*2.4.1 Hemolysins*

*S. aureus* isolates from bovine mastitis produce alpha (α), beta (β), gamma (γ), and delta (δ) hemolysins that cause hemolysis of red blood cells of the host [94] and all are antigenically distinct. α-hemolysin is a pore-forming toxin that binds to a disintegrin and metalloproteinase domain-containing protein-10 (ADAM10) receptor resulting in pore formation and cellular necrosis [95, 96]. It is also known to increase the inflammatory response and decrease macrophage function [97]. α-hemolysin damages the plasma membrane of the epithelial cell resulting in leakages of low-molecular-weight molecules from the cytosol and death of the cell [98]. It is produced by 20–50% of strains from bovine IMI [99]. A study reported that the α-hemolysin might be required for a cell to cell interaction during biofilm formation [100]. β-hemolysin hydrolyzes the sphingomyelin present in the plasma membrane resulting in increased permeability with progressive loss of cell surface charge [101]. It is produced by 75–100% of *S. aureus* strains from bovine IMI [99]. α-hemolysin expression requires specific growth conditions *in vitro* because its growth is inhibited by agar [102]. α-hemolysin producing strains cause complete hemolysis of sheep red blood cells, whereas β-hemolysin producing strains cause partial hemolysis within 24 h of incubation at 37°C [103]. Partial hemolysis caused by β-hemolysin becomes completely lysed after further storage at 4–15°C, which is also expressed as hot-cold lysis [104]. β-hemolysin producing strains are the most frequent isolates from animals [105]. δ-hemolysin causes complete hemolysis of red blood cells of wide range of species including human, rabbit, sheep, horse, rat, guinea pig, and some fish erythrocytes. δ-hemolysin migrates more slowly through agar than the α-hemolysin so the effect takes longer time to express. Double (α- and β-) hemolysin producing strains caused complete hemolysis in the middle with partial hemolysis on the peripheral area around each colony [105]. γ-hemolysin is produced by almost every strain of *S. aureus*, but γ-hemolysin is not identifiable on blood agar plates, due to the inhibitory effect of agar on toxin activity [106].

### *2.4.2 Enterotoxins Enterotoxins*

These toxins are heat stable and can resist pasteurization. *S. aureus* produces staphylococcal enterotoxins A, B, C, D, E, G, H, I, and J–Q as well as toxic shock syndrome toxin 1 (tsst-1) [105, 107, 108]. Enterotoxins can get into the food chain through the consumption of contaminated food and cause food poisoning [109]. Staphylococcal enterotoxins tend to contaminate dairy products and cause foodborne illness [110, 111]. Staphylococcal enterotoxins G to Q (SEG–SEQ ) are prevalent among *S. aureus* isolates from cases of bovine mastitis and are also implicated in the pathogenesis of mastitis. Some of these toxins are known to function as

**155**

[130, 131].

*Bovine Mastitis: Part I*

and yet to be determined.

cells [122, 123].

*DOI: http://dx.doi.org/10.5772/intechopen.93483*

superantigens that cause increased immunological reactivity in the host [110]. Some studies showed that about 20% of *S. aureus* isolates from IMI produce toxic shock syndrome toxin-1 [109, 112]. Toxic shock syndrome toxin causes toxic shock syndrome and can be fatal [113]. Besides the superantigenic effect of enterotoxins, their role in the pathogenesis of mastitis is unknown. It may be specific to each strain or area based on selective pressures in the habitat [114]. Enterotoxin prevalence seems to vary between geographical regions. The strains producing enterotoxin C have been isolated relatively frequently from cases of bovine mastitis [108, 115, 116]. Enterotoxins are believed to have a role in the development of mastitis since *S. aureus* isolates from cases of mastitis had a high prevalence of enterotoxins than isolates from milk of cows without mastitis [117, 118]; however, staphylococcal enterotoxins expressions are controlled by several regulatory elements [119] that respond to a variety of different micro-environmental stimuli and the exact mechanisms by which enterotoxins contribute to the development of mastitis are not clearly known

In addition to specific virulence factors, *Staphylococcus aureus* also possesses different mechanisms or traits such as biofilm formation, adhesion to and invasion into mammary epithelial cells, and formation of small colony variant (SCV) that enable this pathogen to resist host defense mechanisms. The ability of *S. aureus* to invade mammary epithelial cells during mastitis plays a significant role in the pathogenesis of *S. aureus*. Internalized bacteria can hide from the host's immune system inside the host cell and continue to multiply inside the host cell [120]. There may be many mechanisms that *S. aureus* uses to invade into host cells, and each mechanism can be strain dependent. *S. aureus* strains have a fibronectin-binding protein that can link to the fibronectin on the mammary epithelial cell surface. Fibronectin binding protein is thought to be a common way for the bacteria cells to invade bovine mammary epithelial cells. Fibronectin-binding protein-deficient strains cannot invade host cells [121]. The presence of a capsule prevents adherence to epithelial

Adhesion is the first step in the formation of biofilm or the invasion of host cells, which protects the bacteria from the host immune system and facilitates chronic infection [124]. Adhesion is dependent on surface proteins called adhesins, which help the bacterium to recognize and attach to host cells. Staphylococci are coated with a wide variety of surface proteins that help them to adhere to host cells and extracellular matrix components. Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) of the host are the most common surface proteins that are involved in adhesion [124]. The ability to bind to host tissue or the host's cell surface is a pivotal part of the bacteria's pathogenicity because adhesion is

typically the first step in the invasion and biofilm formation [125, 126].

