Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Prevalence of udder pathogens in milk samples from Norwegian dairy cows recorded in a national database in 2019 and 2020.

BACKGROUND: Identification of aetiological agents of mastitis in dairy cattle is important for herd management of udder health. In Norway, results from mastitis diagnostics are systematically recorded in a central database, so that the dairy industry can follow trends in the recorded frequency of udder pathogens and antimicrobial resistance patterns at national level. However, bacteriological testing of milk samples is based on voluntary sampling, and data are therefore subject to some bias. The aim of this study was to examine the prevalence of udder pathogens in Norwegian dairy cows by analysing data from the national routine mastitis diagnostics and to explore how routines for sampling and diagnostic interpretations may affect the apparent prevalence of different bacterial pathogens. We also assessed associations between udder pathogen findings and the barn- and milking systems of the herds. RESULTS: The most frequently detected major udder pathogens among all milk samples submitted for bacterial culture (n = 36,431) were Staphylococcus aureus (24.5%), Streptococcus dysgalactiae (13.3%) and Streptococcus uberis (9.0%). In the subset of samples from clinical mastitis (n = 7598); Escherichia coli (14.5%) was the second most frequently detected pathogen following S. aureus (27.1%). Staphylococcus epidermidis (10.0%), Corynebacterium bovis (9.4%), and Staphylococcus chromogenes (6.0%) dominated among the minor udder pathogens. Non-aureus staphylococci as a group, identified in 39% of the sampling events, was the most frequently identified udder pathogen in Norway. By using different definitions of cow-level bacterial diagnoses, the distribution of minor udder pathogens changed. Several udder pathogens were associated with the barn- and milking system but the associations were reduced in strength when data were analysed from farms with a comparable herd size. S. aureus was associated with tiestall housing, E. coli and S. dysgalactiae were associated with freestall housing, and S. epidermidis was associated with automatic milking systems. Only 2.5% of the 10,675 tested S. aureus isolates were resistant to benzylpenicillin. Among the 2153 tested non-aureus staphylococci, altogether 34% were resistant to benzylpenicillin. CONCLUSIONS: This study presents the recorded prevalence of udder pathogens in Norway over a two-year period and assesses the possible impact of the sampling strategies, diagnostic methods and diagnostic criteria utilized in Norway, as well as associations with different housing and milking systems. The national database with records of results from routine mastitis diagnostics in Norway provides valuable information about the aetiology of bovine mastitis at population level and can reveal shifts in the distribution and occurrence of udder pathogens.

Sensitivity and specificity of bacterial culture, qPCR, and somatic cell count for detection of goats with Staphylococcus aureus intramammary infection using Bayesian latent class models.

Staphylococcus aureus (S. aureus) is an important udder pathogen affecting goat milk production. The ability to detect goats with subclinical mastitis caused by S. aureus is essential in udder health control programs. In Norway, the industry recommends using somatic cell count (SCC) as a screening tool, and conventional bacterial culture (BC) as a confirmatory test for goat milk samples, but a commercial qPCR, Mastitis 4 qPCR (DNA Diagnostics, Risskov, Denmark) is also available. However, few studies have validated the use of these methods for the detection of goats with S. aureus intramammary infection (IMI). Therefore, the objective of this retrospective study was to estimate the diagnostic sensitivity and specificity of BC, qPCR, and SCC for the detection of goats with IMI caused by S. aureus using Bayesian latent class analysis. We analyzed the BC and qPCR results of aseptically collected milk samples and SCC results from milk recording samples from 319 goats from three herds using different SCC cut-offs. At a SCC cut-off of 2000,000 cells/mL, the estimated median prevalence in each herd was 12.7% (95% highest posterior density credible interval [CI] 6.5-19.8), 15.7% (95% CI 9.3-23.0), and 1.5% (95% CI 0.0-4.3). The median sensitivity was 93.0% (95% CI 80.2-100), 93.6% (95% CI 82.3-100) and 78.2% (95% CI 62.3-91.2) for BC, qPCR, and SCC, respectively. The estimated median specificity of BC was 99.5% (99% CI 98.4-100), for qPCR, 98.9% (95% CI 97.5-100), and for SCC 61.5% (95% CI 56.0-67.1). The results show that BC, which is today's standard method for diagnosing IMI, has a high accuracy for detection of goats with S. aureus IMI, but qPCR had a sensitivity and specificity similar to BC, and may act as an alternative.

