Bovine Mastitis Pathogens: Prevalence and Effects on Somatic Cell Count

© 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. Bovine Mastitis Pathogens: Prevalence and Effects on Somatic Cell Count


Introduction
Bovine mastitis is the most prevalent and costly disease, affecting dairy farms worldwide. Economic losses associated with mastitis derive mainly from a decrease in milk production and to a lesser extent, from the culling of chronically infected cows, cost of veterinary treatment, and penalties on milk quality (Seegers et al., 2003). Mastitis is caused by a wide spectrum of pathogenic agents that penetrate the teat canal and multiply in the udder cistern. The majority of mastitis cases are produced by a relatively small group of bacteria, including Staphylococcus aureus, Streptococcus uberis, Mycoplasma spp and Escherichia coli (Calvinho & Tirante, 2005). Bovine mastitis is characterized by inflammation of the mammary gland. The inflammation severity depends on the causative agent and the host response (Bannerman et al., 2004;Barkema et al., 2006;Burvenich et al., 2003;Petzl et al., 2008). Resident and recruited cells together play an essential role in immediate defense against local infection (Rainard & Riollet, 2006). Extensive neutrophil recruitment from the circulation to the lumen of the mammary gland is a hallmark of the early immune response to mammary infection (Thomas et al., 1994;Sordillo & Streicher, 2002;Oviedo-Boyso et al., 2007). When designing mastitisprevention and control programs, it is worthy to take on account the adoption level of mastitis-prevention management practices and control programs as well as the etiology of the intramammary infections (IMI), the herd-level prevalence of contagious mastitis pathogens, and the general factors that influence milk production.

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., Milk Production -An Up-to-Date Overview of Animal Nutrition, Management and Health 360 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), and43% 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)
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 (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, 2004Sampimon 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) (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

Somatic cell counts (SCC)
Throughout the world in the last ten years, udder health programs have been increasing (Godkin et al., 1999;Østerås et al., 1998;Plym 1996a;Plym et al., 1996b;Sargeant et al., 1998), and regarded as a critical production issue on dairy farms. In Europe, the European Economic Community (EEC) since 1998 does not recommend consumption of milk with SCC over 400, 000 cells/ml. In North America the limit has been established at 750, 000 (USA) and in Canada at 500, 000 cells (Sargeant et al., 1998).
Somatic cells are, in great quantity, cells of the immune system (80% in uninfected quarters, and 99% in quarters with mastitis) (Sordillo et al., 1997). They are part of the natural defense mechanisms, including lymphocytes, macrophages, polymorphonuclear and some epithelial cells (Pillai et al., 2001). Somatic cells are therefore a reflection of the inflammatory response to an IMI. Somatic cell counts are often used to distinguish between infected and uninfected quarters according to the general agreement between infection status and the inflammatory response to infection reflected as an increased SCC. As with any diagnostic test, errors will occur when solely depending on a single test. To minimize error, diagnostic test parameters such as sensitivity & specificity are calculated at various cut-off values in the continuum SCC (Schepers et al., 1997). In North America and Europe the SCC for an uninfected quarter is approximately 70, 000 cells. There is of course variation around this mean; its value can increase with age, decreasing milk production and days in milk period (Schepers et al., 1997).
Hence, to be able to distinguish between infected and uninfected quarters a cut-off of approximately 200, 000 to 250, 000 cells is accepted (Dohoo et al., 1991;Laevens et al., 1997;Leslie et al, 1997;Schepers et al, 1997). At this cut-off value, diagnostic sensitivity is approximately 75%, and specificity approximately 90% (Schepers et al., 1997). The 200, 000 cells cut-off is not considered a physiological cell concentration in milk able to distinguish between healthy and unhealthy udders, but it is a practical value under field conditions (minimizing diagnostic error). Erskine et al. 1987, evaluated 32 dairy herds, 16 with low SCC less than or equal to 150, 000 cells/ml and 16 with high SCC greater than or equal to 700, 000 cells/ml. From the 16 herds with low SCC, S. agalactiae was isolated in two herds (12.5%), and S. aureus was isolated from seven herds (44%). Moreover both microorganisms were found in all of the herds with high SCC, a program of post-milking teat dipping and treatment of all cows at the beginning of the non-lactating period was practiced in the herds with low SCC. Whist et al. (2007) reported low SCC in milk from heifers having Streptococcus dysgalactiae IMI and in noninfected glands the results indicated that SCC were high (between 50,000 and 100,000 cells/ml) during the immediate postpartum period, within the next 5 days after calving.

