Streptococcus dysgalactiae ssp. dysgalactiae in Norwegian bovine dairy herds: Risk factors, sources, and genomic diversity.

Despite the importance of Streptococcus dysgalactiae ssp. dysgalactiae (SDSD) as an udder pathogen, the reservoir and epidemiological characteristics of this bacterium are largely unexplored. The aims of this study were to investigate risk factors for SDSD intramammary infections (SDSD-IMI) in Norwegian bovine dairy herds, identify sources of SDSD on animals and in the environment, and elucidate the genetic diversity of SDSD isolates. Data from herd recordings and a questionnaire were used to investigate herd-level risk factors for SDSD-IMI in 359 freestall dairy herds. Seven herds with a suspected high prevalence of SDSD-IMI were visited to sample extramammary sources (e.g., skin, wounds, mucous membranes, and freestall environment). Bacterial isolates were whole-genome sequenced to investigate the distribution of SDSD genotypes within herds and to assess the phylogenetic relationship between SDSD isolates from 27 herds across Norway. Risk factors for high incidence of SDSD-IMI in freestall dairy herds were related to housing, including closed flooring in alleys and rubber mats in cubicle bases. Parlor milking was also a risk factor compared with automatic milking systems. From herd visits, a considerable proportion of extramammary samples were SDSD positive, particularly from wounds and skin of the animals and the cubicle bases. Samples from mucous surfaces (nostrils, rectum, and vagina) and water troughs were least frequently positive. Eight multilocus sequence types (ST) were identified among the sequenced isolates from 27 herds, and phylogenetic analyses revealed 8 clades corresponding to ST. No significant association was identified between sampling site (milk, body sites, and environment) and ST. In 4 of 6 herds from which 5 or more isolates were available, one ST dominated and was found in milk and extramammary samples. One ST (ST453) was found in 15 of 27 herds, which implies that this is a widely distributed and possibly a bovine-adapted strain. Findings in this study suggest that SDSD is a cow-adapted opportunist with potential for contagious transmission, and that the freestall environment is likely to play a role in transmission between cows.

Distribution of somatic cell count and udder pathogens in Norwegian dairy goats.

Compared with dairy cows, goat somatic cell count (SCC) is higher and probably more affected by physiological factors such as parity, stage of lactation, and season. Thus, SCC is believed to be a less precise indicator of intramammary infections in dairy goats, and no consensus exists on SCC thresholds for considering goats as infected. The Norwegian Goat Recording System maintains individual goat production records and results from microbiological analyses of milk samples. In this retrospective observational study, we used recordings over a 10-yr period (2010 to 2020) to describe the association between individual goat SCC and noninfectious factors, as well as intramammary infections. The median SCC in the 1,000,802 milk recordings included in the study was 440,000 cells/mL, and the mode was 70,000 cells/mL. Somatic cell count increased with parity, days in milk, estrus, pasture season, and intramammary infections. The effect of parity and stage of lactation was significantly higher in infected compared with uninfected goats. Staphylococci dominated as causes of intramammary infections, with Staphylococcus aureus as the udder pathogen associated with highest SCC. The most prevalent non-aureus staphylococci were Staphylococcus warneri, Staphylococcus epidermidis, and Staphylococcus caprae. This study provides guidelines for interpretation of goat SCC at different parities and stages of lactations under Norwegian management conditions. We revealed a considerable variation in SCC associated with physiological factors, indicating that the cutoff for identifying infected goats should be a dynamic threshold adjusted for parity, stage of lactation, and season.

Streptococcus agalactiae in the environment of bovine dairy herds--rewriting the textbooks?

