**2. Mastitis pathogens agents**

#### *Staphylococcus aureus*

*Staphylococcus aureus (S. aureus*) colonize the nipple skin, advance through the mammary gland canal into the gland. The IMI with *S. aureus* predominantly cause subclinical mastitis resulting in a chronic infection lingering lifelong (Bannerman *et al*., 2004; Riollet *et al*., 2000; Yang *et al*.,

© 2012 Carrillo-Casas and Miranda-Morales, licensee InTech. This is an open access chapter 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. © 2012 Carrillo-Casas and Miranda-Morales, licensee InTech. This is a paper 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.

2008). During the infection's early stages, the mild damage may be reverted but *S. aureus* infections, in its peracute mastitis presentation generates gangrene and severe tissue damage. In comparison with *Streptococcus agalactiae*, *S. aureus* is more difficult to be eradicated*. S. aureus* infections cause a 45% decrease on milk production per quarter, reflected as a 15% per infected animal (NMC, 1999). The chronic, subclinical infections account for approximately 80% of mastitis related costs, due to reduced milk yield and product quality (Shim *et al*., 2004). In practice, an elevated somatic cell count (SCC), over 300, 000 to 500, 000 cells/ml, indicates high prevalence of infected glands with *S. aureus* in a herd (NMC, 1999).

#### *Streptococcus agalactiae*

*Streptococcus agalactiae* (*S. agalactiae*) causes contagious mastitis, an obligated pathogen of the mammary gland, which is transmitted directly among cows during milking (NMC, 1999). *S. agalactiae* infects the gland cistern and ducts of the mammary gland causing irritation, swelling and subclinical mastitis. The infected cow shows mere clinical signs without abnormalities drawn in milk. However, low production rates and high SCC are usually registered. *S. agalactiae* infections are related to Bulk-tank milk figures around a 1,000, 000 cells/ml on SCC or higher. Currently, these figures are rarely seen because the control measures and milking management had been improving along with better antibiotic treatment (Hillerton & Berry, 2003).

Globally *S. agalactiae* is a low prevalence pathogen. In Canadian bulk milk, its prevalence ranged between 6% in Alberta (Schoonderwoerd *et al.*, 1993), and 43% in Québec (Guillemette *et al*., 1992). In the Prince Edward Island, Keefe *et al*., (1997) determined a herd prevalence of 18%. Furthermore, a study recently performed in Canada (Richard G.M. *et al*., 2010) demonstrated the low prevalence of *S. agalactiae* at 4.4% and in Argentina, in the last 25 years, the mastitis prevalence due to *S. agalactiae* has been 0.3% in the four quarters before-delivery (Calvinho *et al*., 2001).

#### *Mycoplasma* **spp**

*Mycoplasma* spp are highly contagious microorganisms, but less common than *S. agalactiae* and *S. aureus.* Nevertheless, *Mycoplasma* spp damage the secretory tissue, induce the gland fibrosis, abscesses and the lymphatic nodules fibrosis (NMC, 1999). Animals from all ages are susceptible, as well as at any time during lactation. Those in early lactation are more susceptible to *Mycoplasma* infection and it can be isolated from high production animals without signology.

Mycoplasmosis is frequently related to the mastitis outbreak onsets, to the introduction of new animals to a herd, to previous respiratory or articular disease, and to herds with unresponsive mastitis to antibiotic treatment. When at least the recurrent mastitis, a non-signs illness and an unresponsive treatment are observed, a mycoplasma infection is suspected.

