Denis Haine

Characterization of the ability of coagulase-negative staphylococci isolated from the milk of Canadian farms to form biofilms

Mastitis is the most common and detrimental infection of the mammary gland in dairy cows and has a major economic impact on the production of milk and dairy products. Bacterial mastitis is caused by several pathogens, and the most frequently isolated bacterial species are coagulase-negative staphylocci (CNS). Although CNS are considered minor mastitis pathogens, the importance of CNS has increased over the years. However, the mechanism and factors involved in CNS intramammary infection are poorly studied and defined. Biofilms have been proposed as an important component in the persistence of CNS intramammary infection. Biofilms are defined as a cluster of bacteria enclosed in a self-produced matrix. The objectives of this study were to investigate the ability of CNS to form biofilms. A total of 255 mastitis-associated CNS isolates were investigated using a standard microtiter plate biofilm assay. The biofilms of some isolates were also observed by using confocal microscopy. The presence of biofilm-associated genes icaA, bap, aap, embP, fbe, and atlE was determined by PCR in the 255 isolates. The 5 dominant species assayed were Staphylococcus chromogenes (n=111), Staphylococcus simulans (n=53), Staphylococcus xylosus (n=25), Staphylococcus haemolyticus (n=15), and Staphylococcus epidermidis (n=13), and these represented 85% of the isolates. The data gathered were analyzed to identify significant links with the data deposited in the Canadian Bovine Mastitis Research Network database. Overall, Staph. xylosus is the species with the strongest ability to form biofilm, and Staph. epidermidis is the species with the lowest ability to form biofilm. Regardless of the species, the presence of icaA, bap, or the combination of multiple genes was associated with a greater ability to form biofilm. A strong relationship between the strength of a biofilm and days in milk was also noted, and CNS isolated later in the lactation cycle appeared to have a greater ability to form biofilm than those isolated earlier in the lactation cycle. In conclusion, Staph. xylosus is the species with the strongest biofilm formation ability. Furthermore, days in milk and gene combinations are predicted to be the variables with the strongest effect on biofilm formation by CNS.

Zinc as an agent for the prevention of biofilm formation by pathogenic bacteria

Biofilm formation is important for the persistence of bacteria in hostile environments. Bacteria in a biofilm are usually more resistant to antibiotics and disinfectants than planktonic bacteria. Our laboratory previously reported that low concentrations of zinc inhibit biofilm formation of Actinobacillus pleuropneumoniae. The aim of this study is to evaluate the effect of zinc on growth and biofilm formation of other bacterial swine pathogens.

Examining the effect of intramammary infections with minor mastitis pathogens on the acquisition of new intramammary infections with major mastitis pathogens—A systematic review and meta-analysis

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, and the coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium bovis and coagulase-negative staphylococci (CNS). The current literature contains several studies detailing analyses with conflicting results as to whether intramammary infection (IMI) with the minor pathogens decreases, increases, or has no effect on the risk of a quarter acquiring a new intramammary infection (NIMI) with a major pathogen. To investigate the available scientific evidence regarding the effect of IMI with minor pathogens on the acquisition of NIMI with major pathogens, a systematic review and meta-analysis were conducted. The total extant English- and French-language literature in electronic databases was searched and all publications cited by relevant papers were investigated. Results from 68 studies were extracted from 38 relevant papers. Random-effects models were used to investigate the effects of CNS and C. bovis on acquisition of new IMI with any of the major pathogens, as well as individually for the minor pathogens and Staph. aureus. Significant heterogeneity among studies exists, some of which could be accounted for by using meta-regression. Overall, observational studies showed no effect, whereas challenge studies showed strong and significant protective effects, specifically when major pathogens were introduced into the mammary gland via methods bypassing the teat end. Underlying risk can account for several unmeasured factors, and studies with higher underlying risk found more protective effects of minor pathogens. Larger doses of challenge organisms reduced the protective effect of minor pathogens, and studies with more stringent diagnostic criteria for pathogen IMI identified less protection. Smaller studies (those utilizing fewer than 40 cows) also showed a greater protective effect than larger studies.