Adhesion to and invasion into epithelial cells [124], intracellular survival in macrophages [127], and epithelial cells allow them to avoid detection by the host immune system and resist treatment with antibiotics [120]. Due to its poor response to treatments, *S. aureus* infections often become chronic with a low cure rate [128]. Treatment of *Staphylococcus aureus* mastitis with cloxacillin cured only 25% of the clinical cases and 40% of subclinical cases in the study by Tyler and Baggot [129]. *Staphylococcus aureus* also has a known ability to form biofilms [77, 78, 86] and acquire antimicrobial-resistance genes via horizontal resistance gene transfer, which enables this bacterium to develop antimicrobial resistance

The mode of transmission from infected mammary glands or colonized udder skin to healthy mammary glands is through contact during milking procedures with milker's hand, towel, and milking machine [58]. *S. aureus* usually causes subclinical

or chronic infections and is difficult to clear with antibiotic treatment [132].

### *Bovine Mastitis: Part I DOI: http://dx.doi.org/10.5772/intechopen.93483*

*Animal Reproduction in Veterinary Medicine*

**2.4 Secretory factors**

*2.4.1 Hemolysins*

the *ica* gene is not mandatory for biofilm production since *S. aureus* lacking *ica* gene can still produce biofilm through other microbial surface components recognizing

Some of the known secretory virulence factors are toxins which include staphylococcal enterotoxins, non-enteric exfoliative toxins, toxic shock syndrome toxin 1, leucocidin, and hemolysins (alpha, beta, delta, and gamma) [91, 92]. Similarly, enzymes such as coagulase, staphylokinase, DNAase, phosphatase, lipase, phospho-

*S. aureus* isolates from bovine mastitis produce alpha (α), beta (β), gamma (γ), and delta (δ) hemolysins that cause hemolysis of red blood cells of the host [94] and all are antigenically distinct. α-hemolysin is a pore-forming toxin that binds to a disintegrin and metalloproteinase domain-containing protein-10 (ADAM10) receptor resulting in pore formation and cellular necrosis [95, 96]. It is also known to increase the inflammatory response and decrease macrophage function [97]. α-hemolysin damages the plasma membrane of the epithelial cell resulting in leakages of low-molecular-weight molecules from the cytosol and death of the cell [98]. It is produced by 20–50% of strains from bovine IMI [99]. A study reported that the α-hemolysin might be required for a cell to cell interaction during biofilm formation [100]. β-hemolysin hydrolyzes the sphingomyelin present in the plasma membrane resulting in increased permeability with progressive loss of cell surface charge [101]. It is produced by 75–100% of *S. aureus* strains from bovine IMI [99]. α-hemolysin expression requires specific growth conditions *in vitro* because its growth is inhibited by agar [102]. α-hemolysin producing strains cause complete hemolysis of sheep red blood cells, whereas β-hemolysin producing strains cause partial hemolysis within 24 h of incubation at 37°C [103]. Partial hemolysis caused by β-hemolysin becomes completely lysed after further storage at 4–15°C, which is also expressed as hot-cold lysis [104]. β-hemolysin producing strains are the most frequent isolates from animals [105]. δ-hemolysin causes complete hemolysis of red blood cells of wide range of species including human, rabbit, sheep, horse, rat, guinea pig, and some fish erythrocytes. δ-hemolysin migrates more slowly through agar than the α-hemolysin so the effect takes longer time to express. Double (α- and β-) hemolysin producing strains caused complete hemolysis in the middle with partial hemolysis on the peripheral area around each colony [105]. γ-hemolysin is produced by almost every strain of *S. aureus*, but γ-hemolysin is not identifiable on blood agar plates, due to the inhibitory effect of agar on toxin activity [106].

These toxins are heat stable and can resist pasteurization. *S. aureus* produces staphylococcal enterotoxins A, B, C, D, E, G, H, I, and J–Q as well as toxic shock syndrome toxin 1 (tsst-1) [105, 107, 108]. Enterotoxins can get into the food chain through the consumption of contaminated food and cause food poisoning [109]. Staphylococcal enterotoxins tend to contaminate dairy products and cause foodborne illness [110, 111]. Staphylococcal enterotoxins G to Q (SEG–SEQ ) are prevalent among *S. aureus* isolates from cases of bovine mastitis and are also implicated in the pathogenesis of mastitis. Some of these toxins are known to function as

adhesive matrix molecules (MSCRAM) and secreted proteins [89, 90].

lipase, and hyaluronidase serve as virulence factors of *S. aureus* [93].