Mycoplasma infection prevalence at the herd-level is estimated by *Mycoplasma* culture from Bulk Tank Milk (BTM) and has been suggested to be between 1 and 8%in the USA (Fox LK., *et al*., 2005). These monitoring of mycoplasma-mastitis performed through BTM cultures assumes that the appearance of a *Mycoplasma* sp in it indicates that there is at least one cow in the herd affected with mycoplasma and that environmental contamination of the bulk tank by mycoplasma is unlikely, hence a false positive result is discarded. The speciation of mycoplasma mastitis pathogens requires secondary tests, usually only carried out by specialized laboratories from colonies presumptively identified as *Mycoplasma* spp and with specific end point PCR for *M. alkalescens, M. bovigenitalium, M. bovirhinis, M. californicum, M. canadense* and *M. bovis* (Hirose *et al.,* 2001; Kirk JH. *et al*., 1997) applied to determine the genus and specie prevalence from BTM samples collected monthly between 1989 and 1995 from 267 dairy herds. From these *M. bovis, M. canadense, M. californicum , M. bovigenitalium, M. alkalescens*, were retrieved from 209 (78.2%) dairies and they had been identified and reported as potentially pathogenic *Mycoplasma* organisms. Further studies, in the herd level such as, Fox *et al*., (2003) and the Nothwest Dairy Association (NDA), processed milk from 463 herds concluding 93 herds diagnosed as mycoplasma-positive from BTM. *Mycoplasma* was more likely to be present in samples from herds shipping higher milk amounts, therefore mycoplasma is indirectly related to the herd size and the larger the herds are, the higher *mycoplasma* caused mastitis prevalence will be. From the same study, a year later, *Mycoplasma* spp were not detected in any herd. These finding suggested that Mycoplasma caused mastitis can be controlled and eliminated from herds. This observation is supported by the studies done by, Brown *et al*. (1990), who reported that an outbreak of *Mycoplasma bovis* mastitis was controlled by an intensive identification scheme to find infected cows, culling the unproductive ones, and segregating and milking the left under a milking time hygiene procedure, also Bicknell *et al*. (1983) reported similar findings with intensive identification schemes to determine cows with *Mycoplasma bovis* mastitis and successfully managed with segregation and culling. Similar findings were reported by Mackie *et al*. (2000) specifically for *M. californicum* and *M. canadense*. The exception was reported by Jackson and Boughton (1991) who observed that segregation and culling were not necessarily required for controlling a *M. bovigenitalium* outbreak.

#### **Coagulase-negative** *Staphylococci* **(CNS)**

360 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

prevalence of infected glands with *S. aureus* in a herd (NMC, 1999).

*Streptococcus agalactiae* 

treatment (Hillerton & Berry, 2003).

before-delivery (Calvinho *et al*., 2001).

*Mycoplasma* **spp**

2008). During the infection's early stages, the mild damage may be reverted but *S. aureus* infections, in its peracute mastitis presentation generates gangrene and severe tissue damage. In comparison with *Streptococcus agalactiae*, *S. aureus* is more difficult to be eradicated*. S. aureus* infections cause a 45% decrease on milk production per quarter, reflected as a 15% per infected animal (NMC, 1999). The chronic, subclinical infections account for approximately 80% of mastitis related costs, due to reduced milk yield and product quality (Shim *et al*., 2004). In practice, an elevated somatic cell count (SCC), over 300, 000 to 500, 000 cells/ml, indicates high

*Streptococcus agalactiae* (*S. agalactiae*) causes contagious mastitis, an obligated pathogen of the mammary gland, which is transmitted directly among cows during milking (NMC, 1999). *S. agalactiae* infects the gland cistern and ducts of the mammary gland causing irritation, swelling and subclinical mastitis. The infected cow shows mere clinical signs without abnormalities drawn in milk. However, low production rates and high SCC are usually registered. *S. agalactiae* infections are related to Bulk-tank milk figures around a 1,000, 000 cells/ml on SCC or higher. Currently, these figures are rarely seen because the control measures and milking management had been improving along with better antibiotic

Globally *S. agalactiae* is a low prevalence pathogen. In Canadian bulk milk, its prevalence ranged between 6% in Alberta (Schoonderwoerd *et al.*, 1993), and 43% in Québec (Guillemette *et al*., 1992). In the Prince Edward Island, Keefe *et al*., (1997) determined a herd prevalence of 18%. Furthermore, a study recently performed in Canada (Richard G.M. *et al*., 2010) demonstrated the low prevalence of *S. agalactiae* at 4.4% and in Argentina, in the last 25 years, the mastitis prevalence due to *S. agalactiae* has been 0.3% in the four quarters