Many free-stall bovine dairy herds in Norway fail to eradicate Streptococcus agalactiae despite long-term control measures. In a longitudinal study of 4 free-stall herds with automatic milking systems (AMS), milk and extramammary sites were sampled 4 times with 1-2 month intervals. Composite milk, rectal- and vaginal swabs were collected from dairy cows; rectal swabs from heifers and young stock; rectal- and tonsillar swabs from calves; and environmental swabs from the AMS, the floors, cow beds, watering and feeding equipment. A cross sectional study of 37 herds was also conducted, with 1 visit for environmental sampling. Fifteen of the herds were known to be infected with S. agalactiae while the remaining 22 had not had evidence of S. agalactiae mastitis in the preceding 2 years. All samples were cultured for S. agalactiae, and selected isolates (n=54) from positive herds were genotyped by Multi Locus Sequence Typing (MLST). Results show that the bovine gastrointestinal tract and the dairy cow environment are reservoirs of S. agalactiae, and point to the existence of 2 transmission cycles; a contagious transmission cycle via the milking machine and an oro-fecal transmission cycle, with drinking water as the most likely vehicle for transmission. Ten sequence types were identified, and results suggest that strains differ in their ability to survive in the environment and transmit within dairy herds. Measures to eradicate S. agalactiae from bovine dairy herds should take into account the extra-mammary reservoirs and the potential for environmental transmission of this supposedly exclusively contagious pathogen.

Caprine arthritis encephalitis and caseous lymphadenitis in goats: use of bulk tank milk ELISAs for herd-level surveillance.

The objective of this study was to evaluate the diagnostic performance of two ELISA tests applied to bulk tank milk (BTM) as the first part of a two-step test scheme for the surveillance of caprine arthritis encephalitis (CAE) and caseous lymphadenitis (CLA) infections in goats. The herd-level BTM tests were assessed by comparing them to the test results of individual serological samples. The potential for refining the cut-off levels for BTM tests used as surveillance tools in a population recently cleared of infection was also investigated. Data was gathered on serum (nCAE =9702 and nCLA=13426) and corresponding BTM (nCAE=78 and nCLA=123) samples from dairy goat herds enrolled in the Norwegian disease control and eradication programme 'Healthier Goats'. The results showed that the sensitivity and specificity of the CAE ELISA BTM test with respect to detecting ≥2 per cent within-herd prevalence were 72.7 per cent and 86.6 per cent, respectively. For the CLA ELISA BTM the sensitivity and specificity were 41.4 per cent and 81.7 per cent, respectively, for the same goal of detection. The results suggest that BTM testing can be applied as a cost-effective first step for early detection of CAE and CLA infection.

The Norwegian Healthier Goats program--modeling lactation curves using a multilevel cubic spline regression model.

In 2001, the Norwegian Goat Health Service initiated the Healthier Goats program (HG), with the aim of eradicating caprine arthritis encephalitis, caseous lymphadenitis, and Johne's disease (caprine paratuberculosis) in Norwegian goat herds. The aim of the present study was to explore how control and eradication of the above-mentioned diseases by enrolling in HG affected milk yield by comparison with herds not enrolled in HG. Lactation curves were modeled using a multilevel cubic spline regression model where farm, goat, and lactation were included as random effect parameters. The data material contained 135,446 registrations of daily milk yield from 28,829 lactations in 43 herds. The multilevel cubic spline regression model was applied to 4 categories of data: enrolled early, control early, enrolled late, and control late. For enrolled herds, the early and late notations refer to the situation before and after enrolling in HG; for nonenrolled herds (controls), they refer to development over time, independent of HG. Total milk yield increased in the enrolled herds after eradication: the total milk yields in the fourth lactation were 634.2 and 873.3 kg in enrolled early and enrolled late herds, respectively, and 613.2 and 701.4 kg in the control early and control late herds, respectively. Day of peak yield differed between enrolled and control herds. The day of peak yield came on d 6 of lactation for the control early category for parities 2, 3, and 4, indicating an inability of the goats to further increase their milk yield from the initial level. For enrolled herds, on the other hand, peak yield came between d 49 and 56, indicating a gradual increase in milk yield after kidding. Our results indicate that enrollment in the HG disease eradication program improved the milk yield of dairy goats considerably, and that the multilevel cubic spline regression was a suitable model for exploring effects of disease control and eradication on milk yield.

Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

Norwegian mastitis control programme.

This paper describes the methods and results of the Norwegian Mastitis Control Program implemented in 1982. The program has formed an integral part of the Norwegian Cattle Health Services (NCHS) since 1995. The NCHS also have specific programs for milk fever, ketosis, reproduction and calf diseases. The goal of the program is to improve udder health by keeping the bulk milk somatic cell count (BMSCC) low, to reduce the use of antibiotics, to keep the cost of mastitis low at herd level and improve the consumers' attitude to milk products. In 1996, a decision was made to reduce the use of antibiotics in all animal production enterprises in Norway by 25% within five years. Relevant data has been collected through the Norwegian Cattle Herd Recording System (NCHRS); including health records since 1975 and somatic cell count (SCC) data since 1980. These data have been integrated within the NCHRS. Since 2000, mastitis laboratory data have also been included in the NCHRS. Data on clinical disease, SCC and mastitis bacteriology have been presented to farmers and advisors in monthly health periodicals since 1996, and on the internet since 2005. In 1996, Norwegian recommendations on the treatment of mastitis were implemented. Optimal milking protocols and milking machine function have been emphasised and less emphasis has been placed on dry cow therapy. A selective dry cow therapy program (SDCTP) was implemented in 2006, and is still being implemented in new areas. Research demonstrates that the rate of clinical mastitis could be reduced by 15% after implementing SDCTP. The results so far show a 60% reduction in the clinical treatment of mastitis between 1994 and 2007, a reduction in BMSCC from 250,000 cells/ml to 114,000 cells/ml, and a total reduction in the mastitis cost from 0.23 NOK to 0.13 NOK per litre of milk delivered to the processors, corresponding to a fall from 9.2% to 1.7% of the milk price, respectively. This reduction is attributed to changes in attitude and breeding, eradicating bovine virus diarrhoea virus (BVDV) and a better implementation of mastitis prevention programmes.

Relationships between milk culture results and composite milk somatic cell counts in Norwegian dairy cattle.

Associations between test-day composite milk somatic cell counts (CMSCC) and results from quarter milk cultures for various pathogens associated with mastitis, including Staphylococcus aureus, Streptococcus spp., coagulase-negative staphylococci (CNS), were investigated. S. aureus was dichotomized according to sparse (1,500 colony forming units/mL of milk) growth of the bacteria. Quarter milk samples were obtained on between 1 and 4 occasions from 2,714 cows in 354 Norwegian dairy herds, resulting in a total of 3,396 samples. Cows included in the study were randomly selected, without regard to current or previous udder health status. Measures of test-day CMSCC were obtained every second month, and related to 3528 microbiological diagnoses at the cow level. Mixed linear regression models incorporating a compound symmetry covariance structure accounting for repeated test-day CMSCC within cow, and a random effect variable on herd level, was used to quantify the relationship between a positive milk culture and the natural logarithm of test-day CMSCC (LnCMSCC). The material was stratified in time periods before 151 d in milk (DIM) and after 150 DIM. A positive diagnosis for any category of mastitis pathogen was significantly associated with elevated CMSCC. Pathogen positive cows sampled for microbiological diagnosis during the first 150 DIM had higher levels of CMSCC throughout lactation than cows with a positive diagnosis after 150 DIM. Streptococcus spp.-positive milk cultures were associated with steadily elevated values for CMSCC throughout lactation both when sampled before and after 150 DIM. Cows diagnosed with rich growth of S. aureus after 150 DIM experienced a characteristic and sharp increase in CMSCC, but this effect was not observed in cows with a positive diagnosis for rich growth of S. aureus during the first 150 DIM. A considerable increase in CMSCC in cows positive for CNS during the first part of the lactation period was also observed. The practicability of using CMSCC in a diagnostic test to identify cows with a positive milk culture for mastitis pathogens was also assessed. The sensitivity, specificity, and positive predictive values of the tests were regarded as low when sampling for milk culture was conducted, irrespective of cow level characteristics.

