A topological switch in CFTR modulates channel activity and sensitivity to unfolding.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the ß-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Micromotion Analysis of Various Tibial Constructs in Moderate Tibial Defects in Revision Total Knee Arthroplasty.

BACKGROUND: The purpose of this study is to compare the micromotion of various tibial reconstruction strategies including short cemented and long cementless stems with or without metaphyseal augmentation. METHODS: A moderate tibial bone defect was milled into dual density polyurethane test blocks. Mechanical testing was performed on 4 test constructs: (1) short cemented stem (75-mm total length) alone; (2) short cemented stem with a symmetric metaphyseal cone; (3) a press-fit (175-mm total length) diaphyseal engaging tibial construct without a cone, and (4) the same press-fit tibial construct with a metaphyseal cone augment. Micromotion of the baseplate/cone construct with respect to the tibia block was measured during a stair descent loading profile for 10,000 cycles. The peak-to-peak micromotion of these various tibial constructs was compared. Unpaired t-tests were used to evaluate differences in peak-to-peak micromotion among the various tibial constructs tested. An analysis of variance was performed for final validation. RESULTS: The cemented short stem demonstrated similar varus/valgus displacement, internal/external rotation, compression, and lift-off micromotion values under loading compared to a cementless long stem. A tibial cone improved compression and lift-off micromotion for both cemented and cementless constructs. A short 50-mm cemented stem with a cone demonstrated a lower micromotion at the anterior SI location compared to a press-fit 150-mm cementless stem without a tibial cone. CONCLUSIONS: A short cemented tibial component with a cone achieved similar micromotion during simulated stair descent compared to a cementless diaphyseal press-fit implant in cases of moderate tibial defects.

Tibial shaft anatomy differs between Caucasians and East Asian individuals.

PURPOSE: The orientation and distance from the shaft of the femur and tibia to the articular surface centre is important for performing total knee arthroplasty (TKA) with cementless stems. It is important to understand anatomic differences between races to match the tibial and femoral shaft axis to the knee articular surface. Thus, the purpose of this study was to compare knee morphology between Caucasian and East Asian individuals to determine the optimal placement of tibial and femoral stems. METHODS: A retrospective study was conducted on a matched cohort of 50 East Asians (21F, 29M) and 50 Caucasians (21F, 29M) by age and gender. CT scans were obtained in healthy volunteers using < 2-mm slices. The distance from the proximal tibial diaphysis axis to the tibial plateau centre and the distance from the distal femoral diaphysis axis to the centre of distal femoral articular surface were measured separately. Tibial measurements were taken using Akagi's anteroposterior (AP) axis and the widest mediolateral (ML) diameter, and femoral measurements were based on Whiteside's line and the surgical epicondylar axis. RESULTS: The ML distance between the tibial shaft centre and Akagi's line was significantly higher for Asians (9.9 ± 2.7 mm, Caucasians 7.7 ± 3.1 mm, p < 0.001). The distance between the femoral shaft centre and Whiteside's line was lower, although not significantly different (Asians 1.9 ± 1.0 mm, Caucasians 2.2 ± 1.1 mm, n.s.). However, there were no differences in the AP dimension for the femur or tibia comparing Asians to Caucasians in both tibial side (Asians 10.6 ± 3.3 mm vs. Caucasians 10.9 ± 4.0 mm) and femoral side (Asians 18.1 ± 1.7 mm vs. Caucasians 17.5 ± 1.6 mm). CONCLUSION: East Asian individuals have more offset in the ML dimension for the tibia. This finding is clinical relevant, as this dimensional difference should be taken into consideration when designing primary and revision TKA stemmed tibial implants for East Asian individuals. LEVEL OF EVIDENCE: Case-control study, Level III.

Benchmarks for evaluation and comparison of udder health status using monthly individual somatic cell count.

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.

Normes de référence pour l'analyse de la santé mammaire des troupeaux à l'aide des comptages en cellules somatiques individuels mensuels. L'objectif de l'étude consistait à établir des normes de référence reliées aux comptages en cellules somatiques (CCS) individuels mensuels pouvant être utilisées pour l'interprétation de la santé du pis des troupeaux et d'évaluer l'effet de certains facteurs de risque sur la dynamique d'infection intra-troupeau. La moyenne et les percentiles d'indices de santé du pis [proportion de vaches saines, nouvellement infectées (NIMI), guéries et infectées de manière chronique] ont été calculés pour les périodes de lactation et de tarissement en utilisant un CCS ≥ 200 000 cells/mL comme définition d'infection intramammaire. Les proportions moyennes de NIMI et de guérison en lactation ont été établies à 0,11 et 0,27, respectivement. Les objectifs quant à la proportion de vaches saines et infectées de manière chronique au tarissement ont été établis à 0,70 et 0,03. La saison et le CCS moyen du troupeau sont apparus comme des facteurs de risque significatifs.(Traduit par les auteurs).

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms.

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.

Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Conformational Freedom and Topological Confinement of Proteins in Biomolecular Condensates.