*Mycoplasma* spp are highly contagious microorganisms, but less common than *S. agalactiae* and *S. aureus.* Nevertheless, *Mycoplasma* spp damage the secretory tissue, induce the gland fibrosis, abscesses and the lymphatic nodules fibrosis (NMC, 1999). Animals from all ages are susceptible, as well as at any time during lactation. Those in early lactation are more susceptible to *Mycoplasma* infection and it can be isolated from high production animals without signology. Mycoplasmosis is frequently related to the mastitis outbreak onsets, to the introduction of new animals to a herd, to previous respiratory or articular disease, and to herds with unresponsive mastitis to antibiotic treatment. When at least the recurrent mastitis, a non-signs illness and an

Mycoplasma infection prevalence at the herd-level is estimated by *Mycoplasma* culture from Bulk Tank Milk (BTM) and has been suggested to be between 1 and 8%in the USA (Fox LK., *et al*., 2005). These monitoring of mycoplasma-mastitis performed through BTM cultures assumes that the appearance of a *Mycoplasma* sp in it indicates that there is at least one cow in the herd affected with mycoplasma and that environmental contamination of the bulk tank by

unresponsive treatment are observed, a mycoplasma infection is suspected.

Coagulase-negative *Staphylococci* (CNS) are considered opportunistic mastitis pathogens, resident colonizers on the teat skin, rarely causing clinical mastitis (Hogan *et al*., 1987) and are frequently not reported in mastitis studies (Bramley & Dodd, 1984). However, CNS are isolated from cases of subclinical and clinical mastitis and as the cause of IMI in lactating cattle with subclinical prevalence of 31.1% at prepartum and 27.9% postpartum (Hogan, 1997; Fox , 2009). Moreover, CNS are the most frequently isolated pathogens from mastitis in heifers. This bacteria group comprises more than 50 species and subspecies (Pyöräla S. *et al.,* 2008). Coagulase-negative *Staphylococcus* species differ from each other in antimicrobial susceptibility, virulence factors and host response to infection (Birgersson *et al*., 1992; Devriese *et al*., 2002; Taponen S. *et al.*, 2009). Thus, identification of species may be relevant for epidemiological surveys, the assessment of their pathogenic significance and for developing specific management practices to prevent mastitis. Perhaps it could be worthy to study them as individual species. There are many differences regarding the pathogenicity of different species of CNS that are studied with molecular diagnostic techniques (Zadoks & Schukken, 2006).

The most commonly isolated species of CNS from bovine mastitis are *Staphylococcus chromogenes, Staphylococcus epidermitis*, *Staphylococcus hyicus* and *Staphylococcus simulans.*  Prevalence studies have demonstrated that CNS are the bacteria group most frequently isolated

from milk samples with high SCC (Pitkälä *et al*., 2004; Bradley *et al*., 2007; Piepers *et al.*, 2007; Sampimon *et al*., 2009). In mammary quarter infection prevalence ranges between 28.9–74.6% prepartum, and 12.3–45.5% at calving. CNS are the most prevalent cause of subclinical IMI in heifers and coagulase-positive *Staphylococci* (CPS) are the second most prevalent pathogens, while in other studies the environmental mastitis pathogens are more prevalent. Generally, the pathogens that cause mastitis in heifers are the same as those that cause infections in older cows. The risk factors for subclinical mastitis appear to be dependent on the season, herd location, and trimester of pregnancy; all suggesting that management has great impact in the prepartum disease control. Regarding clinical mastitis, the most prevalent mastitis pathogen has been reported to be CNS as well as CPS and environmental mastitis pathogens. Heifers are at a higher risk for clinical mastitis during the periparturient period including those related to diet, intrinsic mammary gland factors such as swelling and milk leaking, and factors associated with management changes and the heifer's introduction the milking herd (Fox, 2009).

