A single center's experience using four different front line mobilization strategies in lymphoma patients planned to undergo autologous hematopoietic cell transplantation.

In an otherwise eligible patient with relapsed lymphoma, inadequate mobilization of hematopoietic stem cells (HSCs) is a limiting factor to proceeding with an autologous hematopoietic cell transplantation (auto-HCT). Multiple strategies have been used to mobilize an adequate number of HSCs with no obvious front-line strategy. We report a single institutional experience mobilizing HSCs using four different approaches in lymphoma patients. We prospectively collected mobilization outcomes on patients planned to undergo auto-HCT at Ohio State University. We report results of first mobilization attempts for all relapsed or refractory lymphoma patients between 2008 and 2014. We identified 255 lymphoma patients who underwent mobilization for planned auto-HCT. The 255 lymphoma patients underwent the following front line mobilization strategies: 95 (37%) G-CSF alone, 38 (15%) chemomobilization (G-CSF+chemotherapy), 97 (38%) preemptive day 4 plerixafor, and 25 (10%) rescue day 5 plerixafor. As expected, there were significant differences between cohorts including age, comorbidity indices, histology, and amount of prior chemotherapy. After controlling for differences between groups, the odds of collecting 2 × 106/kg HSCs on the first day of collection and 5 × 106/kg HSCs in total was the highest in the cohort undergoing chemomobilization. In conclusion, our experience highlights the effectiveness of chemomobilization.

Inter- and intrasubject variability of the inflammatory response to segmental endotoxin challenge in healthy volunteers.

Segmental endotoxin challenge with lipopolysaccharide (LPS) can be used as a pharmacodynamic model to safely induce a transient airway inflammation in the peripheral lung of healthy subjects and to test the anti-inflammatory efficacy of investigational new drugs. In contrast to whole lung LPS challenge only a fraction of the dose is required that can be precisely administered to a specific lung region and a vehicle challenged segment as an intra-subject control can be included. The aim of this study was to assess the intra- and inter-individual variability of the response to segmental LPS challenge for the appropriate design and power calculation of future clinical trials. Two cohorts with 10 subjects each underwent two segmental LPS challenges within five weeks. The inflammatory response was evaluated in bronchoalveolar lavage (BAL) fluid at 6 (cohort 1) and 24 h (cohort 2) both in the LPS and in a vehicle challenged segment, as well as in plasma for up to 26 h post LPS challenge. While the cytokine response was more pronounced at 6 h, the influx of neutrophils and monocytes dominated at 24 h; e.g. neutrophils increased from a median (inter-quartile range, IQR) of 0.14 (0.16) and 0.09 (0.08)x10(4) cells/mL BAL fluid at baseline to 10.2 (17.1) and 19.3 (15.9)x10(4) cells/mL 24 h after the two separate challenges. The within-subject variability was higher than the between-subject variability for most of the markers. However, sample size estimations based on the variability of outcome variables found lower or equal numbers with cross-over designs compared to parallel group designs for cellular markers at 24 h and cytokine variables at 6 h. The segmental LPS challenge model was safe. Future study designs have to balance between burden to the study subjects (4 versus 2 bronchoscopies), variability (within-versus between-subject), and the desired outcome variable (cells versus chemo/cytokine).

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.

Association of coagulase-negative staphylococcal species, mammary quarter milk somatic cell count, and persistence of intramammary infection in dairy cattle.

