MHC-II molecule through its T- cell receptor and become activated and start secreting cytokines, which further stimulate B-cells to produce antibodies. Antibody produced by B-cells released into the blood circulation and depending on type of antibody may be released to the site of infection (e.g., IgG) and opsonize the infecting pathogen and subject them to destruction by opsonophagocytic mechanisms. Antibodies may also remain on mucosal surfaces (e.g., IgA) and bind to invading pathogens and prevent them from binding to host cells or

Intramammary infection (IMI) leads to increased somatic cell count in the milk or mammary secretion. Somatic cells are mainly white blood cells such as granulocytes (neutrophils, eosinophils, and basophils), monocytes or macrophages, and lymphocytes, which are recruited to the mammary glands in response to mammary gland infection to fight off infection. A small proportion of mammary epithelial cells that produce milk are also shed through milk and are included in the somatic cell count. So, somatic cells are white blood cells and mammary epithelial cells. Milk somatic cell count (SCC) increases when there is mammary gland infection (IMI) because of an inflammatory response to clear infection. In general, SCC is also an indicator of milk quality [112–116] because if there are few mammary pathogenic bacteria in the gland, the inflammatory response is less, and somatic cells recruitment into the gland is also low and vice versa. Bulk tank milk (BTM) is milk collected from all lactating dairy cows in a farm into a tank or multiple tanks. So BTSCC is somatic cell counts obtained from milk sample collected from a tank. Intramammary infection may progress to clinical or subclinical mastitis [117]. Clinically infected udder usually treated with antimicrobial, whereas subclinically infected udder may not be diagnosed immediately and treated but remained infected and shedding bacteria through milk throughout lactation. The proportion of cure following treatment of mastitis varies and the variation in cure rate is multifactorial including cow factors (age or parity number, stage of lactation, and duration of infection, etc.), management factors (detection and diagnosis of infection and time from detection to treatment, availability of balanced nutrition, sanitation, etc.), factors related to antimicrobial use patterns (type, dose, route, frequency, and duration), and pathogen factors (type, species, number, pathogenicity or virulence,

The dilution of effector humoral immune responses by large volume of milk coupled with the ability of mastitis causing bacteria to develop resistance to antimicrobials makes the control of mastitis very difficult. Therefore, the development of an alternative preventive tool such as a vaccine, which can overcome these limitations, has been a crucial focus of current research to decrease not only the incidence of mastitis but also the use of antimicrobials in dairy cattle farms. Most vaccination strategies against mastitis have focused on the enhancement of humoral immunity. Development of vaccines that induce an effective cellular

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

**2.1 Intramammary immune mechanisms**

tissue and thereby prevent colonization and infection.

resistance to antimicrobial, etc.) [46, 118].

#### **Table 1.**

*Commercialized and experimental vaccines against major bovine mastitis pathogens.*

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

### **2.1 Intramammary immune mechanisms**

*Animal Reproduction in Veterinary Medicine*

CP conjugated to a protein and incorporated in polymicrospheres and emulsified in

Polysaccharideprotein conjugates

Vaccination with Efb and LukM

**Experimental**

Live *S. uberis/* cutaneous route

GapC or chimeric CAMP factor

Coliform **Commercial**

*leukocidin subunit M.*

*E. coli* J5 Mastiguard® J Vac® Endovac-bovi® (IMMVAC)

UBAC® Extract from

Killed bacterial cells Bacterin of *S. uberis*

FIA

in FIA

*S. uberis* **Commercial**

**Vaccine Vaccine component Protective effect Reference**

Polysaccharideprotein conjugate

SASP or SCSP Surface proteins Induced partial

biofilm-forming strains of *S. uberis*

Killed *S. uberis cells* Bacterin Reduced numbers of

and *S. agalactiae*

*SAAC: slime associated antigenic complex, SASP: Staphylococcus aureus surface proteins, SCSP: Staphylococcus chromogenes surface proteins, CP: Capsular polysaccharide, GapC: Glyceraldehyde-3-phosphate dehydrogenase C, pauA: plasminogen activator protein, FIA: Freund's incomplete adjuvant, Efb: fibrinogen-binding protein, LukM:* 

*Commercialized and experimental vaccines against major bovine mastitis pathogens.*

Inactivated Bacterin Bacterin Partial protection [102]