Evaluation of test characteristics of 2 ELISA tests applied to bulk tank milk and claw-trimming records for herd-level diagnosis of bovine digital dermatitis using latent class analysis.

Bovine digital dermatitis (DD) is an infectious claw disease with a negative effect on animal welfare and production. Treponema spp. is the main causative agent, and infected animals produce specific antibodies. Our aim was to estimate sensitivity (Se) and specificity (Sp) of 2 ELISA research tests, Medicago's ELISA test and GD Animal Health's in-house ELISA test, for detection of DD-associated Treponema antibodies in bulk tank milk. We used bulk tank milk samples from 154 Norwegian dairy cattle herds, 96 from an expected high-prevalence region and 58 from a low-prevalence region. Both tests were evaluated separately against herd-level (aggregated) claw-trimming records extracted from the Norwegian Dairy Herd Recording System using latent class models in a Bayesian analysis. Cutoff values were selected using an explorative approach, and both noninformative priors for all parameters and informative ß priors for distribution of Se and Sp of claw trimming were explored. The estimated (median) true herd-level prevalence of digital dermatitis varied between 24 and 30% in the high-prevalence region and between 3 and 6% in the low-prevalence region. For Medicago's ELISA test, an Se (95% posterior credible interval) of 0.57 (0.32; 0.94) could be achieved without compromising Sp, and for GD Animal Health's in-house ELISA test, an Se of 0.60 (0.37; 0.92) was achieved. Our study showed that both ELISA tests can detect antibodies against DD-associated Treponema spp. in bulk tank milk. However, neither of the 2 ELISA tests produced satisfactory sensitivity without compromising specificity. Based on these results, inspection at claw trimming in a chute is necessary for surveillance and control of DD at the herd level in Norway, although these ELISA tests of bulk tank milk might be a useful supplement.

Salivary IgG levels in neonatal calves and its association to serum IgG: an observational pilot study.

The diagnosis of inadequate transfer of colostrum immunoglobulin G (IgG) to calf serum, often known as failure of passive transfer (<10 g/L IgG1 at 24 to 48 h), necessitates blood sampling from the calf and in some instances the presence of a veterinarian. Sampling saliva is both less invasive and easy for the producer. Previous research has shown that quantification of saliva IgG is possible in juvenile and adult cattle. The objectives of this observational pilot study were to investigate whether IgG can be quantified in neonatal calf saliva, if it is correlated to serum IgG concentrations, and if the indirect quantification of saliva IgG is achievable by use of a digital refractometer. Paired blood and saliva samples were collected from 20 healthy dairy calves aged 1 to 3 d. In these samples, IgG was quantified directly with single radial immunodiffusion and indirectly by use of a digital refractometer indicating Brix % (a subsample of n = 12 saliva samples). A strong positive correlation (r = 0.7, P < 0.001) between saliva IgG (mean ± SD; 0.2 ± 0.11 g/L) and serum IgG (32.1 ± 11.94 g/L) was found. Saliva IgG ranged from the lowest detectable value, 0.1 g/L (n = 6 samples) to 0.6 g/L. Saliva Brix (1.2 ± 0.69%) was not significantly correlated to serum IgG (n = 12, r = 0.43, P = 0.155); however, it was significantly correlated to saliva IgG (n = 12, r = 0.7, P = 0.018) and Brix in serum (n = 12, r = 0.7, P = 0.013). We conclude that IgG was quantifiable in most of the saliva samples. For saliva IgG to be of any value with regards to detecting failure of passive transfer, future studies should investigate methods that can detect IgG <0.1 g/L. The results indicate that saliva IgG can be used to predict serum IgG at levels above 10 g/L, which may warrant further exploration of the use of saliva in the surveillance of failure of passive transfer. The results of the current pilot study did not support the potential usage of a Brix % refractometer to quantify saliva IgG.

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis.

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR® BCV-Ab and SVANOVIR® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high-prevalence area. As none of the tests were considered perfect, estimation of test characteristics was performed using Bayesian latent class models. At the manufacturers' recommended cut-off values, the median sensitivity for the BRSV multiplex and the BRSV ELISA was 94.4 [89.8-98.7 95% Posterior Credibility Interval (PCI)] and 99.8 [98.7-100 95% PCI], respectively. The median specificity for the BRSV multiplex was 90.6 [85.5-94.4 95% PCI], but only 57.4 [50.5-64.4 95% PCI] for the BRSV ELISA. However, increasing the cut-off of the BRSV ELISA increased specificity without compromising sensitivity. For the BCV multiplex we found that by using only one of the three antigens included in the test, the specificity increased, without concurrent loss in sensitivity. At the recommended cut-off this resulted in a sensitivity of 99.9 [99.3-100 95% PCI] and specificity of 93.7 [88.8-97.8 95% PCI] for the multiplex and a sensitivity of 99.5 [98.1-100 95% PCI] and a specificity of 99.6 [97.6-100 95% PCI] for the BCV ELISA.

Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples.

Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR. The within-herd prevalence in the six herds, as estimated by the latent class models ranged from 7.7 to 50.8%. At the recommended cut-off (cycle threshold 37), the sensitivity of the qPCR was significantly higher at 95.3 (95% posterior probability interval [PPI] [84.2; 99.6]) than that of bacteriological culture at 58.2 (95% PPI [43.8; 74.4]). However, bacterial culture had a higher specificity of 99.7 (95% PPI [98.5; 100.0]) compared to the qPCR at 98.5 (95% PPI [94.6; 99.9]). The median estimated negative predictive values of qPCR was consistently higher than those of the BC at all estimated prevalences, and the superiority of the qPCR increased with increasing within-herd prevalence. The median positive predictive values of BC was in general higher than the estimates for the qPCR, however, at the highest prevalence the predictive ability of both tests were similar.

The Norwegian Healthier Goats programme--a financial cost-benefit analysis.

The aim of this study was to evaluate the profitability to dairy goat farmers of participating in the Healthier Goats disease control and eradication programme (HG), which was initiated in 2001 and is still running. HG includes the control and eradication of caprine arthritis encephalitis (CAE), caseous lymphadenitis (CLA) and paratuberculosis (Johne's disease) in Norwegian goat herds. The profitability of participation was estimated in a financial cost-benefit analysis (CBA) using partial budgeting to quantify the economic consequences of infectious disease control through HG versus taking no action. Historical data were collected from 24 enrolled dairy goat herds and 21 herds not enrolled in HG, and supplemented with information from a questionnaire distributed to the same farmers. Expert opinions were collected to arrive at the best possible estimates. For some input parameters there were uncertainty due to imperfect knowledge, thus these parameters were modelled as PERT probability distributions and a stochastic simulation model was built. The CBA model was used to generate distributions of net present value (NPV) of farmers' net cash flows for choosing to enroll versus not enrolling. This was done for three selected milk quota levels of 30000L, 50000L and 70000L, and both for before and after the introduction of a reduced milk price for the non-enrolled. The NPVs were calculated over time horizons of 5, 10 and 20 years using an inflation-adjusted discount rate of 2.8% per annum. The results show that participation in HG on average was profitable over a time horizon of 10 years or longer for quota levels of 50000L and 70000L, although not without risk of having a negative NPV. If farmers had to pay all the costs themselves, participation in HG would have been profitable only for a time horizon beyond 20 years. In 2012, a reduced milk price was introduced for farmers not enrolled in HG, changing the decision criteria for farmers, and thus, the CBA. When the analysis was altered to account for these changes, the expected NPV was positive over five years for the 50000L quota, indicating an increased profitability of enrolling in HG. The sensitivity analysis showed that particular attention should be paid to work load and investment costs when planning for disease control programmes in the future.

Association between isolation of Staphylococcus aureus one week after calving and milk yield, somatic cell count, clinical mastitis, and culling through the remaining lactation.

Cows with isolation of Staphylococcus aureus approximately 1 week after calving and milk yield, somatic cell count (SCC), clinical mastitis (CM), and culling risk through the remaining lactation were assessed in 178 Norwegian dairy herds. Mixed models with repeated measures were used to compare milk yield and SCC, and survival analyses were used to estimate the hazard ratio for CM and culling. On average, cows with an isolate of Staph. aureus had a significantly higher SCC than culture-negative cows. If no post-milking teat disinfection (PMTD) was used, the mean values of SCC were 42,000, 61,000, 68,000 and 77,000 cells/ml for cows with no Staph. aureus isolate, with Staph. aureus isolated in 1 quarter, in 2 quarters and more than 2 quarters respectively. If iodine PMTD was used, SCC means were 36,000; 63,000; 70,000 and 122,000, respectively. Primiparous cows testing positive for Staph. aureus had the same milk yield curve as culture-negative cows, except for those with Staph. aureus isolated in more than 2 quarters. They produced 229 kg less during a 305-d lactation. Multiparous cows with isolation of Staph. aureus in at least 1 quarter produced 94-161 kg less milk in 2nd and >3rd parity, respectively, and those with isolation in more than 2 quarters produced 303-390 kg less than multiparous culture-negative animals during a 305-d lactation. Compared with culture-negative cows, the hazard ratio for CM and culling in cows with isolation of Staph. aureus in at least 1 quarter was 2.0 (1.6-2.4) and 1.7 (1.5-1.9), respectively. There was a decrease in the SCC and in the CM risk in culture-negative cows where iodine PMTD had been used, indicating that iodine PMTD has a preventive effect on already healthy cows. For cows testing positive for Staph. aureus in more than 2 quarters at calving, iodine PMTD had a negative effect on the CM risk and on the SCC through the remaining lactation.

