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Abstract

The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 × 102 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 × 103 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

The culture of a sample of bulk tank milk may be a useful technique by which to screen herds for major mastitis pathogens. Staphylococcus aureus and Streptococcus agalactiae, if identified on a culture of a sample of bulk milk, reliably indicate infection of the udder. Environmental bacteria, such as the other streptococci and coliforms, are unlikely to be indicative of the proportion of cows infected with these organisms.

Samples of bulk milk are readily obtainable and can be rapidly and inexpensively cultured to screen large numbers of herds for mastitis-causing bacteria, yet the performance of the test has only recently been formally assessed for its ability to correctly classify herds according to infection status.

A single culture of bulk tank milk has been found to be a test with low sensitivity and high specificity for determining the presence of S. agalactiae or S. aureus in the herd. This means that many infected herds will be called negative, but few uninfected herds will be classified as positive.

The literature assessing the performance of bulk tank milk culture in comparison with other mastitis screening tests, the use of bulk milk culture for prevalence surveys, and factors affecting these results is discussed.

Antibiotic resistance patterns of the major groups of bovine mastitis pathogens (Streptococcus agalactiae, other streptococci, Staphylococcus aureus, and Staphylococcus epidermidis) were examined by determining the minimum inhibitory concentration (MIC) of 13 different antibiotics against bacterial isolates from dairy cattle. The bacterial strains were obtained from milk samples from each cow in 21 New York state dairy herd surveys. In 12 herd surveys (high antibiotic-use group), all 365 cows received antibiotic infusions into the udder at the cessation of each lactation cycle. The 324 animals in the other nine herd surveys (low antibiotic-use group) did not routinely receive antibiotics during the nonlactation period. The MICs from the two groups were compared by calculating for each bacterial group the average MIC, the antibiotic concentration necessary to inhibit 90% of the isolates, and the antibiotic concentration necessary to inhibit 50% of the isolates. Increased resistance to all 13 antibiotics was observed with Streptococcus agalactiae isolates from the high antibiotic use herds. However, there was relatively little difference between the two groups in the resistance patterns of the other bacterial species examined. The most important finding of the study was the identification of a multiple beta-lactam resistance phenotype in Streptococcus agalactiae.

The effect of fresh whole milk without pasteurization or other pretreatment on in vitro antibacterial activity of selected compounds was determined in broth dilution. The milk was collected by hand directly from dairy goats, or by syringe or cannula from bovine quarters showing low bacterial counts. Antibacterial activity was determined in 50% (v/v) milk-broth medium against sensitive mastitis-etiologic strains of Streptococcus agalactiae and Staphylococcus aureus. The indicator salt 2,3,5-triphenyltetrazolium chloride was incorporated in the milk broth medium to determine inoculum growth. Contaminant interference was circumvented through early as well as late readings and comparisons with uninoculated control tubes, with and without the test compounds. Application of the method with more than 75 compounds, including nitrofurans, antibiotics, and other chemicals uncovered marked degrees of milk interference. The method warrants routine use among preliminary screens to relate in vitro with in vivo observations of antimicrobial activity. Similar procedures may be used with serum, skim milk, or mastitis-milk media for separating effects due to protein, lipid, or other elements in product evaluation.

The study purpose was to validate PetrifilmsTM (3M Microbiology, 2005) against standard culture methods in the diagnosis of bovine mastitis organisms in Kenya. On 128 smallholder dairy cattle farms in Kenya, between June 21, 2010 and August 31, 2010, milk samples from 269 cows that were positive on California Mastitis Test (CMT) were cultured using standard laboratory culture methods and PetrifilmsTM (Aerobic Count and Coliform Count –3M Microbiology, 2005), and results were compared. Staphylococcus aureus was the most common bacterium isolated (73 % of samples). Clinical mastitis was found in only three cows, and there were only two Gram-negative isolates, making it impossible to examine the agreement between the two tests for Gram-negative- or clinical mastitis samples. The observed agreement between the standard culture and PetrifilmTM (3M Microbiology, 2005) results for Gram-positive isolates was 85 %, and there was fair agreement beyond that expected due to chance alone, with a kappa (κ) of 0.38. Using culture results as a gold standard, the PetrifilmsTM had a sensitivity of 90 % for Gram-positive samples and specificity of 51 %. With 87 % of CMT-positive samples resulting in Gram-positive pathogens cultured, there was a positive predictive value of 93 % and a negative predictive value of 43 %. PetrifilmsTM should be considered for culture of mastitis organisms in developing countries, especially when Gram-positive bacteria are expected.

