Partial prediction of the duration and the clinical status of Staphylococcus aureus bovine intramammary infections based on the phenotypic and genotypic analysis of isolates.

Staphylococcus aureus is a bacterial pathogen causing bovine intramammary infections (IMIs) often leading to chronic clinical or subclinical mastitis. Predicting the outcome of S. aureus IMIs (duration and clinical vs subclinical) based on the characterization of isolates would help to make better case management decisions. For this purpose, 583 S. aureus isolates from series of quarter milk samples were characterized by genotypic tests (detection of virulence genes seg, tst, lukM), epidemiological typing (spa type) and by a phenotypic test (biofilm production). VNTR typing (variable number of tandem repeats) was used to establish persistence of the same S. aureus strain in each series of sequential isolates. This allowed to associate each strain to a clinical/subclinical status and to validate the duration of infection. We found differences in the distribution of spa types between the strains from clinical and subclinical cases. Prevalence of lukM was also higher in strains from clinical cases than in strains from subclinical cases. A Kaplan-Meier analysis was then used to determine factors influencing the duration of the infection. Considering a multivariable model of the logistic regression, time to elimination was shorter with the strains of the subclinical lactation series compared to the clinical series (series with at least one clinical case). Strains from the spa type t359 and t529 were less likely to persist compared to those of spa type t13410. In sum, strain characterization including determination of the spa type helps to predict duration of infection and the clinical or subclinical outcome of S. aureus IMIs.

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.

Prediction of bulk tank somatic cell count violations based on monthly individual cow somatic cell count data.

The regulatory limit in Canada for bulk tank somatic cell count (BTSCC) was recently lowered from 500,000 to 400,000 cells/mL. Herd indices based on changes in cow somatic cell count over 2 consecutive months (e.g., proportion of healthy or chronically infected cows, cows cured, and new intramammary infection rate) could be used as predictors for BTSCC violations. The objective of this study was to develop a predictive model for exceeding the limit of 400,000 cells/mL in the next month using these herd indices. Dairy Herd Improvement (DHI) data were used from 924 dairy herds in Québec, Canada. Test-day BTSCC was estimated by dividing the sum of all cows' DHI test-day somatic cell count times DHI test-day milk production by the total volume of milk produced by the herd on that test-day. In total, 986 of 8,681 (11.4%) estimated BTSCC exceeded 400,000 cells/mL. The final predictive model included 6 variables: mean herd somatic cell score at the current test-month, proportion of cows >500,000 cells/mL at the current test-month, proportion of healthy cows during lactation at the current test-month, proportion of chronically infected cows at the current test-month, average days in milk at the current test-month, and annual mean daily milk production. The optimized sensitivity and specificity of the model were 76 and 74%, respectively. The positive predictive value and negative predictive value were 25 and 95%, respectively. This low positive predictive value and high negative predictive value demonstrated that the model was less accurate at predicting herds that would violate the estimated BTSCC threshold but very accurate at identifying herds that would not. In addition, the area under the curve for the receiver operating characteristic curve was 0.82, suggesting that the model had excellent discrimination between test-months that did and did not exceed 400,000 cells/mL. An internal validation was completed using a bootstrapped resampling-based estimation method and confirmed that the final model provided a validated estimate of predictive accuracy. This model could be used to monitor and advise clients on impending risks of exceeding the BTSCC limit.

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.

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

AIMS: 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. METHODS AND RESULTS: To determine the effect of zinc on biofilm formation, biofilms were grown with or without zinc in 96-well plates and stained with crystal violet. At micromolar concentrations (0-250 µ mol l(-1)), zinc weakly inhibited bacterial growth and it effectively blocked biofilm formation by A. pleuropneumoniae, Salmonella Typhymurium and Haemophilus parasuis in a dose-dependent manner. Additionally, biofilm formation of Escherichia coli, Staphylococcus aureus and Streptococcus suis was slightly inhibited by zinc. However, zinc did not disperse preformed biofilms. To determine whether zinc inhibits biofilm formation when poly-N-acetylglucosamine (PGA) is present, PGA was detected with the lectin wheat germ agglutinin. Only A. pleuropneumoniae and Staph. aureus biofilms were found to contain PGA. CONCLUSION: Zinc used at nonbactericidal concentrations can inhibit biofilm formation by several Gram-negative and Gram-positive bacterial swine pathogens. SIGNIFICANCE AND IMPACT OF STUDY: The antibiofilm activity of zinc could provide a tool to fight biofilms, and the nonspecific inhibitory effect may well extend to other important human and animal bacterial pathogens.

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.

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.

Domain-specific cognitive effects of tramiprosate in patients with mild to moderate Alzheimer's disease: ADAS-cog subscale results from the Alphase Study.