Bulk tank milk (BTM) SCC
BTM SCC is a general indicator of the udder health in a herd and it is also regarded as an indirect measure of milk quality (Schukken et al. 2003). Elevated SCC, are correlated with changes in milk composition, casein and more serum-derived whey proteins, as well as increased proteolytic and lipolytic activities (Auldist & Hubble, 1998). SCC may, however, vary greatly depending on factors such as number of lactations, stage of lactation, season and milking frequency (Harmon, 1994;Pyörälä, 2003). In BTM, where the total volume of milk will dilute effects from affected quarters, SCC appears to be less sensitive and specific as a biomarker for milk quality, e.g. suitability for cheese production (Leitner et al., 2006).
Bulk tank milk SCC assist in directing milk quality control programs and assist with the identification of risk factors in herds. The production of milk with low bacterial counts starts at the farm and is influenced by many procedures related to farm management practices. At the farm level, microbial contamination of BTM occurs through three main sources; bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis organisms within the udder (Murphy & Boor, 2000). The levels and types of microorganisms in BTM provide valuable information on the hygienic conditions during the steps of milk production. The microbiological count methods are used to monitor hygienic quality of raw milk including the total aerobic count (TAC). TAC is the most common method for the assessment of bacterial quality of raw milk, it estimates the total number of bacteria present at the farm´s pickup time, providing an overall hygienic milk-quality measure; however, it is limited for the identification of the bacteria contamination source. An alternative has been the standard plate count (SPC) and the preliminary incubation count (PIC), a selective count is measuring psychotropic bacteria, which will grow and multiply under improper refrigeration conditions. These organisms can create undesirable odors and off-flavors. Many psychotropic bacteria can also produce heat-stable enzymes that will survive pasteurization degrading and reducing milk and milk products during shelf-life (Hayes & Boor, 2001). The laboratory pasteurization count (LPC), another selective count, estimates the number of thermoduric bacteria, mainly from the surfaces of poorly cleaned farm equipment that will survive a laboratory-scale batch pasteurization process. Thermoduric bacteria have been associated with spoilage of pasteurized milk. The Coliform count (CC) measures the number of coliform bacteria in milk, organisms primarily coming from the cow's environment, therefore high CC will give an estimation of the production sanitary status and practices. Coliforms can also incubate on residual films of improperly cleaned milking equipment (Reinemann et al., 2003).
The results from a case-control study indicated that TAC and PIC were mostly related to cow and stall hygiene, whereas LPC and CC were related to equipment hygiene (Elmoslemany et al., 2009;Jayarao et al., 2004), and included among the bacteria groups associated with bovine IMI are Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp, Streptococcus spp, Escherichia coli, and SCN.

Prevalence of mastitis pathogens and somatic cell counts
In Mexico, the prevalence of mastitis pathogens in BTM SCC from 224 milk samples of 112 herds was as follows; Mycoplasma spp were isolated from 62 herds (55%), S. aureus from 34 cattle barns, S. uberis and CNS were isolates from milk from 42 herds (37.5%) and from 43 (38.3%) bulk tank milk samples. The geometric mean of SCCs was 465, 000 cells/ml. No significant differences were observed in SCCs between Mycoplasma spp, S. aureus and Streptococcus spp positive and negative herds (P>0.5) (Miranda-Morales, et al., 2008).
In Latin America, few data had been carried out regarding microorganism's prevalence and SCC in cases of clinical and subclinical mastitis. Nonetheless, regarding bovine mastitis, Calvinho et al, (2005) assessed the primary pathogens prevalence, and its relation with the general udder health status in Argentina from 1983 to 2001. The subclinical mastitis showed a prevalence of 25.3% of S. aureus in the 80's and through the years it has been decreasing until a level of 13.9% in 2000. This situation was also observed regarding S. agalactiae, which has been reducing its prevalence from 8.8 to 1.6%; Streptococcus spp from 19.3 to 6.5% and coliforms from 2.7 to 2%. The prevalence observed for the same pathogens causing clinical mastitis, were low prevalence levels for S. aureus , S. agalactiae and coliforms respectively from 34.45 to 29.2%, 13 to 3.9%, and from 20 to 4.4%. In contrast the CNS, S. dysgalactiae and Streptococcus spp registered rising prevalence from 2.1 to 12.7%, 1.7 to 15.9%, and from 6.4 to 19.8% respectively. This situation was also seen among SCC registering levels of 400, 000 to 900, 000 cells/ml in the 80's, since after a sustained decrease in the SCC from BTM in recent years; in 2004 ranging around 300, 000 cells/ml, and in 2005, an average of 384, 000 cells/ml (SAGPyA, 2005). The producers have been implemented systematically control programs based on hygiene and antibiotic therapy, there has been a decrease in the prevalence of contagious pathogens and environmental relative increase, however it should be noted that the SCC values remain high compared with those of countries with high dairy development.
In Perú, Ortiz, et al., 2006, assessed the SCC in dairy herds of different technological levels in Arequipa, milk samples were collected twice in 2005. The stables were stratified according to their technological level in high, medium and low. The general average of SCC were 505 x 103 x 103 ± 150 cells/ml, and significant differences between technology levels were identified as SCC were 353, 559 and 603 x 103 cells/ml for high, medium and low, respectively (p <0.05), feature explained by the dilution of somatic cells in a greater volume of milk and a more rational application of best practices to prevent and control mastitis in the most sophisticated stables. On the other hand, limited access to training adversely affects low-technology. In a study by Moraga et al (1994)  , 000 cells/ml, therefore milk quality requirements are barely meet except for some regions that had achieved SCC levels of < 200, 000 cells/ml, and low prevalence of mastitis associated pathogens. Therefore, herd overall studies are mandatory for mastitis control programs including duration of lactation, season, milk production and parity. But will also be guided by the prevalence of mastitis pathogens and by the, geographic region and production practice.