Phagocytosis by bovine polymorphonuclear granulocytes of seven capsular polysaccharide type 5 Staphylococcus aureus strains isolated from mastitis [corrected] was investigated by means of luminol-dependent chemiluminescence. Bacteria were grown on four different agar media (brain heart infusion, Columbia broth, modified staphylococcus medium 110, and skim milk) and were opsonized by normal bovine serum. When compared to growth on brain heart infusion agar, Columbia agar, and modified staphylococcus medium 110 agar, growth on skim milk agar rendered five of the strains more resistant to opsonization. The other two strains were resistant in all culture media used. Short periods of incubation in milk after growth on brain heart infusion agar did not augment resistance to phagocytosis, indicating that mere adsorption of milk components on bacteria was not responsible. The variability of the chemiluminescence response of polymorphonuclear leukocytes was pronounced among strains with each growth medium except milk. Growth on modified staphylococcus medium 110 and on milk agar favored the masking of teichoic acid, as shown by inagglutinability with rabbit antiserum. Interestingly, agglutination by a monoclonal antibody to capsular polysaccharide type 5 was optimal when bacteria were grown on skim milk agar. This suggests that capsular polysaccharide participated in the masking effect. These findings indicate that masking of the bacterial target of most of the naturally acquired opsonins present in normal bovine serum occurred when bacteria grew in the presence of milk, resulting in an increased resistance to phagocytosis by polymorphonuclear leukocytes.

A latex agglutination test system (Rapid Mastitis Test [RMT]; Immucell, Portland, Maine) containing reagents for the identification of Staphylococcus aureus and Streptococcus agalactiae from bovine intramammary infections was evaluated with 527 staphylococcal and 267 streptococcal isolates. The RMT Staphylococcus aureus reagent detected 94.2% of 242 Staphylococcus aureus isolates, 80% of 25 Staphylococcus intermedius isolates, and 42.8% of 21 tube coagulase-positive Staphylococcus hyicus isolates. All Streptococcus agalactiae isolates were correctly identified by the RMT Streptococcus agalactiae reagent. Cross-reactions were observed with one Streptococcus dysgalactiae and three Streptococcus uberis strains. The RMT was found to be an acceptable method for the detection of Staphylococcus aureus and Streptococcus agalactiae isolated from bovine mammary glands. The occurrence of coagulase-positive staphylococci other than Staphylococcus aureus requires biochemical testing for species level identification.

Bovine mastitis caused by Mycoplasma agalactiae subsp. bovis was first diagnosed in 16 of 55 cows in an Ontario herd in Feburary 1972. A total of 182 of 598 (30.4%) cows from 33 of 64 (51.5%) farms in widely separated areas of the province were culturally positive. Herd incidence varied from 15 to 40% with one closed herd having an incidence of 61%. Four herds were investigated culturally and serologically by the growth inhibition test for 15 months. In the acute phase the organism was present in the milk in extremely high numbers and could still be isolated from a few cows after eight to 12 months. The sera from 89.5% of the animals with clinical mycoplasma mastitis produced a zone of surface "film" and/or colony inhibition and some cows remained positive for six to 12 months. The disease was experimentally reproduced with a pure culture of the organism isolated from the milk of a cow from one of the herds.

Streptococcus agalactiae continues to be a major cause of subclinical mastitis in dairy cattle and a source of economic loss for the industry. Veterinarians are often asked to provide information on herd level control and eradication of S. agalactiae mastitis. This review collects and collates relevant publications on the subject. The literature search was conducted in 1993 on the Agricola database. Articles related to S. agalactiae epidemiology, pathogen identification techniques, milk quality consequences, and control, prevention, and therapy were included. Streptococcus agalactiae is an oblique parasite of the bovine mammary gland and is susceptible to treatment with a variety of antibiotics. Despite this fact, where state or provincial census data are available, herd prevalence levels range from 11% (Alberta, 1991) to 47% (Vermont, 1985). Infection with S. agalactiae is associated with elevated somatic cell count and total bacteria count and a decrease in the quantity and quality of milk products produced. Bulk tank milk culture has, using traditional milk culture techniques, had a low sensitivity for identifying S. agalactiae at the herd level. New culture methods, using selective media and large inocula, have substantially improved the sensitivity of bulk tank culture. Efficacy of therapy on individual cows remains high. Protocols for therapy of all infected animals in a herd are generally successful in eradicating the pathogen from the herd, especially if they are followed up with good udder hygiene techniques.

