Coagulase Negative Staphylococci: Major or Minor Bovine Mastitis Pathogen?
ACVIM 2008
John R. Middleton, DVM, PhD, DACVIM
Columbia, MO, USA

Introduction

Staphylococci are a diverse group of gram-positive cocci which can infect the cow's mammary gland. For the purposes of diagnosing intramammary infection (IMI), staphylococci are usually classified based on their ability to clot plasma, known as the coagulase test, as either coagulase positive or coagulase negative. The most common coagulase positive staphylococci (CPS) isolated from the cow's mammary gland are Staphylococcus aureus. Staphylococcus aureus are considered major mastitis pathogens because many strains are contagious, harbored in the cow's mammary gland, and spread from cow-to-cow at the time of milking. In contrast, coagulase negative staphylococci (CNS) have historically been classified as minor mastitis pathogens and are rarely, if ever, further differentiated when diagnosing an IMI. The assumption has been that, as a group, CNS only cause minor elevations in milk somatic cell count (SCC) and control measures appear to be uniformly efficacious against members of this group. However, there has been a recent resurgence of interest in the role of different species of CNS in mammary gland inflammation. The goal of this paper is to review the current state of knowledge on CNS IMI and explore the potential role of the different CNS species in mammary gland inflammation.

Coagulase Negative Staphylococcal Species

There are at least 39 species and 17 subspecies in the genus Staphylococcus. Coagulase negative staphylococci commonly isolated from milk include S. chromogenes, S. hyicus, S. warneri, S. epidermidis, S. simulans, S. xylosus, and S. sciuri. Staphylococcus xylosus and S. sciuri can be found in the cow's environment, while S. hyicus and S. chromogenes tend to inhabit the teat canal and mammary gland. The remaining CNS species tend to be normal inhabitants of the teat skin. The frequency of occurrence of each of the species of CNS in milk varies from herd-to-herd, with most herds having a predominant CNS species.3-5 Staphylococcus chromogenes, S. simulans, and S. epidermidis seem to be the predominant species isolated in most studies.

Speciation Techniques

Historically few labs have speciated staphylococci beyond CPS and CNS. Any speciation that has been performed was done using phenotypic methods. Characteristics evaluated included colonial morphology, hemolysis, antimicrobial resistance, and biochemical reactivity. A number of commercial biochemical test kits are marketed for speciating staphylococci and are based on fermentation of a series of sugars by the bacterial isolate. However, these test kits are usually validated for staphylococci isolated from humans. In addition, phenotypic traits are not uniformly expressed by a given isolate and hence problems with reproducibility of results can be encountered. While studies report 80% or greater typeability for CNS isolates using these test kits, most of these studies use a phenotypic technique as the reference method. Modern molecular techniques such as sequencing of housekeeping genes have demonstrated that phenotypic speciation techniques provide poor typeability of isolates and frequent misclassification of staphylococcal species. Hence, current research is focusing on genotypic speciation of isolates.

Current Understanding of CNS Mastitis

While many CNS species have been isolated from cow's milk, our understanding of the role of individual species in IMI and mammary gland inflammation is limited. As a group, CNS are the most common organism isolated from cow's milk with as many as 50% of the IMIs in a herd being caused by CNS. In addition, some studies indicate that the prevalence of CNS IMI is on the rise. One study showed an increase in prevalence of subclinical CNS IMI in Dutch dairies from 16.2% to 42.2% and an increase in the isolation of CNS from clinical mastitis cases from 7.3% to 14.1% between 1999 and 2004.1 Despite such data, Schukken and others reported that, in dairy cattle in New York State, CNS IMI only resulted in a moderate increase in milk SCC. Only about 2% of herds would experience a high bulk tank SCC (>400,000 cells/ml) problem due to CNS.2 However, this study considered CNS as a group without species differentiation. It could be that cattle in the 2% of herds experiencing a high milk SCC were infected with a more virulent species or strain, but this cannot be ascertained from the collected data.