Genotypic and phenotypic characterization of Staphylococcus aureus causing persistent and nonpersistent subclinical bovine intramammary infections during lactation or the dry period

Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be nonpersistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type tbl 2645 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type tbl 2645 were less likely to cause a persistent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than tbl 2645 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada.

Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens

Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1 cfu/0.01 mL and ≥10 cfu/0.01 mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR did not concur with any apparent differences between Se estimates of the 2 tests. Further work should be done to extend these results to other PCR-based tests for detecting bacterial species in milk samples, for which presented results could be used as prior parameter distributions. Limits of sample handling and storage and the potential existence of substances in clinical case samples that may interfere with PCR reactions also are worth further investigation.

Herd-level Mastitis-Associated Costs on Canadian Dairy Farms

Mastitis imposes considerable and recurring economic losses on the dairy industry worldwide. The main objective of this study was to estimate herd-level costs incurred by expenditures and production losses associated with mastitis on Canadian dairy farms in 2015, based on producer reports. Previously, published mastitis economic frameworks were used to develop an economic model with the most important cost components. Components investigated were divided between clinical mastitis (CM), subclinical mastitis (SCM), and other costs components (i.e., preventive measures and product quality). A questionnaire was mailed to 374 dairy producers randomly selected from the (Canadian National Dairy Study 2015) to collect data on these costs components, and 145 dairy producers returned a completed questionnaire. For each herd, costs due to the different mastitis-related components were computed by applying the values reported by the dairy producer to the developed economic model. Then, for each herd, a proportion of the costs attributable to a specific component was computed by dividing absolute costs for this component by total herd mastitis-related costs. Median self-reported CM incidence was 19 cases/100 cow-year and mean self-reported bulk milk somatic cell count was 184,000 cells/mL. Most producers reported using post-milking teat disinfection (97%) and dry cow therapy (93%), and a substantial proportion of producers reported using pre-milking teat disinfection (79%) and wearing gloves during milking (77%). Mastitis costs were substantial (662 CAD per milking cow per year for a typical Canadian dairy farm), with a large portion of the costs (48%) being attributed to SCM, and 34 and 15% due to CM and implementation of preventive measures, respectively. For SCM, the two most important cost components were the subsequent milk yield reduction and culling (72 and 25% of SCM costs, respectively). For CM, first, second, and third most important cost components were culling (48% of CM costs), milk yield reduction following the CM events (34%), and discarded milk (11%), respectively. This study is the first since 1990 to investigate costs of mastitis in Canada. The model developed in the current study can be used to compute mastitis costs at the herd and national level in Canada.

Diagnosing Intramammary Infection: Controlling Misclassification Bias in Longitudinal Udder Health Studies

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.

Early-lactation extended pirlimycin therapy against naturally acquired Staphylococcus aureus intramammary infections in heifers: A randomized controlled trial

The primary objective of the current study was to evaluate cure rate following an early-lactation extended intramammary pirlimycin treatment on heifers naturally infected by Staphylococcus aureus. The secondary objective was to assess Petrifilm Staph Express (3M Microbiology, St. Paul, MN) count plate characteristics when used in a protocol for early-lactation detection of infected quarters in heifers. Milk samples were collected from heifers (n = 946) in the first few days following calving (mean = 5 d). Heifers with laboratory-confirmed S. aureus intramammary infection (n = 72) were randomly allocated into 2 groups. The treatment group (n = 54 quarters from 38 heifers) received an intramammary infusion of 50 mg of pirlimycin once per day for 8 consecutive days in infected quarters. The control group (n = 44 quarters from 34 heifers) did not receive any treatment. Treatment success was defined as having negative culture results for S. aureus in all 3 post-treatment quarter milk samples collected on d 17, 24, and 31 post-treatment. Treatment group mammary quarters showed a statistically significant higher cure rate (64.8%) compared with the control group (34.1%). A total of 38% of quarters identified as S. aureus-positive using the Petrifilm Staph Express count plate were in fact identified as non-aureus staphylococci on routine laboratory-based bacteriological culture. The current study demonstrates that a higher cure rate for S. aureus IMI can be achieved in dairy heifers if an extended treatment protocol is put in place soon after calving. Use of Petrifilm Staph Express count plate for identification of S. aureus-infected heifers could lead to unnecessary treatments because of false-positive results.