CP types 5, 8 and 336 Cows in both groups

cells

protection

Induced increased titers in serum and milk

Reduce clinical signs, bacterial count, temperature, daily milk yield losses and increased the number of quarters with isolation and somatic cell count <200,000 cells/mL of milk

homologous *S. uberis*

Parenteral vaccination has no effect on streptococcal mastitis

only on the homologous

in milk, duration of IMI and resulted in fewer clinical symptoms

in milk

strain

inflammation

Live *S. uberis* Some protective effect

Bacterin Reduce bacterial counts

Protein Reduction in

PauA protein Partial protection [108]

produced increased concentrations of IgG1, IgG2 antibodies, hyperimmune sera from immunized cows increased phagocytosis, decreased bacterial adherence to epithelial

[99]

[100]

[101]

[84]

[103]

[104, 105]

[106]

[107]

[82, 83, 109–111]

*Mastitis Pathogen*

**190**

**Table 1.**

Intramammary immunity can be induced locally in the mammary gland or systemically in the body and cross from the body into the mammary glands. Mammary gland pathogen that enters through teat opening interact with host innate defense system primarily with macrophages in the mammary gland. Macrophages recognize invading pathogens through its pattern recognition receptors (PRR) which binds to pathogen associated molecular patterns (PAMPs) and engulf and break down the foreign pathogen into small peptides and load on to MHC-II molecules move to the supramammary lymph nodes and display on its surface to the T cells. Naïve T cells bind with peptide on MHC-II molecule through its T- cell receptor and become activated and start secreting cytokines, which further stimulate B-cells to produce antibodies. Antibody produced by B-cells released into the blood circulation and depending on type of antibody may be released to the site of infection (e.g., IgG) and opsonize the infecting pathogen and subject them to destruction by opsonophagocytic mechanisms. Antibodies may also remain on mucosal surfaces (e.g., IgA) and bind to invading pathogens and prevent them from binding to host cells or tissue and thereby prevent colonization and infection.

Intramammary infection (IMI) leads to increased somatic cell count in the milk or mammary secretion. Somatic cells are mainly white blood cells such as granulocytes (neutrophils, eosinophils, and basophils), monocytes or macrophages, and lymphocytes, which are recruited to the mammary glands in response to mammary gland infection to fight off infection. A small proportion of mammary epithelial cells that produce milk are also shed through milk and are included in the somatic cell count. So, somatic cells are white blood cells and mammary epithelial cells. Milk somatic cell count (SCC) increases when there is mammary gland infection (IMI) because of an inflammatory response to clear infection. In general, SCC is also an indicator of milk quality [112–116] because if there are few mammary pathogenic bacteria in the gland, the inflammatory response is less, and somatic cells recruitment into the gland is also low and vice versa. Bulk tank milk (BTM) is milk collected from all lactating dairy cows in a farm into a tank or multiple tanks. So BTSCC is somatic cell counts obtained from milk sample collected from a tank.

Intramammary infection may progress to clinical or subclinical mastitis [117]. Clinically infected udder usually treated with antimicrobial, whereas subclinically infected udder may not be diagnosed immediately and treated but remained infected and shedding bacteria through milk throughout lactation. The proportion of cure following treatment of mastitis varies and the variation in cure rate is multifactorial including cow factors (age or parity number, stage of lactation, and duration of infection, etc.), management factors (detection and diagnosis of infection and time from detection to treatment, availability of balanced nutrition, sanitation, etc.), factors related to antimicrobial use patterns (type, dose, route, frequency, and duration), and pathogen factors (type, species, number, pathogenicity or virulence, resistance to antimicrobial, etc.) [46, 118].

The dilution of effector humoral immune responses by large volume of milk coupled with the ability of mastitis causing bacteria to develop resistance to antimicrobials makes the control of mastitis very difficult. Therefore, the development of an alternative preventive tool such as a vaccine, which can overcome these limitations, has been a crucial focus of current research to decrease not only the incidence of mastitis but also the use of antimicrobials in dairy cattle farms. Most vaccination strategies against mastitis have focused on the enhancement of humoral immunity. Development of vaccines that induce an effective cellular

immune response in the mammary gland has not been well investigated. The ability to induce cellular immunity, especially neutrophil activation and recruitment into the mammary gland, is one of the key strategies in the control of mastitis, but the magnitude and duration of increased cellular recruitment into the mammary gland leads to a high number of somatic cells and poor-quality milk. So, effective balanced humoral and cellular immunity that clear intramammary infection in a short period of time is required. Several vaccine studies were conducted over the years under controlled experimental and field trials. The major bacterial bovine mastitis pathogens that have been targeted for vaccine development are *S. aureus*, *S. uberis,* and *E. coli* [119]. Most of these experimental and some commercial vaccines are Bacterins which are inactivated whole organism, and some vaccines contained subunits of the organism such as surface proteins [100], toxins, or polysaccharides.