The emergence of biomolecular condensation and liquid-liquid phase separation (LLPS) introduces a new layer of complexity into our understanding of cell and molecular biology. Evidence steadily grows indicating that condensates are not only implicated in physiology but also human disease. Macro- and mesoscale characterization of condensates as a whole have been instrumental in understanding their biological functions and dysfunctions. By contrast, the molecular level characterization of condensates and how condensates modify the properties of the molecules that constitute them thus far remain comparably scarce. In this minireview we summarize and discuss the findings of several recent studies that have focused on structure, dynamics, and interactions of proteins undergoing condensation. The mechanistic insights they provide help us identify the relevant properties nature and scientists can leverage to modulate the behavior of condensate systems. We also discuss the unique environment of the droplet surface and speculate on effects of topological constraints and physical exclusion on condensate properties.

The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems.

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

Evaluation of the California Mastitis Test as a precalving treatment selection tool for Holstein heifers.

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.

Domain-interface dynamics of CFTR revealed by stabilizing nanobodies.

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.

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.

Effect of precalving intramammary treatment with pirlimycin in nulliparous Holstein heifers.

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.

Prevalence and incidence density of Mycobacterium leprae and Trypanosoma cruzi infections within a population of wild nine-banded armadillos.

A total of 415 wild 9-banded armadillos from the East Atchafalaya River Levee (Point Coupee, LA) were collected over 4 years to estimate the incidence and prevalence of Mycobacterium leprae and Trypanosoma cruzi and to discern any relationship between the 2 agents. M. leprae infections were maintained at a high steady prevalence rate year to year averaging 19%. T. cruzi antibody prevalence remained relatively low, averaging 3.9%, and varied markedly between years. Prevalence rates were independent, with only 3 armadillos coinfected with both agents. M. leprae incidence density ranged from 0.47 to 3.5 cases per 1,000 animal-days, depending on case definition, confirming active intense transmission of M. leprae among armadillos. No incident T. cruzi cases were found. These infections seem to occur independently and may be used in comparisons to understand better factors that may influence transmission of these agents.

Climatic and demographic determinants of American visceral leishmaniasis in northeastern Brazil using remote sensing technology for environmental categorization of rain and region influences on leishmaniasis.

Remote sensing (RS) permits evaluation of spatial and temporal variables that can be used for vector-borne disease models. A Landsat Thematic Mapper scene covering Canindé, Ceará in northeastern Brazil (September 25, 1986) was spectrally enhanced and classified using ERDAS (Atlanta, GA) Imagine for 873 4-km2 areas. The population and number of cases of American visceral leishmaniasis (AVL) were determined for each 4-km2 area. Relative risk (RR) ratios were calculated for climate, demographic, and case data recorded for 17 years by the Municipality of Conidé. The RR of AVL for a child less than 10 years old from the foothills relative to non-foothill residency was 4.0 (95% confidence limit = 3.5, 4.5). The RR of AVL in children was 9.1 during a time when the three-year rolling rain average (current year plus two previous year's precipitation) was between 40 and 60 cm relative to rain greater than 100 cm. The results suggest that features detected by RS techniques combined with climatic variables can be used to determine the risk of AVL in northeastern Brazil.

Efficacy of extended intramammary ceftiofur therapy against mild to moderate clinical mastitis in Holstein dairy cows: a randomized clinical trial.

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.

Peu d'études ont investigué l'efficacité d'une thérapie prolongée avec du ceftiofur et aucune n'a examiné attentivement une thérapie prolongée dans le cas de mammite clinique se produisant naturellement. L'objectif de la présente étude était de comparer l'efficacité d'une thérapie intra-mammaire prolongée d'une durée de 8 jours avec du ceftiofur à la thérapie standard de 2 jours pour le traitement de mammite clinique légère à modérée survenant naturellement chez des vaches laitières en lactation. Des vaches de race Holstein provenant de 22 troupeaux laitiers (n = 241) ont été réparties de manière aléatoire dans les 2 groupes de traitement. Pour chaque cas de mammite, 125 mg d'hydrochlorure de ceftiofur furent administrés par voie intra-mammaire une fois par jour pour 2 ou 8 jours. La guérison clinique, 21 jours après le dernier traitement, était de 89 % (98/110) dans chaque groupe. La guérison bactériologique 21 jours après le dernier traitement pour les groupes 2 et 8 jours était respectivement 32 % (15/47) et 61 % (25/41) pour toutes les bactéries (P = 0,007), 64 % (9/14) et 82 % (9/11), respectivement, pour les streptocoques (P = 0,50) et 0 % (0/20) et 47 % (9/19), respectivement pour Staphylococcus aureus (P = 0,0004). Il n'y avait aucune différence statistiquement significative entre les groupes pour les nouvelles infections intra-mammaires. Globalement, une thérapie prolongée au ceftiofur augmenta la guérison lorsque comparée à une thérapie de 2 jours pour le traitement de mammite clinique légère à modérée survenant naturellement chez les vaches laitières en lactation.(Traduit par Docteur Serge Messier).

Draft Genome Sequence of Bovine Mastitis Isolate Staphylococcus agnetis CBMRN 20813338.

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.

The expression of a putative exotoxin and an ABC transporter during bovine intramammary infection contributes to the virulence of Staphylococcus aureus.

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.

Management practices associated with the bulk-milk prevalence of Staphylococcus aureus in Canadian dairy farms.

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 Québec 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.

Development and validation of a bilingual questionnaire for measuring udder health related management practices on dairy farms.

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.