The prevalence of IMI with CNS has been increasing in North America, Europe and Latin America (Calvinho *et al*., 2001, Jánosi1 & Baltay, 2004; Sampimon *et al.*, 2009; Pantoja, *et al*., 2009) (Table 1 and Table 2). CNS are the most frequently isolated pathogen group from IMI in The Netherlands, estimated as 10.8% at the quarter level and 34.4% at the cow level. Fourteen species of CNS were identified and the most relevant were *Staphylococcus chromogenes* (30.3%) *Staphylococcus epidermidis* (12.9%) and *Staphylococcus capitis* (11.0%) and prevalence of CNS IMI was higher in heifers than in older cows. Geometric mean quarter SCC of CNS-positive quarters was 109,000 cells/ml, which was approximately twice as high as culture-negative quarters. Quarters infected with *S. chromogenes*, *S. capitis* and *Staphylococcus xylosus* had a higher SCC (P < 0.05) than culture negative quarters, while quarters that were culture-positive for *S. epidermidis* and *Staphylococcus hyicus* tended to higher SCC than culture-negative quarters. An increased prevalence of CNS-IMI is associated with the herd-level variables such as a taped source of drinking water, single dry-cows housing, monthly SCC measure, veterinary udder health monitoring, outdoors season pasturing, percentage of milk contaminated stalls, and bulk milk SCC (BMSCC) > 250,000 cells/ml. Currently the prevalence of CNS-IMI is already high in heifers around their first calving (Borm *et al*., 2006), the lower prevalence of CNS in multiparous cows may be explained by the fact that in the 80% of the farms included in this study, the practice of antibiotic dry off and post-milking teat disinfection applied twice a day during lactation was used. Also pasturing during the outdoor season was associated with an increased prevalence of CNS-IMI, and the summer period is related to active flies, especially the horn fly *Haematobia irritans* which can transmit *S. aureus* (Owens *et al*., 1998) and possibly transmits CNS.


#### Bovine Mastitis Pathogens: Prevalence and Effects on Somatic Cell Count 363


(\*)Coagulase-negative *Staphylococci* (CNS)

362 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

management changes and the heifer's introduction the milking herd (Fox, 2009).

(Owens *et al*., 1998) and possibly transmits CNS.

*Streptococcus agalactiae* 

*Mycoplasma spp* 

Iran - - 48.75% - - - Ghazaei,

Mexico 9.92% Infante., *et* 

Environmental *Streptococcus spp* 

CN*S\** Environmental pathogens

Reference

2006

*al*., 1999

Country *Staphylococcus* 

*aureus* 

from milk samples with high SCC (Pitkälä *et al*., 2004; Bradley *et al*., 2007; Piepers *et al.*, 2007; Sampimon *et al*., 2009). In mammary quarter infection prevalence ranges between 28.9–74.6% prepartum, and 12.3–45.5% at calving. CNS are the most prevalent cause of subclinical IMI in heifers and coagulase-positive *Staphylococci* (CPS) are the second most prevalent pathogens, while in other studies the environmental mastitis pathogens are more prevalent. Generally, the pathogens that cause mastitis in heifers are the same as those that cause infections in older cows. The risk factors for subclinical mastitis appear to be dependent on the season, herd location, and trimester of pregnancy; all suggesting that management has great impact in the prepartum disease control. Regarding clinical mastitis, the most prevalent mastitis pathogen has been reported to be CNS as well as CPS and environmental mastitis pathogens. Heifers are at a higher risk for clinical mastitis during the periparturient period including those related to diet, intrinsic mammary gland factors such as swelling and milk leaking, and factors associated with