### **2.2 Vaccine trials against** *Staphylococcus aureus* **mastitis**

*Staphylococcus aureus* is one of the most common contagious mastitis pathogens, with an estimated incidence rate ranging from 43–74% [25, 38, 56, 120, 121]. *Staphylococcus chromogenes* is another increasingly reported coagulase-negative *Staphylococcus* species with an estimated quarter incidence rate of 42.7% characterized by high somatic cell counts [122–128]. In a study on conventional and organic Canadian dairy farms, coagulase-negative *Staphylococcus species* were found in 20% of the clinical samples [129]. Recently, mastitis caused by coagulase-negative *Staphylococcus species* increasingly became more problematic in dairy herds [125, 127, 130, 131].

Several staphylococcal vaccine efficacy trials showed that vaccination with Bacterin vaccines induced increased antibody titers in the serum and milk that are associated with partial protection [75–77, 80, 132–134] or no protection at all [78, 79, 81]. However, effective intramammary immune mechanisms against staphylococcal mastitis is still poorly understood. None of the commercially available Bacterin vaccines protects new intramammary infection [75, 77, 80, 81]. Dependence on antibiotics for the prevention and treatment of mastitis is not sustainable because of limited success [46, 47] and the emergence of antimicrobialresistant bacteria that are major threat to human and animal health [72–74].

Despite several mastitis vaccine trials conducted against *S. aureus* mastitis [75, 77, 80, 88, 89, 91, 93–95, 97–99, 133] all field trials have either been unsuccessful or had limited success. There are two commercial vaccines for *Staphylococcus aureus* mastitis on the market, Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the United States and Startvac® (Hipra S.A, Girona, Spain) in Europe and Canada [78]. None of these vaccines confer protection under field trials as well as under controlled experimental studies [75, 77, 80, 81]. Several field trials and controlled experimental studies have been conducted testing the efficacy of Lysigin® and Startvac®, and results from those studies have shown some interesting results, namely a reduced incidence, severity, and duration of mastitis in vaccinated cows compared to non-vaccinated control cows [75–77]. Contrary to these observations, other studies failed to find an effect on improving udder health or showed no difference between vaccinated and non-vaccinated control cows [78, 79]. None of these Bacterin-based vaccines prevents new *S. aureus* IMI [75, 77, 80, 81]. Differences found in these studies are mainly due to methodological differences (vaccination schedule, route of vaccination, challenge model, herd size, time of lactation, etc.) in testing the efficacy of these vaccines. It is critically important to have a good infection model that mimics natural infection and a model that has 100% efficacy in causing infection. Without a good challenge model, the results from vaccine efficacy will be inaccurate.

**193**

control cows.

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

The Startvac® (Hipra, Girona, Spain) is the commercially available vaccine in Europe and is a polyvalent vaccine that contains *E. coli* J5 and *S. aureus* strain SP140 [119]. In a field trial, Freick et al. [78] compared the efficacy of Startvac® with Bestvac® (IDT, Dessau-Rosslau, Germany) another herd-specific autologous commercial vaccine in a dairy herd with a high prevalence of *S. aureus* and found that the herd prevalence of *S. aureus* mastitis was lower in the Startvac® and Bestvac® vaccinated cows compared to the control cows. However, there were no other differences in terms of improvement of udder health. These authors [78] concluded that vaccination with Startvac® and Bestvac® did not improve udder health. In another field efficacy study on Startvac® in the UK, Bradley et al. [75] found that Startvac® vaccinated cows had clinical mastitis with reduced severity and higher milk produc-

Similarly, Schukken et al. [77] evaluated effect of Startvac® on the development of new IMI and the duration of infections caused by *S. aureus* and CNS. These authors [77] found that vaccinated cows had decreased incidence rate and a shorter duration of *S. aureus* and CNS mastitis. Piepers et al. [76], also tested the efficacy of Startvac® through vaccination and subsequent challenge with a heterologous killed *S. aureus* strain and found that the inflammatory response in the vaccinated cows was less severe compared to the control cows. These authors [76] suggested that Startvac® elicited a strong Th2 immune response against *S. aureus* in vaccinated cows and was more effective at clearing bacteria compared to the control cows. Contrary to these observations, Landin et al. [135], evaluated the effects of Startvac® on milk production, udder health, and survival on two Swedish dairy herds with *S. aureus* mastitis problems and found no significant differences between the Startvac® vaccinated and non-vaccinated control cows on the health param-