OBJECTIVES: Tramiprosate (homotaurine, ALZHEMEDTM) was recently investigated for its efficacy, safety and disease-modification effects in a Phase III clinical study in mild to moderate Alzheimer's disease (AD) patients (the Alphase study). The primary cognitive endpoint measure of that study was the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog). To characterize potential cognitive benefits of tramiprosate, the present study describes exploratory analyses performed on scores obtained from the specific ADAS-cog subscales in order to determine whether specific domains of cognition may be differentially affected by tramiprosate, which would not have been evident from the measure's total score. DESIGN: Multi-center, double-blind, randomized, placebo-controlled study. SETTING: 67 investigative sites in the United States and Canada. PARTICIPANTS: A total of 1,052 patients were randomized. INTERVENTIONS: Patients were randomized to receive twice a day Placebo (n=353), tramiprosate 100 mg (n=352) and tramiprosate 150 mg (n=347). MEASUREMENTS: ADAS-cog assessments were conducted every three months over the 78-week study period. Exploratory analyses were performed by comparing ADAS-cog subscale scores between Placebo and each active treatment arm at each visit. RESULTS: The findings of this analysis revealed statistically significant differences or statistical trends in favour of tramiprosate on six ADAS-cog subscales, namely Following Commands, Language Comprehension, Ideational Praxis, Object Naming, Remembering Test Instructions, and Spoken Language Ability. Differences in favor of Placebo were only observed on the Constructional Praxis subscale. CONCLUSION: This exploratory analysis suggests that tramiprosate may have some benefit on memory, language and praxis skills in mild to moderate AD individuals. Future clinical studies of tramiprosate should include specialized neuropsychological tests to validate its effects within these cognitive domains.

Effect of tramiprosate in patients with mild-to-moderate Alzheimer's disease: exploratory analyses of the MRI sub-group of the Alphase study.

OBJECTIVES: The efficacy, safety and disease-modification of tramiprosate (homotaurine)were investigated in a recently completed large-scale Phase III clinical study in patients with mild to moderate Alzheimer's disease (AD), the Alphase study. Disease-modification was assessed using longitudinal volumetric MRI (vMRI) measurements of the hippocampus in a subgroup of patients. The present study describes the vMRI, cognitive and clinical results obtained in this subgroup. DESIGN: Multi-center, double-blind, randomized, placebo-controlled study in a subset of the 1052 patients of the Alphase study. SETTING: 51 vMRI investigative sites in the United States and Canada. PARTICIPANTS: A total of 508 patients underwent vMRI scanning. Of these, 312 provided scan pairs for assessing hippocampus volume changes and were included in the analyses. INTERVENTIONS: Patients were randomized to receive Placebo BID (n = 109), tramiprosate 100 mg BID (n = 103), or tramiprosate 150 mg BID (n = 100) for 78 weeks. MEASUREMENTS: Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) and Clinical Dementia Rating-Sum-of-boxes CDR-SB assessments were conducted at Baseline and at Weeks 13, 26, 39, 52, 65 and 78. Exploratory analyses were performed using similar First and Final mixed-effects repeated-measures models that were used for the analysis of the entire patient dataset. RESULTS: Psychometric score results showed numerical trends in favour of tramiprosate that did not reach statistical significance. While there were no statistically significant group differences in hippocampus volume using the First modeling approach, a significant dose-response reduction in hippocampus volume change was found in the Final models. Moreover, there was a marginally significant overall treatment main effect and a significant slope difference in favour of tramiprosate according to the Final model analysis of the ADAS-cog scores. ADAS-cog scores analyzed according to this model also revealed differences in favor of the tramiprosate 150 mg group at weeks 26 and 52, with marginally significant differences at Weeks 13 and 39. Slope analyses of ADAS-cog score changes showed significant differences in favor of the 150 mg BID group, and when both active groups were combined, in comparison to the placebo group. No between-group differences with respect to changes to each visit in the CDR-SB were observed with either modeling approach. Although there was a similar dose-response relationship observed in the hippocampus volume and ADAS-cog Final model analyses, the overall changes in psychometric scores and hippocampus volume were not significantly correlated. CONCLUSION: Exploratory analysis of the vMRI subgroup suggests that tramiprosate slows hippocampal atrophy, and reveals some evidence of a beneficial effect on cognition. The clinical validity of the vMRI biomarker is discussed.

Herd-level seroprevalence and risk-mapping of bovine hypodermosis in Belgian cattle herds.

Our objective was to determine the seroprevalence of Hypoderma spp. and to develop a spatial model describing the risk surface of warble-fly infection in Belgian cattle herds (adjusting for herd size, herd type, local temperature, rainfall, relative air humidity and land-cover). This survey was carried out in 390 selected herds of all types (dairy, mixed and beef) from December 1997 to March 1998, which were included in a national infectious bovine rhinotracheitis and paratuberculosis (Johne's-disease) survey. All animals >24 months old were blood sampled and an ELISA was used on pooled serum samples (10 animals per pool). The herd seroprevalence was 48.7% (95% confidence interval: 43.6-53.8); positive herds were mainly in the south of the country and along the North Sea coast. The logistic multiple-regression model of herd-level seropositivity indicated that mixed-type and beef-cattle herds have more than four-fold and two-fold increases in the odds of being Hypoderma-positive, respectively, compared with dairy herds.