Using imperfect tests may lead to biased estimates of disease frequency and of associations between risk factors and disease. For instance in longitudinal udder health studies, both quarters at risk and incident intramammary infections (IMI) can be wrongly identified, resulting in selection and misclassification bias, respectively. Diagnostic accuracy can possibly be improved by using duplicate or triplicate samples for identifying quarters at risk and, subsequently, incident IMI. The objectives of this study were to evaluate the relative impact of selection and misclassification biases resulting from IMI misclassification on measures of disease frequency (incidence) and of association with hypothetical exposures. The effect of improving the sampling strategy by collecting duplicate or triplicate samples at first or second sampling was also assessed. Data sets from a hypothetical cohort study were simulated and analyzed based on a separate scenario for two common mastitis pathogens representing two distinct prevailing patterns. Staphylococcus aureus, a relatively uncommon pathogen with a low incidence, is identified with excellent sensitivity and almost perfect specificity. Coagulase negative staphylococci (CNS) are more prevalent, with a high incidence, and with milk bacteriological culture having fair Se but excellent Sp. The generated data sets for each scenario were emulating a longitudinal cohort study with two milk samples collected one month apart from each quarter of a random sample of 30 cows/herd, from 100 herds, with a herd-level exposure having a known strength of association. Incidence of IMI and measure of association with exposure (odds ratio; OR) were estimated using Markov Chain Monte Carlo (MCMC) for each data set and using different sampling strategies (single, duplicate, triplicate samples with series or parallel interpretation) for identifying quarters at risk and incident IMI. For S. aureus biases were small with an observed incidence of 0.29 versus a true incidence of 0.25IMI/100 quarter-month. In the CNS scenario, diagnostic errors in the two samples led to important selection (40IMI/100 quarter-month) and misclassification (23IMI/100 quarter-month) biases for estimation of IMI incidence, respectively. These biases were in opposite direction and therefore the incidence measure obtained using single sampling on both the first and second test (29IMI/100 quarter-month) was exactly the true value. In the S. aureus scenario the OR for association with exposure showed little bias (observed OR of 3.1 versus true OR of 3.2). The CNS scenario revealed the presence of a large misclassification bias moving the association towards the null value (OR of 1.7 versus true OR of 2.6). Little improvement could be brought using different sampling strategies aiming at improving Se and/or Sp on first and/or second sampling or using a two out of three interpretation for IMI definition. Increasing number of samples or tests can prevent bias in some situations but efforts can be spared by holding to a single sampling approach in others. When designing longitudinal studies, evaluating potential biases and best sampling strategy is as critical as the choice of test.

The objectives of this study were to propose benchmarks for the interpretation of herd udder health using monthly individual somatic cell counts (SCC) from dairy herds in Quebec, Canada and to evaluate the association of risk factors with intramammary infection (IMI) dynamics relative to these benchmarks. The mean and percentiles of indices related to udder infection status [e.g., proportion of healthy or chronically infected cows, cows cured and new IMI (NIMI) rate] during lactation and over the dry period were calculated using a threshold of ≥ 200 000 cells/mL at test day. Mean NIMI proportion and proportion of cows cured during lactation were 0.11 and 0.27. Benchmarks of 0.70 and 0.03 for healthy and chronically infected cows over the dry period were proposed. Season and herd mean SCC were risk factors influencing IMI dynamics during lactation and over the dry period.

Few studies have investigated the efficacy of extended ceftiofur therapy and none have focused on extended therapy for naturally occurring clinical mastitis. The objective of this study was to compare the efficacy of extended intramammary ceftiofur therapy of 8 d duration with a standard 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows. Holstein cows from 22 dairy herds (n = 241) were randomly allocated to the 2 treatment groups. For each case of mastitis, 125 mg of ceftiofur hydrochloride was administered intramammary once a day for 2 or 8 d. Clinical cure, 21 d after the last treatment, was 89% (98/110) in each group. Bacteriological cure 21 d after the last treatment for the 2- and 8-day regimens were 32% (15/47) and 61% (25/41), respectively, for all bacteria (P = 0.007), 64% (9/14) and 82% (9/11), respectively, for streptococci (P = 0.50), and 0% (0/20) and 47% (9/19), respectively, for Staphylococcus aureus (P = 0.0004). There were no statistical differences between groups for new intramammary infections. Overall, ceftiofur extended therapy increased cure when compared to a 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows.