An experimental *S. aureus* vaccine made up of a combination of plasmids encoding fibronectin-binding motifs of fibronectin-binding protein (FnBP) and clumping factor A (ClfA), and plasmid encoding bovine granulocyte-macrophagecolony stimulatory factor, was used as a vaccine with a subsequent challenge with bacteria to test its protective effects [95]. These authors (Shkreta et al. 2004) found that their experimental vaccine-induced immune responses in the heifers that were partially protective upon experimental challenge [95]. Another controlled experimental vaccine efficacy study was conducted on the slime associated antigenic complex (SAAC) which is an extracellular component of *Staphylococcus aureus,* as vaccine antigen in which one group of cows were vaccinated with a vaccine containing a low amount of SAAC and another group with a high amount of SAAC and the unvaccinated group served as a control [136]. Upon intramammary infusion (challenge) with *S. aureus*, no difference in the occurrence of mastitis among all three groups despite the fact that the vaccine with high SAAC content induced higher production of antibodies compared to the vaccine with a low amount of SAAC [136]. Similarly, Pellegrino et al. [137], vaccinated dairy cows with an avirulent mutant strain of *S. aureus* and subsequently challenged with *S. aureus* 20 days after the second vaccination which resulted in no significant differences in the number of somatic cell count (SCC) or number of bacteria shedding through milk despite increased IgG antibody titer in the vaccinated cows compared to the

Some of the constraints affecting the successful development of effective mastitis

vaccines are strain variation, the presence of exopolysaccharide (capsule, slime, biofilm) layer in most pathogenic strains of bacteria (*Staph. aureus*, *Strep. uberis*) which does not allow recognition of antibody-coated bacteria by phagocytic cells, dilution of immune effectors by milk [138, 139], the interaction between milk components and immune effectors [140] that reduce their effectiveness, and the ability

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

tion compared to non-vaccinated control cows [75].

eters they evaluated.

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

The Startvac® (Hipra, Girona, Spain) is the commercially available vaccine in Europe and is a polyvalent vaccine that contains *E. coli* J5 and *S. aureus* strain SP140 [119]. In a field trial, Freick et al. [78] compared the efficacy of Startvac® with Bestvac® (IDT, Dessau-Rosslau, Germany) another herd-specific autologous commercial vaccine in a dairy herd with a high prevalence of *S. aureus* and found that the herd prevalence of *S. aureus* mastitis was lower in the Startvac® and Bestvac® vaccinated cows compared to the control cows. However, there were no other differences in terms of improvement of udder health. These authors [78] concluded that vaccination with Startvac® and Bestvac® did not improve udder health. In another field efficacy study on Startvac® in the UK, Bradley et al. [75] found that Startvac® vaccinated cows had clinical mastitis with reduced severity and higher milk production compared to non-vaccinated control cows [75].

Similarly, Schukken et al. [77] evaluated effect of Startvac® on the development of new IMI and the duration of infections caused by *S. aureus* and CNS. These authors [77] found that vaccinated cows had decreased incidence rate and a shorter duration of *S. aureus* and CNS mastitis. Piepers et al. [76], also tested the efficacy of Startvac® through vaccination and subsequent challenge with a heterologous killed *S. aureus* strain and found that the inflammatory response in the vaccinated cows was less severe compared to the control cows. These authors [76] suggested that Startvac® elicited a strong Th2 immune response against *S. aureus* in vaccinated cows and was more effective at clearing bacteria compared to the control cows. Contrary to these observations, Landin et al. [135], evaluated the effects of Startvac® on milk production, udder health, and survival on two Swedish dairy herds with *S. aureus* mastitis problems and found no significant differences between the Startvac® vaccinated and non-vaccinated control cows on the health parameters they evaluated.

An experimental *S. aureus* vaccine made up of a combination of plasmids encoding fibronectin-binding motifs of fibronectin-binding protein (FnBP) and clumping factor A (ClfA), and plasmid encoding bovine granulocyte-macrophagecolony stimulatory factor, was used as a vaccine with a subsequent challenge with bacteria to test its protective effects [95]. These authors (Shkreta et al. 2004) found that their experimental vaccine-induced immune responses in the heifers that were partially protective upon experimental challenge [95]. Another controlled experimental vaccine efficacy study was conducted on the slime associated antigenic complex (SAAC) which is an extracellular component of *Staphylococcus aureus,* as vaccine antigen in which one group of cows were vaccinated with a vaccine containing a low amount of SAAC and another group with a high amount of SAAC and the unvaccinated group served as a control [136]. Upon intramammary infusion (challenge) with *S. aureus*, no difference in the occurrence of mastitis among all three groups despite the fact that the vaccine with high SAAC content induced higher production of antibodies compared to the vaccine with a low amount of SAAC [136]. Similarly, Pellegrino et al. [137], vaccinated dairy cows with an avirulent mutant strain of *S. aureus* and subsequently challenged with *S. aureus* 20 days after the second vaccination which resulted in no significant differences in the number of somatic cell count (SCC) or number of bacteria shedding through milk despite increased IgG antibody titer in the vaccinated cows compared to the control cows.