Bacterial L forms were isolated from milk samples of dairy cattle infected experimentally with Staphylococcus aureus. Initially, bacterial L forms were induced in vitro from 12 of 44 S. aureus strains isolated from bovine mastitis. Cows were experimentally infected in two experiments with strains shown in vitro to be easily inducible to L form and with S. aureus Newbould 305. Each quarter of the mammary gland was infected with either 10(3) or 10(6) CFU of the test strains. Treatment was initiated with 100,000 U of penicillin G per quarter at the first signs of clinical mastitis. Milk samples were collected daily and cultured on bovine blood agar and PPLO agar (Difco Laboratories, Detroit, Mich.) with 10% horse serum and 5% NaCl. Staphylococcal L forms were isolated from milk samples collected from infected glands in both experiments after antibiotic therapy. Glands with the highest concentrations of leukocytes and bacteria were most likely to yield L forms in milk samples after treatment was initiated. Cows harboring L forms typically yielded parental organisms after cessation of antibiotic therapy. No detectable changes occurred in antibiotic susceptibilities, coagulase production, or biochemical activities in strains induced to L form followed by reversion to the parental form. These results demonstrated that L forms can occur during treatment of bovine mastitis and that L forms may be one explanation for the poor response of staphylococcal bovine mastitis to antibiotic therapy.

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A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs.

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Streptococcus agalactiae remains an important pathogen of dairy herds in Québec, but data about antigenic characteristics of this microorganism are sparse. This study was conducted to determine the variety of S. agalactiae serotypes in dairy herds in Québec. Two hundred and ninety-five isolates cultured from the milk of individual cows from 7 regions of Québec were serotyped. Sixty-two percent of the isolates were untypeable. Among the 38% of typeable isolates, serotype III was found most frequently.

In conclusion, the heterogeneity found among antigenic determinants of isolates from bovine milk suggests that an immunological method for the detection of S. agalactiae performed directly on bovine milk would not be a practical approach.

Six milking cows were inoculated with bovine and human T-mycoplasmas and control materials into the udder via the teat canal. Control materials produced only a slight transient cell response in the milk. Bovine T-mycoplasmas produced clinical mastitis in nine out of ten quarters inoculated. Seven developed clinical mastitis together with visible changes in the milk, excretion of T-mycoplasmas and greatly increased cell counts in the milk. In three of these quarters, in two different cows, milk secretion ceased completely. Two quarters in a different cow showed visible milk changes, excretion of T-mycoplasmas and increased cell counts. Two quarters were inoculated with human T-mycoplasmas and neither produced any signs of mastitis.

Infection of the udder with T-mycoplasmas did not stimulate high-titre serum antibody levels as measured by the metabolic inhibition test, but whey samples gave high titres in two of the cows that were able to control and resolve the infection.

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This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.

Staphylococcus aureus isolates from bovine milk were assessed for capsule or slime production. When pure S. aureus cultures in milk were inoculated directly into serum-soft agar constituted with a modified staphylococcus 110 medium, 100% of the isolates grew with diffuse colony morphology. Diffuse colony morphology was rapidly lost on subculture and was more rapidly lost in brain heart infusion-serum-soft agar. No evidence was seen for encapsulation in India ink preparations or by the clumping factor test. It was concluded that freshly isolated S. aureus strains produce slime, not true capsules. During examination of the 84 milk samples that grew staphylococci in addition to S. aureus (27.4%), a significant number of coagulase-negative staphylococcal species were encountered and identified by conventional tests as S. simulans (41.7%), S. xylosus (11.9%), S. epidermidis (3.6%), S. saprophyticus (3.6%), S. hyicus (2.9%), S. cohnii (1.2%), S. haemolyticus (1.2%), and S. warneri (1.2%). Five isolates (6.0%) were not identified. Attempts were also made to identify the isolates by the API Staph-Ident system, which gave an overall accuracy of 45.2%. The susceptibilities of the isolates to a variety of antibiotics were determined, and they appeared to be less resistant than human clinical isolates.