ABSTRACT Presented here is a draft genome sequence for Staphylococcus agnetis CBMRN 20813338, isolated from a lactating dairy cow with subclinical mastitis. The genome is approximately 2,416 kb and has 35.79% G+C content. Analysis of the deduced open reading frame (ORF) set identified candidate virulence attributes in addition to potential molecular targets for species identification.

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n = 394) and Klebsiella species (n = 139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR) = 26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR = 162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR = 5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR = 8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR = 0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR = 3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.

Since August 2012, the upper tolerance limit for bulk-tank somatic cell count (BTSCC) in Canada is 400,000 c/mL. Among Quebec's dairy herds in 2011, 13% of BTSCC analyses exceeded that limit and were at risk of financial penalties. Herd indices on the basis of individual-cow somatic cell count (SCC) variations around a threshold of 200,000 c/mL between 2 consecutive months in lactation have been described (proportion of cows cured, new 'intramammary infection [NIMI], healthy or chronic) and could be used to monitor the dynamics of herd udder health. The objective of this study was to use individual-cow udder health indices for the preceding 3-month period to develop a predictive model to estimate the risk of exceeding the BTSCC limit of 400,000 c/mL the following month.

Staphylococcus aureus is a leading cause of intramammary infections (IMI) and bovine mastitis is an important disease for the dairy industry. As this bacterium probably expresses specific genes for establishment of IMI, we studied the transcriptional profile of four S. aureus strains recovered from experimentally infected cows. Microbial RNA was extracted from bacteria isolated from milk, reverse-transcribed and labeled for hybridization to sub-genomic microarrays to detect candidate genes for further investigations. Several S. aureus genes were expressed during IMI; some were detected in samples from more than one strain, more than one cow and at more than one time point during infection. A selection of four genes showing strong expression and with putative functions in pathogenesis was further studied by qPCR. By comparing the expression in different media in vitro, we found that gene SACOL2171 was induced by iron restriction whereas the expression of the transcriptional regulator SACOL2325 and the ABC transporter SACOL0718-720 (vraFG) were induced by milk. In addition, the putative exotoxin SACOL0442 seemed to require the intramammary environment for expression. Gene-disrupted mutants for SACOL0720 and SACOL0442 showed no growth defect in vitro but were attenuated during bovine IMI, causing infections with significant reductions in bacterial and somatic cell counts. The milk from the mammary quarters infected with these mutants also showed better appearance and composition than milk from quarters infected with the wild type. In conclusion, we have identified genes that are most likely important for S. aureus IMI. These represent novel candidates to include in a vaccine.

When designing mastitis-prevention and control programs, it is important to know the level of adoption of mastitis-prevention management practices and control programs and the herd-level prevalence of contagious mastitis pathogens. Our objectives were to estimate: (1) adoption of recommended mastitis-preventive management on Canadian dairy farms; (2) herd-level prevalence of contagious mastitis pathogens on Canadian dairy farms; and (3) associations of certain management practices with the isolation of Staphylococcus aureus from the bulk tank milk from Canadian dairy farms.In total, 226 farms participating in dairy herd improvement milk recording were randomly selected. All participating farms in British Columbia had free-stall barns and 85% of farms in Quebec had tie-stall barns. Post-milking teat disinfection was practised on 96% of the farms and 72% had implemented blanket dry-cow treatment.Weighted and province-stratified prevalence of Streptococcus agalactiae and Staph. aureus in bulk tank milk was 4% (95% confidence interval: 0-12%) and 74% (95% confidence interval: 61-86%), respectively. Highest Staph. aureus prevalence was found in Nova Scotia (91%) and lowest prevalence in British Columbia (38%). No Mycoplasma spp. were isolated, but detection of Mycoplasma spp. could have been hampered by the frozen shipment and storage of the milk samples.Management practices associated with a lower probability of isolating Staph. aureus were blanket dry-cow treatment and believing that a nutritionist is important in mastitis data review. Having the milking equipment checked by an independent technician at least once a year and rubber mats or mattresses in the free-stall barns were associated with an increased probability of isolating Staph. aureus from the bulk tank Most of Canadian dairy farms adopted important mastitis-prevention practices, such as post-milking teat disinfection and drying off all cows with antibiotics; however, improvements can still be made. A few management practices were associated with the prevalence of Staph. aureus in bulk tank milk, such as dry-cow treatment and barn type for the lactating cows. Further work has to be carried out to investigate to what extent the associations are causal. (C) 2010 Elsevier B.V. All rights reserved.