Risk factors associated with colostrum quality in Norwegian dairy cows.

The objectives of the present study were to evaluate colostrum quality in Norwegian dairy cows based on IgG content, and to identify associations between possible risk factors and low colostral IgG. A longitudinal cross-sectional survey on calf health in Norway was performed between June 2004 and December 2006. The participating dairy herds were randomly selected among herds registered in the Norwegian Dairy Herd Recording System as having at least 15 cow years. The participating farmers were requested to sample 10 mL of colostrum from the first milking after calving from 12 cows that had calved during the defined project period of 365 d. Colostrum samples from 1,250 cows from 119 herds were collected. The material consisted of 451, 337, 213, and 249 samples collected from cows in their first, second, third, and fourth parity or more, respectively. Analysis was performed on IgG content by using single radial immunodiffusion. Mixed models with herd as a cluster were fit by using grams of IgG per liter of colostrum as the dependent variable for the statistical analyses. The IgG content in the colostrum sampled ranged from 4 to 235 g/L, with a median of 45.0 g of IgG/L, with the 10th, 25th, 75th, and 90th percentiles being 23.1, 31.4, 63.6, and 91.6 g of IgG/L, respectively. Altogether, 57.8% of the samples contained less than the desired 50 g of IgG/L of colostrum. Cows in their fourth parity or more were found to have significantly higher levels of IgG per liter of colostrum than cows in their first or second parity. Colostrum from cows in their second parity had the lowest level of IgG. Cows calving during the winter months (December, January, and February) produced colostrum with a significantly lower IgG content compared with cows calving in any other season of the year. Somatic cell count, measured after calving, was significantly higher in cows producing colostrum of inferior quality compared with those producing high-quality colostrum. Of the total variation in colostrum quality, 13.7% could be explained by cluster effects within herd. The variation in IgG content in colostrum produced by Norwegian dairy cows indicates a need for improved colostrum quality control and subsequent adjustment of the colostrum feeding regimen to ensure a protective immunological status for newborn calves.

Relationships between milk culture results and milk yield in Norwegian dairy cattle.

Associations between test-day milk yield and positive milk cultures for Staphylococcus aureus, Streptococcus spp., and other mastitis pathogens or a negative milk culture for mastitis pathogens were assessed in quarter milk samples from randomly sampled cows selected without regard to current or previous udder health status. Staphylococcus aureus was dichotomized according to sparse (< or =1,500 cfu/mL of milk) or rich (>1,500 cfu/mL of milk) growth of the bacteria. Quarter milk samples were obtained on 1 to 4 occasions from 2,740 cows in 354 Norwegian dairy herds, resulting in a total of 3,430 samplings. Measures of test-day milk yield were obtained monthly and related to 3,547 microbiological diagnoses at the cow level. Mixed model linear regression models incorporating an autoregressive covariance structure accounting for repeated test-day milk yields within cow and random effects at the herd and sample level were used to quantify the effect of positive milk cultures on test-day milk yields. Identical models were run separately for first-parity, second-parity, and third-parity or older cows. Fixed effects were days in milk, the natural logarithm of days in milk, sparse and rich growth of Staph. aureus (1/0), Streptococcus spp. (1/0), other mastitis pathogens (1/0), calving season, time of test-day milk yields relative to time of microbiological diagnosis (test day relative to time of diagnosis), and the interaction terms between microbiological diagnosis and test day relative to time of diagnosis. The models were run with the logarithmically transformed composite milk somatic cell count excluded and included. Rich growth of Staph. aureus was associated with decreased production levels in first-parity cows. An interaction between rich growth of Staph. aureus and test day relative to time of diagnosis also predicted a decline in milk production in third-parity or older cows. Interaction between sparse growth of Staph. aureus and test day relative to time of diagnosis predicted declining test-day milk yields in first-parity cows. Sparse growth of Staph. aureus was associated with high milk yields in third-parity or older cows after including the logarithmically transformed composite milk somatic cell count in the model, which illustrates that lower production levels are related to elevated somatic cell counts in high-producing cows. The same association with test-day milk yield was found among Streptococcus spp.-positive pluriparous cows.