This study was conducted to evaluate the association between subclinical intramammary infection (IMI) with coagulase-negative staphylococci (CNS), mammary quarter milk somatic cell count (SCC), and persistence of IMI in dairy cattle. Convenience samples of CNS isolates harvested from milk samples of subclinically infected mammary quarters collected between 4 and 2wk before drying-off, between 2wk before drying-off and the day of drying-off, within 24h after calving, between 1 and 2wk after calving, and during lactation were evaluated. Isolates were obtained from the Canadian Bovine Mastitis Research Network culture bank and were identified to the species level using rpoB gene sequencing. Cow and quarter-level data were obtained from the Canadian Bovine Mastitis Research Network database and used for statistical analyses. In addition, for mammary quarters that had more than one isolation of the same CNS species at different time points, the isolates were evaluated using pulsed-field gel electrophoresis to identify persistent IMI. Milk SCC was compared between mammary quarters infected with different CNS species and to a cohort of uninfected mammary quarters. A total of 877 isolates from 643 mammary quarters of 555 cows on 89 Canadian dairy farms were identified to the species level. Twenty different species were identified, with Staphylococcus chromogenes being the most common species identified (48% of isolates), followed by Staphylococcus simulans (19%) and Staphylococcus xylosus (10%). Of the 20 species identified, only 9 species were found in persistently infected quarters. Milk SCC was significantly higher in the CNS-infected mammary quarters than in the uninfected control quarters for 8 of the 20 species studied. Also, mean SCC differed significantly between mammary quarters infected with different CNS species. Within a given species, a high degree of variability was noted in milk SCC. These data corroborate recent data from Europe with regard to the predominance of certain species of CNS (e.g., Staph. chromogenes). In addition, some species of CNS appear to have a greater effect on milk SCC. Finally, some CNS species are associated with persistent IMI suggesting that some species (e.g., Staph. chromogenes and Staph. simulans) are better host-adapted, whereas others may have an environmental reservoir.

An effective mobilization strategy for lymphoma patients after failed upfront mobilization with plerixafor.

In an otherwise eligible patient, inadequate mobilization of PBSCs is a limiting factor to proceeding with an auto-ASCT. In such situations, plerixafor is commonly added to improve PBSC collection yields along with cytokine (G-CSF alone) or chemomobilization (chemotherapy+G-CSF). Individually, both strategies are proven to be safe and effective. Here we report six patients who underwent successful mobilization with combination chemomobilization plus plerixafor after upfront failure of cytokine mobilization plus plerixafor. The median CD34(+) cell yield after chemomobilization was 2.48 × 10(6)/kg (range 0.99-8.49) after receiving one to two doses of plerixafor. All patients subsequently underwent ASCT without major unforeseen toxicities and engrafted successfully. No significant delays in time to neutrophil recovery were observed. Our experience highlights the safety and effectiveness of chemomobilization with plerixafor after G-CSF plus plerixafor (G+P) failure and suggests this is a viable salvage strategy after initial failed G+P mobilization.

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.

Antimicrobial resistance profiles of common mastitis pathogens on Canadian dairy farms.

Monitoring of antimicrobial resistance (AMR) in bacteria has clinical and public health significance. The present study determined prevalence of AMR in common mastitis pathogens Staphylococcus aureus, including methicillin-resistant Staph. aureus (MRSA; n=1,810), Escherichia coli (n=394), and Klebsiella species (n=139), including extended-spectrum ß-lactamase (ESBL)-producing E. coli and Klebsiella species, isolated from milk samples on 89 dairy farms in 6 Canadian provinces. Minimum inhibitory concentrations (MIC) were determined using the Sensititer bovine mastitis plate (Trek Diagnostic Systems Inc., Cleveland, OH) and a National Antimicrobial Resistance Monitoring System gram-negative panel containing antimicrobials commonly used for mastitis treatment and control. Denim blue chromogenic agar and real-time PCR were used to screen and confirm MRSA, respectively. Resistance proportion estimates ranged from 0% for cephalothin and oxacillin to 8.8% for penicillin in Staph. aureus isolates, and 15% of the resistant Staph. aureus isolates were multidrug resistant. One MRSA isolate was confirmed (prevalence: 0.05%). Resistance proportion estimates ranged from 0% for ceftriaxone and ciprofloxacin to 14.8% for tetracycline in E. coli, and 0% for amikacin, ceftiofur, ciprofloxacin, and nalidixic acid to 18.6% for tetracycline in Klebsiella species isolates. Further, 62.8 and 55% of the resistant E. coli and Klebsiella species isolates were multidrug resistant, respectively. Resistance to >5 and >2 antimicrobials was most common in E. coli and Klebsiella species isolates, respectively, and no ESBL producers were found. Prevalence of AMR in bovine mastitis pathogens was low. Most gram-negative udder pathogens were multidrug resistant; MRSA was rarely found, and ESBL E. coli and Klebsiella species isolates were absent in Canadian milk samples.

Evaluation of minor pathogen intramammary infection, susceptibility parameters, and somatic cell counts on the development of new intramammary infections with major mastitis pathogens.