**154**

*2.4.2 Enterotoxins Enterotoxins*

superantigens that cause increased immunological reactivity in the host [110]. Some studies showed that about 20% of *S. aureus* isolates from IMI produce toxic shock syndrome toxin-1 [109, 112]. Toxic shock syndrome toxin causes toxic shock syndrome and can be fatal [113]. Besides the superantigenic effect of enterotoxins, their role in the pathogenesis of mastitis is unknown. It may be specific to each strain or area based on selective pressures in the habitat [114]. Enterotoxin prevalence seems to vary between geographical regions. The strains producing enterotoxin C have been isolated relatively frequently from cases of bovine mastitis [108, 115, 116].

Enterotoxins are believed to have a role in the development of mastitis since *S. aureus* isolates from cases of mastitis had a high prevalence of enterotoxins than isolates from milk of cows without mastitis [117, 118]; however, staphylococcal enterotoxins expressions are controlled by several regulatory elements [119] that respond to a variety of different micro-environmental stimuli and the exact mechanisms by which enterotoxins contribute to the development of mastitis are not clearly known and yet to be determined.

In addition to specific virulence factors, *Staphylococcus aureus* also possesses different mechanisms or traits such as biofilm formation, adhesion to and invasion into mammary epithelial cells, and formation of small colony variant (SCV) that enable this pathogen to resist host defense mechanisms. The ability of *S. aureus* to invade mammary epithelial cells during mastitis plays a significant role in the pathogenesis of *S. aureus*. Internalized bacteria can hide from the host's immune system inside the host cell and continue to multiply inside the host cell [120]. There may be many mechanisms that *S. aureus* uses to invade into host cells, and each mechanism can be strain dependent. *S. aureus* strains have a fibronectin-binding protein that can link to the fibronectin on the mammary epithelial cell surface. Fibronectin binding protein is thought to be a common way for the bacteria cells to invade bovine mammary epithelial cells. Fibronectin-binding protein-deficient strains cannot invade host cells [121]. The presence of a capsule prevents adherence to epithelial cells [122, 123].

Adhesion is the first step in the formation of biofilm or the invasion of host cells, which protects the bacteria from the host immune system and facilitates chronic infection [124]. Adhesion is dependent on surface proteins called adhesins, which help the bacterium to recognize and attach to host cells. Staphylococci are coated with a wide variety of surface proteins that help them to adhere to host cells and extracellular matrix components. Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) of the host are the most common surface proteins that are involved in adhesion [124]. The ability to bind to host tissue or the host's cell surface is a pivotal part of the bacteria's pathogenicity because adhesion is typically the first step in the invasion and biofilm formation [125, 126].

Adhesion to and invasion into epithelial cells [124], intracellular survival in macrophages [127], and epithelial cells allow them to avoid detection by the host immune system and resist treatment with antibiotics [120]. Due to its poor response to treatments, *S. aureus* infections often become chronic with a low cure rate [128]. Treatment of *Staphylococcus aureus* mastitis with cloxacillin cured only 25% of the clinical cases and 40% of subclinical cases in the study by Tyler and Baggot [129]. *Staphylococcus aureus* also has a known ability to form biofilms [77, 78, 86] and acquire antimicrobial-resistance genes via horizontal resistance gene transfer, which enables this bacterium to develop antimicrobial resistance [130, 131].

The mode of transmission from infected mammary glands or colonized udder skin to healthy mammary glands is through contact during milking procedures with milker's hand, towel, and milking machine [58]. *S. aureus* usually causes subclinical or chronic infections and is difficult to clear with antibiotic treatment [132].

## *2.4.3 Streptococcus agalactiae*

The most important virulence factor of *S. agalactiae* is the capsular polysaccharide [133], which protects this bacterium from being engulfed by macrophages and subsequently phagocytosed [133]. Another virulence factor of *S. agalactiae* is the Rib protein, which confers resistance to proteases. Emaneini et al. [133] found that the Rib encoding gene (*rib)* was detected in 89% of the isolates from bovine origin. *Streptococcus agalactiae* causes persistent infections that are usually difficult to clear without antibiotic treatment [134]. Though *Streptococcus agalactiae* is highly contagious, it has good response to treatment with antibiotics, which makes it possible to eliminate from herds with current mastitis control measures [129]. Since the adoption of hygienic milking practices, the incidence of mastitis caused by *S. agalactiae* has dramatically decreased and is now rarely observed in dairy herds [135].

#### *2.4.4 Mycoplasma mastitis*

Mastitis caused by *Mycoplasma* spp. is a growing concern in the United States. It is believed that this organism has been underreported due to the difficulty of isolation by culture method [136]. The incidence of *Mycoplasma* mastitis varies across the globe, with a 3.2% prevalence rate in the United States that may increase to 14.4% in larger herd size of greater than 500 cows [47, 48, 62, 137]. A risk factor for *Mycoplasma* mastitis increase with herd size, and most of the *Mycoplasma* mastitis cases are subclinical infections with outbreaks linked to asymptomatic carriers [138]. Pathogenesis of most *Mycoplasma* spp. infection is characterized by adherence to and internalization into host cells resulting in colonization of the host with immune modulation without causing severe disease [138]. *Mycoplasma* species lack a cell wall, thus not sensitive to beta-lactam antibiotics, but showed sensitivity to non-beta-lactam antibiotics [139].