Mastitis, inflammation of the mammary gland, can be caused by a wide range of organisms, including gram-negative and gram-positive bacteria, mycoplasmas and algae. Many microbial species that are common causes of bovine mastitis, such as Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae and Staphylococcus aureus also occur as commensals or pathogens of humans whereas other causative species, such as Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae or Staphylococcus chromogenes, are almost exclusively found in animals. A wide range of molecular typing methods have been used in the past two decades to investigate the epidemiology of bovine mastitis at the subspecies level. These include comparative typing methods that are based on electrophoretic banding patterns, library typing methods that are based on the sequence of selected genes, virulence gene arrays and whole genome sequencing projects. The strain distribution of mastitis pathogens has been investigated within individual animals and across animals, herds, countries and host species, with consideration of the mammary gland, other animal or human body sites, and environmental sources. Molecular epidemiological studies have contributed considerably to our understanding of sources, transmission routes, and prognosis for many bovine mastitis pathogens and to our understanding of mechanisms of host-adaptation and disease causation. In this review, we summarize knowledge gleaned from two decades of molecular epidemiological studies of mastitis pathogens in dairy cattle and discuss aspects of comparative relevance to human medicine.

Staphylococcus aureus is a common causative agent of bovine mastitis in dairy herds. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals as well as the community is a significant and costly public health concern. S. aureus-related bovine mastitis is a common reason for therapeutic and/or prophylactic use of antibiotics on dairy farms. In this study, herd prevalence of S. aureus, including MRSA, was estimated from bulk tank milk (BTM) from Minnesota farms. A total of 150 pooled BTM samples from 50 farms, collected over 3 seasons (spring, summer, and fall of 2009), were assessed. Herd prevalence of methicillin-susceptible S. aureus (MSSA) was 84%, while MRSA herd prevalence was 4%. A total of 93 MSSA isolates and 2 MRSA isolates were recovered from 150 BTM samples. Antibiotic susceptibility testing of S. aureus isolates showed pansusceptibility in 54 isolates, resistance to a single antibiotic class in 21 isolates, resistance to two antibiotic classes in 13 isolates, and resistance to ≥3 antibiotics classes and thus multidrug resistance in 5 isolates. The two MRSA isolates displayed resistance to β-lactams, cephalosporins, and lincosamides and were multiresistant. Staphylococcal protein A gene (spa) typing identified spa types t529 and t034 most frequently among methicillin-susceptible isolates, while t121 was observed in MRSA isolates. Seven isolates, including the two MRSA isolates, produced staphylococcal enterotoxins B, C, D, and E on overnight culture. MRSA isolates were further genotyped using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Of the 2 MRSA isolates, one had a composite genotype profile of MLST ST 5-PFGE USA100-unknown spa type, which has been reported among hospital-associated MRSA isolates, while the second isolate carried the MLST ST 8-PFGE USA300-spa type t121 genotype, commonly identified among community-associated MRSA isolates. These results suggest that MRSA genotypes associated with hospitals and community can be isolated from milk at very low rates.

The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

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Staphylococcus aureus is one of the most important causal agents of bovine mastitis. Population studies on bovine Staphylococcus aureus isolates have identified considerable genetic heterogeneity among strains causing mastitis. The aim of this study was to investigate the contribution of different clonal complexes and the occurrence of virulence factors and resistance determinants within bovine Staphylococcus aureus isolates.

A total of 189 Staphylococcus aureus isolates obtained from milk samples of 34 dairy herds in the German Federal State of Thuringia were characterised by microarray technology.

The isolates were assigned to eleven different clonal complexes with CC151, CC479 and CC133 being dominant (together 80.5%). The methicillin resistance gene mecA was found in four isolates (2.1%), which belonged to CC398. Enterotoxin genes could be detected in 79.3% of analysed Staphylococcus aureus and 19 isolates (10.1%) harboured a distinct allele of the toxic shock syndrome toxin gene, tst-RF122. The most striking finding of the present study was that almost all except one isolate (151/152) belonging to CC151, CC479 and CC133 harboured the leukocidin genes lukF-P83 and lukM, whereas no further isolates from other lineages possessed these genes.

The consistent occurrence of lukF-P83/lukM in the dominating clonal complexes suggests an essential role of this leukocidin in the etiology of bovine mastitis.

Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.