Some of the constraints affecting the successful development of effective mastitis vaccines are strain variation, the presence of exopolysaccharide (capsule, slime, biofilm) layer in most pathogenic strains of bacteria (*Staph. aureus*, *Strep. uberis*) which does not allow recognition of antibody-coated bacteria by phagocytic cells, dilution of immune effectors by milk [138, 139], the interaction between milk components and immune effectors [140] that reduce their effectiveness, and the ability

*Animal Reproduction in Veterinary Medicine*

**2.2 Vaccine trials against** *Staphylococcus aureus* **mastitis**

polysaccharides.

immune response in the mammary gland has not been well investigated. The ability to induce cellular immunity, especially neutrophil activation and recruitment into the mammary gland, is one of the key strategies in the control of mastitis, but the magnitude and duration of increased cellular recruitment into the mammary gland leads to a high number of somatic cells and poor-quality milk. So, effective balanced humoral and cellular immunity that clear intramammary infection in a short period of time is required. Several vaccine studies were conducted over the years under controlled experimental and field trials. The major bacterial bovine mastitis pathogens that have been targeted for vaccine development are *S. aureus*, *S. uberis,* and *E. coli* [119]. Most of these experimental and some commercial vaccines are Bacterins which are inactivated whole organism, and some vaccines contained subunits of the organism such as surface proteins [100], toxins, or

*Staphylococcus aureus* is one of the most common contagious mastitis pathogens,

*Staphylococcus* species with an estimated quarter incidence rate of 42.7% characterized by high somatic cell counts [122–128]. In a study on conventional and organic Canadian dairy farms, coagulase-negative *Staphylococcus species* were found in 20% of the clinical samples [129]. Recently, mastitis caused by coagulase-negative *Staphylococcus species*

Several staphylococcal vaccine efficacy trials showed that vaccination with Bacterin vaccines induced increased antibody titers in the serum and milk that are associated with partial protection [75–77, 80, 132–134] or no protection at all [78, 79, 81]. However, effective intramammary immune mechanisms against staphylococcal mastitis is still poorly understood. None of the commercially available Bacterin vaccines protects new intramammary infection [75, 77, 80, 81]. Dependence on antibiotics for the prevention and treatment of mastitis is not sustainable because of limited success [46, 47] and the emergence of antimicrobial-

resistant bacteria that are major threat to human and animal health [72–74]. Despite several mastitis vaccine trials conducted against *S. aureus* mastitis [75, 77, 80, 88, 89, 91, 93–95, 97–99, 133] all field trials have either been unsuccessful or had limited success. There are two commercial vaccines for *Staphylococcus aureus* mastitis on the market, Lysigin® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) in the United States and Startvac® (Hipra S.A, Girona, Spain) in Europe and Canada [78]. None of these vaccines confer protection under field trials as well as under controlled experimental studies [75, 77, 80, 81]. Several field trials and controlled experimental studies have been conducted testing the effi-

interesting results, namely a reduced incidence, severity, and duration of mastitis in vaccinated cows compared to non-vaccinated control cows [75–77]. Contrary to these observations, other studies failed to find an effect on improving udder health or showed no difference between vaccinated and non-vaccinated control cows [78, 79]. None of these Bacterin-based vaccines prevents new *S. aureus* IMI [75, 77, 80, 81]. Differences found in these studies are mainly due to methodological differences (vaccination schedule, route of vaccination, challenge model, herd size, time of lactation, etc.) in testing the efficacy of these vaccines. It is critically important to have a good infection model that mimics natural infection and a model that has 100% efficacy in causing infection. Without a good challenge model, the results

and results from those studies have shown some

with an estimated incidence rate ranging from 43–74% [25, 38, 56, 120, 121]. *Staphylococcus chromogenes* is another increasingly reported coagulase-negative

increasingly became more problematic in dairy herds [125, 127, 130, 131].

**192**

cacy of Lysigin® and Startvac®,

from vaccine efficacy will be inaccurate.

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 incidence of mastitis in dairy cows.