Questionnaires are frequently used instruments to collect data in epidemiological studies. In countries where more than one language is spoken, the development of a questionnaire in more than one language is needed. The objective of this study was to develop and test the repeatability and validity of English and French versions of a personal interview-format questionnaire designed to capture udder health related management practices used on dairy farms. A standardized protocol was used to develop and translate the research instrument. Equivalence of the English and French questionnaires was assessed using a cross-over study design with 24 bilingual dairy producers completing both versions on three different occasions in a randomly assigned sequence. Repeatability of the questionnaire was evaluated using the test–retest method with the same questions being asked on two different occasions to 88 dairy producers participating in the National Cohort of Dairy Farms of the Canadian Bovine Mastitis Research Network. Validity of the questions related to milking procedures and general housing was assessed using on-farm observations as a gold standard. Measures of agreement were calculated using kappa, quadratic-weighted Kappa and concordance correlation coefficients for categorical, ordinal and continuous variables, respectively. Sensitivity and specificity estimates were computed for the validity analysis. The overall equivalence of the English and French versions of the questionnaire was adequate; agreement measures when administered twice in the same language were not significantly higher than when administered in each language. Similarly, questionnaire overall repeatability was good. When accounting for prevalence bias, Kappa and CCC estimates ranged from 0.40 to 0.92 for 27 of the 29 items evaluated in the questionnaire, with 18 items yielding agreement estimates greater than 0.60. Finally, milking procedures and general housing questions validity was excellent with mean sensitivity and specificity of 86% and 92%, respectively. Although the overall evaluation of the instrument was satisfactory, specific doubtful items were identified. This illustrates the need to address questionnaire reliability as even rigorously designed and pre-tested questions can have poor repeatability or validity. Our results indicate that the developed English and French questionnaires can be used simultaneously to accurately measure the udder health related management practices used on Canadian dairy farms. This questionnaire is adaptable for use in other developed dairy industry populations. The questionnaire is freely available online at www.mastitisnetwork.org under the “Publications/others” section.

The objective of this study was to evaluate the California Mastitis Test (CMT) and a portable electrical conductivity meter for diagnosing precalving intramammary infection (IMI) in Holstein heifers. A total of 428 dairy heifers from 23 dairy herds were enrolled between 6 and 12 days before the expected calving date from June 2002 to June 2003. Mammary secretions were tested by both diagnostic methods and by bacterial culture for evidence of IMI. California Mastitis Test was considered negative if the score was negative, trace or 1 and was considered positive otherwise. Two cut-off points were evaluated for milk electrical conductivity (>5 and >6.5 mS/cm). From this study, an overall proportion of 69% of heifers had precalving IMI and the overall heifer prevalence of major pathogen IMI was 16.8%. At the quarter level, sensitivity and specificity of CMT (68.9% and 68.4%, respectively) and milk conductivity >5 mS/cm (41.0% and 65.2%, respectively) or >6.5 mS/cm (25.2% and 83.3%, respectively) to identify all IMI were low. However, the heifer level sensitivity and specificity of CMT for major pathogens were 91.0% (81.5-96.6) and 27.5% (22.8-32.6), respectively. Using a cut-off point of 5 mS/cm, the heifer level sensitivity and specificity for major pathogens was 68.7% (56.2-79.4) and 44.1% (38.7-49.6), respectively. A conductivity cut-off value of 6.5 mS/cm decreased the sensitivity and increased the specificity to 53.7% (41.1-66.0) and 59.5% (54.0-64.8), respectively. California Mastitis Test and milk electrical conductivity are not good predictors of major pathogen IMI in heifers during the last 2 weeks before calving. However, the negative predictive values at quarter or heifer level were high and the heifer false negative rate was 6-14% using CMT or conductivity, respectively. Therefore, these measures could be useful for screening out heifers or quarters that are unlikely to have a major pathogen IMI.