Selection and Misclassification Biases in Longitudinal Studies

Using imperfect tests may lead to biased estimates of disease frequency and measures of association. Many studies have looked into the effect of misclassification on statistical inferences. These evaluations were either within a cross-sectional study framework, assessing biased prevalence, or for cohort study designs, evaluating biased incidence rate or risk ratio estimates based on misclassification at one of the two time-points (initial assessment or follow-up). However, both observations at risk and incident cases can be wrongly identified in longitudinal studies, leading to selection and misclassification biases, respectively. The objective of this paper was to evaluate the relative impact of selection and misclassification biases resulting from misclassification, together, on measures of incidence and risk ratio. To investigate impact on measure of disease frequency, data sets from a hypothetical cohort study with two samples collected one month apart were simulated and analyzed based on specific test and disease characteristics, with no elimination of disease during the sampling interval or clustering of observations. Direction and magnitude of bias due to selection, misclassification, and total bias was assessed for diagnostic test sensitivity and specificity ranging from 0.7 to 1.0 and 0.8 to 1.0, respectively, and for specific disease contexts, i.e., disease prevalences of 5 and 20%, and disease incidences of 0.01, 0.05, and 0.1 cases/animal-month. A hypothetical exposure with known strength of association was also generated. A total of 1,000 cohort studies of 1,000 observations each were simulated for these six disease contexts where the same diagnostic test was used to identify observations at risk at beginning of the cohort and incident cases at its end. Our results indicated that the departure of the estimates of disease incidence and risk ratio from their true value were mainly a function of test specificity, and disease prevalence and incidence. The combination of the two biases, at baseline and follow-up, revealed the importance of a good to excellent specificity relative to sensitivity for the diagnostic test. Small divergence from perfect specificity extended quickly to disease incidence over-estimation as true prevalence increased and true incidence decreased. A highly sensitive test to exclude diseased subjects at baseline was of less importance to minimize bias than using a highly specific one at baseline. Near perfect diagnostic test attributes were even more important to obtain a measure of association close to the true risk ratio, according to specific disease characteristics, especially its prevalence. Low prevalent and high incident disease lead to minimal bias if disease is diagnosed with high sensitivity and close to perfect specificity at baseline and follow-up. For more prevalent diseases we observed large risk ratio biases towards the null value, even with near perfect diagnosis.

Contextual herd factors associated with cow culling risk in Québec dairy herds: A multilevel analysis

Several health disorders, such as milk fever, displaced abomasum, and mastitis, as well as impaired reproductive performance, are known risk factors for the removal of affected cows from a dairy herd. While cow-level risk factors are well documented in the literature, herd-level associations have been less frequently investigated. The objective of this study was to investigate the effect of cow- and herd-level determinants on variations in culling risk in Québec dairy herds: whether herd influences a cows culling risk. For this, we assessed the influence of herd membership on cow culling risk according to displaced abomasum, milk fever, and retained placenta. A retrospective longitudinal study was conducted on data from dairy herds in the Province of Québec, Canada, by extracting health information events from the dairy herd health management software used by most Québec dairy producers and their veterinarians. Data were extracted for all lactations starting between January 1st and December 31st, 2010. Using multilevel logistic regression, we analysed a total of 10,529 cows from 201 herds that met the inclusion criteria. Milk fever and displaced abomasum were demonstrated to increase the cow culling risk. A minor general herd effect was found for the culling risk (i.e. an intra-class correlation of 1.0% and median odds ratio [MOR] of 1.20). The proportion of first lactation cows was responsible for this significant, but weak herd effect on individual cow culling risk, after taking into account the cow-level factors. On the other hand, the herds average milk production was a protective factor. The planning and management of forthcoming replacement animals has to be taken into consideration when assessing cow culling risks and herd culling rates.