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium spp. and coagulase-negative staphylococci (CNS). The current literature comprises several studies (n=38) detailing analyses with conflicting results as to whether intramammary infections (IMI) with the minor pathogens decrease, increase, or have no effect on the risk of a quarter acquiring a new IMI (NIMI) with a major pathogen. The Canadian Bovine Mastitis Research Network has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to further investigate the effect of IMI with minor pathogens on the acquisition of new major pathogen infections (defined as a culture-positive quarter sample in a quarter that had been free of that major pathogen in previous samples in the sampling period). Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used to this end (n=80,397 quarter milk samples). The effects of CNS and Corynebacterium spp. on the major mastitis pathogens Staph. aureus, Strep. uberis, Strep. dysgalactiae, and coliform bacteria (Escherichia coli and Klebsiella spp.) were investigated using risk ratio analyses and multilevel logistic regression models. Quarter-, cow- and herd-level susceptibility parameters were also evaluated and were able to account for the increased susceptibility that exists within herds, cows and quarters, removing it from estimates for the effects of the minor pathogens. Increased quarter-level susceptibility was associated with increased risk of major pathogen NIMI for all pathogens except the coliforms. Increased somatic cell count was consistently associated with elevated risk of new major pathogen infections, but this was assumed to be a result of low sensitivity of bacteriology to diagnose major pathogen NIMI expediently and accurately. The presence of CNS in the sample 2 samplings before the occurrence of a NIMI increased the odds of experiencing a Staph. aureus NIMI 2.0 times, making the presence of CNS a risk factor for acquiring a Staph. aureus NIMI. Even with this extensive data set, power was insufficient to make a definitive statement about the effect of minor pathogen IMI on the acquisition of major pathogen NIMI. Definitively answering questions of this nature are likely to require an extremely large data set dedicated particularly to minor pathogen presence and NIMI with major pathogens.

Epidemiology of coagulase-negative staphylococci intramammary infection in dairy cattle and the effect of bacteriological culture misclassification.

Objectives of this study were to identify the manageable risk factors associated with the lactational incidence, elimination, and prevalence of coagulase-negative staphylococci (CNS) intramammary infections (IMI) while taking into account the difficulties inherent to their diagnosis. A second objective was to evaluate the effect of CNS IMI misclassification in mastitis research. A cohort of 90 Canadian dairy herds was followed throughout 2007 to 2008. In each herd, series of quarter milk samples were collected from a subsample of cows and bacteriological culture was performed to identify prevalent, incident, and eliminated CNS IMI. Practices used on farms were captured using direct observations and a validated questionnaire. The relationships between herd CNS IMI prevalence and herd incidence and elimination rates were explored using linear regression. Manageable risk factors associated with the prevalence, incidence, or elimination of CNS IMI were identified via Bayesian analyses using a latent class model approach, allowing adjustment of the estimates for the imperfect sensitivity and specificity of bacteriological culture. After adjustment for the diagnostic test limitations, a mean CNS IMI quarter prevalence of 42.7% [95% confidence interval (CI): 34.7, 50.1] and incidence and elimination rates of 0.29 new IMI/quarter-month (95% CI: 0.21, 0.37) and 0.79 eliminated IMI/quarter-month (95% CI: 0.66, 0.91), respectively, were observed. Considerable biases of the estimates were observed when CNS IMI misclassification was ignored. These biases were important for measures of association with risk factors, were almost always toward the null value, and led to both type I and type II errors. Coagulase-negative staphylococci IMI incidence appeared to be a stronger determinant of herd IMI prevalence than IMI elimination rate. The majority of herds followed were already using blanket dry cow treatment and postmilking teat disinfection. A holistic approach considering associations with all 3 outcomes was used to interpret associations between manageable risk factors and CNS IMI. Sand and wood-based product bedding showed desirable associations with CNS IMI compared with straw bedding. Quarters of cows that had access to pasture during the sampling period had lower odds of acquiring a new CNS IMI and of having a prevalent CNS IMI. Many practices showed an association with only one of the CNS outcomes and should, therefore, be considered with caution.