Milk from mammary quarters infected with CNS tends to have higher SCC than milk from mammary quarters that yield no bacterial growth.6 Some data suggest that the degree of udder inflammation induced by different CNS species varies according to the infective species with S. hyicus being the most inflammatory in one study.7 Reports suggest that CNS may persist throughout lactation6,8 although these studies did not fingerprint or speciate the CNS causing these persistent infections. Other studies suggest that many CNS infections may be cleared from the mammary gland by non-lactating (dry) cow intramammary antibiotic therapy9, while other reports suggest that new infections occur shortly after calving.10 Prevalence of CNS IMI seems to be higher in first-calf heifers around the time of calving than in multiparous lactating cattle.

What is missing from most of this research is characterization of CNS IMI at the species or genotype level. Currently control and treatment strategies for CNS mastitis are targeted at CNS as a uniform group. Data to date suggest that intramammary antibiotics are uniformly efficacious in clearing CNS IMI from prepartum heifers, lactating cows, and cows during the non-lactating (dry) period. Routine milking time hygiene applied to control other mastitis pathogens has been considered adequate in controlling CNS IMI. However, only one study has evaluated the association between CNS species, genotype and treatment outcome.11 This study demonstrated no differences between genotypes with regard to Beta-lactamase production, severity of mastitis, or response to treatment. Data of this kind is limited and more research is needed to completely define the role of species and genotype on mammary infection, inflammation and outcome of control and treatment strategies. Previous research has shown differences in pathogenicity between strains (genotypes) of S. aureus12 and differences in pathogenicity between species or strains CNS would not be unexpected, but there is a paucity of data to prove or refute such a hypothesis because CNS have not been routinely differentiated to the species level. On average CNS only cause a moderate increase milk SCC, but there may be outliers within these data which represent species or strains that are more or less pathogenic.

Re-Evaluating CNS Mastitis

Data collected as part of ongoing studies at the University of Missouri show that CNS as a group can contribute significantly to bulk milk SCC (Table 1). While contribution to bulk milk SCC may simply reflect prevalence, these data demonstrate that prevalence of CNS varies between herds as does the percent contribution of CNS infected cattle to bulk milk SCC. In both herds there is a wide range of individual cow SCCs in cattle infected with CNS. The wide range could be due to cow-to-cow variation in response to intramammary infection or may be due to differences in species or genotype of CNS causing the IMI. These data also show that cattle may be infected with CNS and another mastitis pathogen at the same time raising the question as to whether some CNS species act synergistically with other mastitis pathogens.

Table 1. Prevalence of CNS in four-quarter composite milk samples collected from cattle on two Missouri dairy farms.

Data are categorized by contribution to bulk milk SCC, geometric mean SCC induced in all cows with CNS, and ranges of individual SCCs from cows with CNS IMI.

Dairy

Pathogen*

Prevalence

% Contribution
to bulk milk
SCC

Geometric mean
individual cow
SCC x 1,000

Range:
individual cow
SCC x 1000

A

CNS

33%

27%

120

14-1599

CNS + Other Pathogen

18%

17%

87

17-1392

Cumulative

54%

44%

107

14-1599

B

CNS

26%

18%

226

22-5972

CNS + Other Pathogen

3%

6%

689

152-4851

Cumulative

29%

24%

254

22-5972

*Four-quarter composite milk sample cultures. CNS + Other Pathogen represent cultures which yielded CNS and another mastitis pathogen. Because the cultures are composite the other pathogen may have been from the same quarter as the CNS IMI or from another quarter.

Currently our laboratory is evaluating the role of CNS species and genotype within species on mammary gland inflammation. In a recent study we collected mammary quarter foremilk samples from all lactating mammary quarters of all cows on the University of Missouri dairy monthly for 19 months. Using these data we have identified 190 mammary quarter CNS IMIs. To date, we have speciated CNS isolates from the beginning and end of each IMI from 82 of 190 mammary quarter CNS IMIs. Staphylococci were identified to the species level using rpoB gene sequencing (Table 2). Mammary quarter milk SCC data associated with these infections has yet to be analyzed. In addition, we will perform pulsed-field gel electrophoresis (PFGE) on each of the isolates to evaluate strain differences within species. PFGE will also allow us to confirm chronicity of infection and evaluate any differences in pathogenicity between strains within a given species of CNS. The preliminary data presented below demonstrate that S. chromogenes is the predominant species isolated from milk of cows on the University of Missouri dairy and there are differences in chronicity of infection between species. A number of mammary quarters in this data set were co-infected with CNS and environmental streptococci. Future studies will evaluate whether a particular species of CNS is more likely to occur in quarters concurrently infected with streptococci. Previous studies suggest that colonization of the teat apices with S. chromogenes can protect mammary quarters from infection with S. aureus.13 Hence, synergistic and antagonistic effects of infection or colonization with certain species of CNS is of interest.