Streptococcus dysgalactiae isolates at calving and lactation performance within the same lactation.

The objective of the study was to investigate the association between early lactation Streptococcus dysgalactiae isolates and milk yield, somatic cell count (SCC), clinical mastitis, and culling in the same lactation. The 178 commercial dairy herds were randomly placed into 3 penicillin- or penicillin-dihydrostreptomycin-based dry-cow treatments and 3 different postmilking teat disinfection groups-negative control, iodine, or external teat sealant. All cows were sampled in early lactation, and Strep. dysgalactiae-positive and culture-negative cows were followed throughout the remainder of the lactation. Mixed models, including repeated measurements, with test-day observation as dependent variable, were used to compare milk yield, SCC, and available milk quality variables throughout the remaining lactation. Survival analyses, using a positive frailty model to account for any herd random effects, were used to estimate the hazard ratio for clinical mastitis and culling. Streptococcus dysgalactiae-positive cows had a significantly higher SCC throughout the lactation compared to culture-negative cows. For primiparous or multiparous cows, respectively, the differences in the geometric mean SCC between Strep. dysgalactiae-positive and culture-negative cows was 197,000 or 280,000 cells/mL at the beginning of the lactation, 24,000 or 46,000 cells/mL in mid lactation, and 39,000 or 111,000 cells/mL at the end of the lactation. Streptococcus dysgalactiae-positive primiparous or multiparous cows produced 334 or 246 kg less milk, respectively, during a 305-d lactation compared with culture-negative cows. Compared with culture-negative cows, the hazard ratios for clinical mastitis in Strep. dysgalactiae-positive cows were 2.3 (1.9 to 2.9) and 1.6 (1.3 to 2.0) for culling. For cows with both Strep. dysgalactiae and Staphylococcus aureus isolates, the hazard ratio for culling significantly increased to 2.5 (1.9 to 3.2).

Clinical mastitis in Norwegian herds after a combined selective dry-cow therapy and teat-dipping trial.

The objective of this study was to see if introduction of a 2-yr combined selective dry-cow therapy and teat-dipping trial would reduce clinical mastitis (CM) events in 164 Norwegian dairy herds. Three different penicillin or penicillin/dihydrostreptomycin-based dry-cow treatments, and 3 different teat-dipping regimens (negative control, iodine teat dip, or an external teat sealant) were independently and randomly allocated to each herd. Complete lactations both before and during the trial were investigated. Altogether, 1,005 CM cases were recorded in the lactations before the trial and 924 cases were recorded during the trial. Bacteriological milk samples were available from 784 of the 924 CM cases during the trial. Among these, Staphylococcus aureus were isolated from 47.4%, Streptococcus dysgalactiae from 22.5%, Escherichia coli from 10.7%, and coagulase-negative staphylococci from 6.3%. In addition, 12.5% cases were bacteriological negative, and the remainder of the CM cases were caused by other microbes. The different models were analyzed using Cox regression analysis with PROC PHREG and a positive stable frailty model in the SAS macro. Separate models were made for cows housed in tie-stalls and free-stalls. Parity had a significant impact on the CM risk in both type of stalls. Older cows (parity > 3) had the highest hazard ratio of contracting CM in tie-stalls (1.68) and free-stalls (2.18) compared with parity 1. The CM risk decreased significantly (13%) in tie-stalls and by 18% in free-stalls. In tie-stalls, iodine-dipped cows had a significantly lower chance (21%) of getting CM compared with the negative control and the use of external teat sealant. The same trend was seen in free-stalls; however, the differences were not significant. Compared with CM before the trial, the reduction of CM was 15% during the trial.