Risk factors for isolation of Staphylococcus aureus or Streptococcus dysgalactiae from milk culture obtained approximately 6 days post calving.

Milk culture results at approximately 6 d post calving were assessed in a 2-year retrospective single-cohort study in 178 Norwegian herds. A combined teat dipping and selective antibiotic therapy trial was performed in these herds where cows with composite milk somatic cell count (CMSCC) >100,000 cells/ml before drying-off (geometric mean of the last three CMSCC test-days) and isolation of Staphylococcus aureus or Streptococcus dysgalactiae were selected for either short-acting lactation antibiotic treatment or long-acting dry cow antibiotic treatment. Milk culture results at approximately 6 d post-calving were available from 437 treated cows and 3061 non-treated cows before drying-off and separate multivariable logistic regression models were ran for these two groups. Risk factors associated with isolation of Staph. aureus 6 d post calving for non-treated cows were CMSCC >400,000 cells/ml before drying-off v. <400,000 cells/ml (Odd ratio (OR) = 2.4) and clinical mastitis (CM) in the previous lactation v . non-treated (OR=1.5). Risk factors associated with Staph. aureus 6 d post calving for treated cows was a CMS > 200,000 cells/ml before drying-off v. <200,000 cells/ml (OR=2.3) and CM in the previous lactation verus non-treated (OR=1.7). For non-treated cows it was 1.7 times more likely to isolate Str. dysgalactiae 6 d post-calving if the CMSCC was > 50,000 cells/ml compared with <50,000 cells/ml. For treated cows it was 3.7-5.8-times more likely to isolate Str. dysgalactiae 6 d post calving if given short-acting lactation formula at quarter level compared with long-acting dry cow formula used at cow level. Regular use of iodine post-milking teat disinfection (PMTD) did not influence the isolation of Staph. aureus 6 d post calvin, but it was less likely to isolate Str. dysgalactiae 6 d post calving if iodine PMTD was used regularly rather than irregularly. The external teat sealant had no effect on either of the two bacteria. This study indicates that the CMSCC limit for sampling cows before drying-off can be reduced to 50,000 cells/ml in herds with a Str. dysgalactiae problem. Iodine PMTD should also be recommended in these herds. Cows with a CMSCC > 400,000 cells/ml prior to drying-off should receive long-acting dry cow formula irrespective of the milk result.

Staphylococcus aureus and Streptococcus dysgalactiae in Norwegian herds after introduction of selective dry cow therapy and teat dipping.

The objective was to promote a reduction in the prevalence of Staphylococcus aureus and Streptococcus dysgalactiae after 2 years of selective dry cow therapies and teat dipping/external teat sealant implementation. Three different dry cow treatments, one long-acting and two short-acting penicillin-based products were tested at herd level together with a negative control teat dipping group, an iodine teat dipping group and DryFlextrade mark, an external teat sealant. The regimes were independently randomly allocated to 178 dairy herds. Yearly bacteriological quarter milk samples were collected from all cows at the beginning of the trial, after 1 year and 2 years. At herd level, a total of 15% of the herds showed no Staphylococcus aureus isolates after 2 years, compared with 5% at the beginning. The distribution of Streptococcus dysgalactiae infected herds remained the same after 2 years. At cow level, there were no significant differences in the reduction of Staph. aureus between the different dry cow therapies and teat dipping regimes. But there was a significant reduction of Str. dysgalactiae in the iodine teat dipping group compared with Dryflextrade mark and the negative control group. The proportional rate of Staph. aureus positive quarters was reduced from 65.9% to 54.9% after 2 years. As for Str. dysgalactiae, an increase was observed from 14.2% to 15.2%.