The prevalence of IMI with CNS has been increasing in North America, Europe and Latin America (Calvinho *et al*., 2001, Jánosi1 & Baltay, 2004; Sampimon *et al.*, 2009; Pantoja, *et al*., 2009) (Table 1 and Table 2). CNS are the most frequently isolated pathogen group from IMI in The Netherlands, estimated as 10.8% at the quarter level and 34.4% at the cow level. Fourteen species of CNS were identified and the most relevant were *Staphylococcus chromogenes* (30.3%) *Staphylococcus epidermidis* (12.9%) and *Staphylococcus capitis* (11.0%) and prevalence of CNS IMI was higher in heifers than in older cows. Geometric mean quarter SCC of CNS-positive quarters was 109,000 cells/ml, which was approximately twice as high as culture-negative quarters. Quarters infected with *S. chromogenes*, *S. capitis* and *Staphylococcus xylosus* had a higher SCC (P < 0.05) than culture negative quarters, while quarters that were culture-positive for *S. epidermidis* and *Staphylococcus hyicus* tended to higher SCC than culture-negative quarters. An increased prevalence of CNS-IMI is associated with the herd-level variables such as a taped source of drinking water, single dry-cows housing, monthly SCC measure, veterinary udder health monitoring, outdoors season pasturing, percentage of milk contaminated stalls, and bulk milk SCC (BMSCC) > 250,000 cells/ml. Currently the prevalence of CNS-IMI is already high in heifers around their first calving (Borm *et al*., 2006), the lower prevalence of CNS in multiparous cows may be explained by the fact that in the 80% of the farms included in this study, the practice of antibiotic dry off and post-milking teat disinfection applied twice a day during lactation was used. Also pasturing during the outdoor season was associated with an increased prevalence of CNS-IMI, and the summer period is related to active flies, especially the horn fly *Haematobia irritans* which can transmit *S. aureus*

**Table 1.** Pathogen prevalence in Bovine Milk from some productive regions


**Table 2.** Somatic cell count (SCC×1000 cells/ml) associated with the mastitis causing microorganism in different countries.

## **Environmental mastitis pathogens**

*Streptococcus* spp are among the outstanding environmental pathogens as well as *E. coli* and *Corynebacterium* spp. Environmental *Streptococcus* spp are present in dairy herds causing clinical and subclinical mastitis, its presence has been exacerbated due to the increasing implementation of control strategies against contagious pathogens such as *Staphylococcus aureu*s. These programs had reduced the contagious mastitis incidence, however, they had

low effect on the mastitis caused by *Streptococcus* spp, catalase-negative cocci, and by environmental coliform bacteria which affect the udder. Among *Streptococcus* spp, *Streptococcus uberis* (*S. uberis*) is the most frequent as bovine udder pathogen (Olde Riekerink *et al.*, 2008). Moreover, the dairy environment is a determinant factor for mastitis development due to *S. uberis* and *Streptococcus dysgalactiae* subsp. *dysgalactiae (S. dysgalactiae*), and stabled dairies are in greater risk than those held in open pastures (NMC, 1999). Other *Streptococcus* spp related in lesser amount to bovine mastitis are *Streptococcus parauberis* (*S. parauberis*), *Streptococcus salivarius* (*S. salivarius*), and *Streptococcus sanguinis* (*S. sanguinis*) (Whitman, 2009). Some Enterococcus such as *Enterococcus faecium* (*E. faecium*), *Enterococcus faecalis* (*E. faecalis*), *Enterococcus saccharolyticus* (*E. saccharolyticus*) (Østerås, *et al*., 2006). *Aerococcus viridans* (*A. viridans*) has been also related to mastitis but its role has not been elucidated yet (Devriese *et al*., 1999; Zadoks *et al*., 2004). In Hungary, Jánosi1 and Baltay, (2004) determined that the environmental caused mastitis by *Streptococcus* sp and *E. coli* had a prevalence of 12.8% and 6.8 % respectively.

The environmental pathogens, by themselves, are not enough frequent and persistent to cause mastitis or as a significant elevation of somatic cells counts (SCC) of bulk milk (values over 400,000 cells/ml). However, 66% of mastitis caused by environmental *Streptococci* and 85% of those caused by coliform bacteria, display clinical presentation. Therefore, losses due to this type of mastitis can reach substantial amounts even in herds with low SCC (<300,000 cells/ml), mainly due to a high incidence of clinical mastitis as it has been estimated around a 46% of clinical mastitis per year in herds with bulk milk SCC counts of less than 200,000 cells/ml