Relationships between milk culture results and treatment for clinical mastitis or culling in Norwegian dairy cattle.

In quarter milk samples from 2,492 randomly sampled cows that were selected without regard to their current or previous udder health status, the relationships between the following outcome variables were studied: treatment of clinical mastitis; the joint event of either treatment or culling for mastitis; culling for all reasons; culling specifically for mastitis; and the covariates of positive milk culture for Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococcus spp., or other pathogens, or of negative culture for mastitis pathogens. Microbiological diagnoses were assigned at the cow level, and altogether 3,075 diagnoses were related to the outcome variables. The relation between the absence of pathogens and rich (>1,500 cfu/mL of milk) or sparse (

Milk culture results in a large Norwegian survey--effects of season, parity, days in milk, resistance, and clustering.

A nationwide random computerized assignment survey that included 3,538 sets of 4 quarter milk samples from 2,834 dairy cows was conducted during 2000. Every fifth cow from every 50th herd was randomly selected for sampling and culture during each quarter of the year. Milk culture results of pathogens known to be related to mastitis were recorded regardless of whether mastitis had been indicated by any inflammatory measure or not. Farmers were blinded to all test results to minimize any potential interventions that might be prompted by the results. The most prevalent isolate was Staphylococcus aureus, which was identified in 8.2% of the quarter milk samples. More than 15 colony-forming units/0.01 mL of Staph. aureus were found in 4.3% of the quarter milk samples, whereas 3.5% had only 1 to 3 colony-forming units/0.01 mL. Streptococcus dysgalactiae, coagulase-negative staphylococci (CNS), and Streptococcus uberis were isolated from 1.2, 3.3, and 0.4% of quarter milk samples, respectively. No isolates were found in 76.6% of the quarter milk samples tested. Among individual cows, 22.2% had an isolate of Staph. aureus in > or = 1 quarter. Only Strep. dysgalactiae exhibited a higher prevalence with increased parity. Prevalence of Staph. aureus decreased throughout days in milk, but prevalence of Strep. dysgalactiae increased. There was a strong seasonal effect; the highest prevalence of Strep. dysgalactiae and CNS was observed during April and May (late indoor season), and the highest prevalence of Staph. aureus and Strep. uberis was observed during June and July (the outdoor season). A substantial within-cow clustering effect was found for Strep. dysgalactiae, Staph. aureus, and CNS. Additionally, a within-herd effect was found for Strep. uberis, penicillin-resistant Staph. aureus, total Staph. aureus, and CNS. No within-county cluster effect was found. Lastly, both Staph. aureus and CNS exhibited a surprisingly high seasonal effect regarding the prevalence of resistance to penicillin G. Penicillin resistance of Staph. aureus was likely due to higher prevalence of Staph. aureus as a whole, but for CNS, there was also an additional increase caused by a higher proportional rate of penicillin resistance during the late indoor season.

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.

Development and evaluation of an immunomagnetic separation-ELISA for the detection of Staphylococcus aureus thermostable nuclease in composite milk.

An ELISA method based on monodisperse magnetic beads was developed for the detection of Staphylococcus aureus thermostable nuclease (TNase) in composite milk, wherein S. aureus TNase is captured by magnetic beads coated with monoclonal antibodies directed against TNase and subsequently detected by an enzyme-labelled MAb against the same antigen. Sensitivity of the test was approximately 1 ng TNase, which corresponds to the amount of TNase produced and secreted by approximately 10(5) S. aureus per ml. The Immuno Magnetic Separation (IMS)-ELISA detected TNase in samples from which no S. aureus could be demonstrated on culture. The total test time is 3 h and can be performed either on preserved or fresh milk. The method may be automated.