Comparison of three methods by which salmonellae may be isolated and enumerated from dried albumen, direct inoculation of enrichment media, centrifugation of samples, and pre-enrichment in noninhibitory media, reveals pre-enrichment to be the method of choice.

The superiority of pre-enrichment manifests itself in replicate aliquots of the same sample by producing a statistically significant increase in numbers of isolations of salmonellae and in empirical use with various albumen samples by consistently higher values of most probable numbers (MPN).

The primary factor involved in this superiority appears to be the greater ability of small numbers of salmonellae to initiate growth in the nonselective mannitol purple sugar broth than in the inhibitory enrichment media.

The method of analysis recommended entails inoculation of mannitol broth pre-enrichment medium, transfer of 24-hr culture aliquots to tetrathionate broth, and streaking on brilliant green agar for isolation of salmonellae.

Mastitis is a common disease in dairy cattle and is an inflammatory response of the breast tissue to bacterial attack to this tissue. Mastitis causes considerable loss to the dairy industry, among the several bacterial pathogens that can cause mastitis; Staphylococcus aureus is probably the most lethal agent because it causes chronic and deep infection in the mammary glands that is extremely difficult to cure. Several virulence factors including coagulase gene are produced by S. aureus and may contribute to its pathogenicity. This study was conducted to investigate the coagulase gene polymorphism of S. aureus isolated from clinical and sub-clinical bovine mastitis milk samples in Isfahan and Chaharmahal va Bakhtiari provinces of Iran. Amplification of the coagulase gene from 86 S. aureus strains isolates by specific primers showed 31 specimens contained 970 bp fragment, and 11 strains contained 730 bp fragment relevant to coa gene (coagulase) in PCR. After enzymatic digestion with AluI, 31 specimens contained three bands: 320, 490, and 160 bp (genotype I) and 11 specimens contained two bands: 490 and 240 bp (genotype VIII) in the RFLP.

Staphylococcus aureus is a major pathogen associated with mastitis, a disease affecting both women and dairy cows. The longitudinal profiles of bovine peripheral blood and mammary gland lymphocyte phenotypes in response to S. aureus-induced mastitis were investigated in dairy cows. Increased percentage of CD4 lymphocytes in the mammary gland between 1 and 8 days post-inoculation, increased milk CD4 protein density per cell between 1-8 days post-inoculation, and a statistically significant negative correlation between post-inoculation bacterial counts in milk and blood lymphocyte CD4 protein density were found. Together with blood and milk leukocyte counts, the milk lymphocyte CD4/CD8 ratio and the milk lymphocyte CD4 protein density were more informative indicators than milk somatic cell counts and bacteriology for identification of early vs. late inflammatory phases. These findings suggest that CD4+ lymphocytes play a protective role in the early stages of S. aureus-induced mastitis.

Bovine milk is generally considered to be almost devoid of complement, on the basis of undetectable hemolytic activity, unless inflammation recruits plasma components. This study examines the deposition of complement components on a mastitis-causing isolate of Streptococcus agalactiae by enzyme-linked immunosorbent assay (ELISA). Neat milk from mid-lactating, uninflamed mammary glands (normal milk) effected marked C3 deposition on bacteria. Kinetic studies showed a protracted lag period (30 to 45 min) preceding C3 deposition, which required about 2 h to reach a maximum. Experiments with diluted serum suggested that this slow C3 deposition resulted mainly from the low concentration of certain components of complement in milk. Bacteria incubated in neat milk readily bound bovine conglutinin, indicating the presence of iC3b. Elution of covalently bound C3 fragments with hydroxylamine confirmed the deposition of C3b and iC3b on bacteria. Deposition of C4 on bacteria was not detected in neat milk, suggesting that C3 deposition did not result from the activation of the classical pathway. This was not the result of a lack of antibodies. However, C4 deposition could be obtained by adding purified bovine C1q to normal milk, and C3 deposition was accelerated, suggesting the participation of the classical pathway. The deposition of C1q on antibody-sensitized bacteria was impeded by milk compared with that of C1q diluted in phosphate-buffered saline. Concentrations of C1q in normal milk were very low, ranging from 150 to 250 ng/ml. Overall, these findings indicate that C1q was a limiting factor of the classical pathway in normal milk. The capacity of milk to deposit C3 fragments on mastitis-causing S. agalactiae prompts further studies to investigate its role in opsonophagocytosis.