Table 2. CNS species identification based on rpoB gene sequences of CNS isolates from 82 infected mammary quarters on a single dairy farm.

Chronicity of infection is based on monthly mammary quarter milk samples collected from all lactating quarters of all cows over a 19 month period.

CNS species

Number (%)

Median (range)
length of IMI

S. chromogenes

34 (41)

2 (1-11)

S. cohnii ssp. Cohnii

11 (13)

1 (1-3)

S. epidermidis

10 (12)

2.5 (1-10)

S. arlettae

8 (10)

2 (1-3)

S. simulans

8 (10)

1 (1-10)

S. equorum

3 (4)

2 (1-3)

Others (S. xylosus, S. hominis,
S. haemolyticus, S. auricularis
)

8 (10)

1 (1-7)

Work by others14 suggests that some species of CNS possess some of the virulence factors commonly associated with S. aureus. Of particular interest is the ability of CNS to carry the mecA gene which codes for methicillin resistance in S. aureus. Transfer of the mecA from CNS to other staphylococcal species, particularly S. aureus, is of concern because methicillin-resistant staphylococci are usually resistant to a large number of commonly used antibiotics in human and veterinary medicine.

Conclusions

The importance of individual CNS species in bovine mastitis can clearly be debated. Historical evidence suggests that CNS are a homogenous group of mastitis pathogens which have a minor influence on milk SCC. However, much of these data were generated without attention to the species involved. In addition, studies which speciated CNS relied on phenotypic characterization of isolates. Modern molecular techniques allow for more specific definitions of species and technologies such as PFGE allow characterization of strains within a given species of CNS. Evidence exists to suggest that the prevalence of CNS is on the rise on some farms presumably because we have successfully controlled major pathogen IMIs with current environmental and contagious mastitis control procedures. It remains that dairy farmers are paid premiums for low bulk milk SCC, and while mastitis pathogens other than CNS are thought to play a bigger role in elevating bulk milk SCC, CNS cannot be discounted as simply a minor pathogen.6 Work at the University of Missouri and at other centers in the US, Canada, and Europe is currently aimed at defining the role of CNS species and genotype on mammary infection and inflammation using modern speciation and molecular fingerprinting technologies. Results of this work will determine whether future studies focused on species specific control and treatment of CNS IMI are needed.

References

1.  Sampinon et al. Tijdschr Diergeneeskd 2007;132:200.

2.  Schukken Y, et al. Proc Heifer Mastitis Conference, Ghent, Belgium June 24-26, 2007:21.

3.  BirgerssonA, et al. Vet Microbiol 1992;31:181.

4.  Matthews KR, et al. J Dairy Sci 1992;75:1835.

5.  Todhunter et al., 1993.

6.  Rainard P, et al. Vet Res Commun 1990;14:193.

7.  Van der Meer C, et al. Tijdschr Diergeneeskd 1993;118:332.

8.  Timms LL, Schultz LH. J Dairy Sci 1987;70:2648.

9.  Harmon RJ, et al. J Dairy Sci 1986;69:843.

10. Bramley J, et al. Current Concepts of Bovine Mastitis 4th ed. Natl Mast Counc, Madison, WI 1998:12.

11. Taponen S, et al. Vet Microbiol 2006;115:199.

12. Middleton JR, et al. Vet Rec 2002;150:411.

13. De Vliegher S, et al. Vet Microbiol 2003;92:245.

14. Mevius DJ, et al. Proc Heifer Mastitis Conference, Ghent, Belgium June 24-26, 2007:27.

Speaker Information
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John Middleton, DVM, PhD, DACVIM
University of Missouri
Columbia, MO


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