A clinical trial was conducted to determine whether prepartum intramammary pirlimycin reduces the proportion of nulliparous heifers with intramammary infection (IMI) during early lactation and improves milk production. Quarter milk samples were collected from 428 heifers, systematically allocated to treatment and control groups, 6 to 12 d before the expected calving date and 2 to 8 d after calving. At the prepartum visit, heifers in the treatment group (n = 219) received an infusion of pirlimycin hydrochloride in all 4 quarters; the control heifers (n = 209) received no infusions. Intramammary infection was detected in 69% of the heifers and 33% of the quarters before calving. After calving, the proportion of treated heifers with IMI was significantly lower than the proportion of control heifers (31% versus 45%). Staphylococcus aureus was isolated from 10% of the heifers and 3% of the quarters before calving. After calving, the proportion of IMIs due to S. aureus was significantly lower in the treated heifers than in the control heifers (5.6% versus 10%). Antibiotic treatment increased the percentage of cures and prevented new IMIs caused by gram-positive bacteria after calving. The incidence of new IMIs caused by gram-negative bacteria and yeast was higher among treated heifers than among control heifers. There was no overall effect of treatment on milk production, but there was a significant interaction effect of treatment and the interval between treatment and calving. An increase of 302 kg of milk was observed when antibiotic treatment was applied more than 1 wk before calving. Treatment did not affect the milk somatic cell count on the 1st 3 test days after calving.

201 (5101) MASTITIS RESEARCH FOR MAXIMUM INDUSTRY BENEFIT: CANADIAN BOVINE MASTITIS RESEARCH NETWORK Daniel Scholl1, Grant Tomita1, Serge Messier1, Émile Bouchard1, Herman Barkema2, Pierre Lacasse3, Xin Zhao4 1Université de Montréal, CP 5000, 3200 Sicotte, Saint Hyacinthe, Québec, J2S 7C6, Canada; 2UPEI, Atlantic Veterinary College, 550, Univ. Ave., Charlottetown, Prince-Edward Island, C1A 4P3, Canada; 3Dairy and Swine R&D Centre, PO Box 90, 2000 Route 108E, Lennoxville, J1M 1Z3, Canada; 4McGill University, Department of Animal Science, 21 111 Lakeshore road, Sainte-Anne de Bellevue, Quebec, H9X 3V9, Canada Bovine mastitis has been researched for decades, but is still the costliest disease of dairy cattle. Researchers and the dairy industry in Canada are forming a mastitis research network to address this problem. The Network is a partnership between scientists and members of the industry, and provides a framework for concerted research collaboration and proactive knowledge and technology transfer. Thirty-four scientists from eight institutions conduct a concerted and collaborative research program under the themes of mastitis prevention, elimination and monitoring. There is a core research platform comprising a national cohort of dairy farms, a mastitis pathogen strain-bank, a network of diagnostic laboratories, and a biosecurity level 2 facility. By planning and executing research cooperatively, resources are utilized efficiently, trainees benefit from multi-disciplinary experiences, duplication of research is prevented, and relevance to the dairy producers is maintained. The Network promotes a balance of applied and theoretical research including epidemiological strategies for mastitis control, novel technologies for enhancing host resistance, and alternatives to traditional antimicrobial therapy. A key to the Network's success is the successfully transfer of knowledge and technology to dairy producers. For knowledge transfer, the Network will disseminate existing and new mastitis control information into existing channels that are normally accessed by dairymen in Canada. The Network will collaborate with knowledge transfer professionals and the National Mastitis Council to articulate mastitis management standards for Canadian dairy farmers and promote implementation of those standards through field based knowledge transfer activities. The Network will provide a framework for dialogue and monitoring of progress and impediments to implementation of control practices. The Network offers opportunities for international scientific collaboration, the multidisciplinary training of highly qualified personnel, and provides an advanced educational curriculum for domestic and international students.