Herd-level association between antimicrobial use and antimicrobial resistance in bovine mastitis Staphylococcus aureus isolates on Canadian dairy farms.

Surveillance of antimicrobial use and resistance is needed to manage antimicrobial resistance in bacteria. In this study, data were collected on antimicrobial use and resistance in Staphylococcus aureus (n=562), isolated from intramammary infections and (sub)clinical mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and the Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Dairy producers were asked to deposit empty drug containers into specially provided receptacles, and antimicrobial drug use rate was calculated to quantify antimicrobial use. Minimum inhibitory concentrations were determined using the Sensititer bovine mastitis plate system (TREK Diagnostic Systems Inc., Cleveland, OH), containing antimicrobials commonly used for mastitis treatment and control. Multivariable logistic regression models were built to determine herd-level risk factors of penicillin, ampicillin, pirlimycin, penicillin-novobiocin combination, tetracycline and sulfadimethoxine resistance in Staph. aureus isolates. Intramammary administration of the penicillin-novobiocin combination for dry cow therapy was associated with penicillin and ampicillin resistance [odds ratio (OR): 2.17 and 3.10, respectively]. Systemic administration of penicillin was associated with penicillin resistance (OR: 1.63). Intramammary administration of pirlimycin for lactating cow mastitis treatment was associated with pirlimycin resistance as well (OR: 2.07). Average herd parity was associated with ampicillin and tetracycline resistance (OR: 3.88 and 0.02, respectively). Average herd size was also associated with tetracycline resistance (OR: 1.02). Dairy herds in the Maritime region had higher odds of penicillin and lower odds of ampicillin resistance than dairy herds in Québec (OR: 2.18 and 0.19, respectively). Alberta dairy herds had lower odds of ampicillin and sulfadimethoxine resistance than dairy herds in Québec (OR: 0.04 and 0.08, respectively). Ontario dairy herds had lower odds of tetracycline and sulfadimethoxine resistance than dairy herds in Québec (OR: 0.05 and 0.33, respectively). Herd-level use of certain antimicrobials administered for mastitis treatment and control, such as intramammary penicillin and pirlimycin as well as systemically administered penicillin and florfenicol, was positively associated with antimicrobial resistance in bovine mastitis pathogens in the field conditions. Differences in antimicrobial resistance outcomes across 4 regions of Canada were observed.

Antimicrobial use on Canadian dairy farms.

Antimicrobial use (AMU) data are critical for formulating policies for containing antimicrobial resistance. The present study determined AMU on Canadian dairy farms and characterized variation in AMU based on herd-level factors such as milk production, somatic cell count, herd size, geographic region and housing type. Drug use data were collected on 89 dairy herds in 4 regions of Canada, Alberta, Ontario, Québec, and the Maritime provinces (Prince Edward Island, New Brunswick, and Nova Scotia) for an average of 540 d per herd. Dairy producers and farm personnel were asked to deposit empty drug containers into specially provided receptacles. Antimicrobial use was measured as antimicrobial drug use rate (ADUR), with the unit being number of animal defined-daily doses (ADD)/1,000 cow-days. Antimicrobial drug use rates were determined at farm, region, and national level. Combined ADUR of all antimicrobial classes was 14.35 ADD/1,000 cow-days nationally. National level ADUR of the 6 most commonly used antimicrobial drug classes, cephalosporins, penicillins, penicillin combinations, tetracyclines, trimethoprim-sulfonamide combinations, and lincosamides were 3.05, 2.56, 2.20, 1.83, 0.87, and 0.84 ADD/1,000 cow-days, respectively. Dairy herds in Ontario were higher users of third-generation cephalosporins (ceftiofur) than in Québec. Alberta dairy herds were higher users of tetracyclines in comparison to Maritimes. Antimicrobial drug use rate was higher via systemic route as compared with intramammary and other routes of administration (topical, oral, and intrauterine). The ADUR of antimicrobials used intramammarily was higher for clinical mastitis treatment than dry cow therapy. For dry cow therapy, penicillin ADUR was greater than ADUR of first-generation cephalosporins. For clinical mastitis treatment, ADUR of intramammary penicillin combinations was greater than ADUR of cephapirin. Herd-level milk production was positively associated with overall ADUR, ADUR of systemically administered ceftiofur, cephapirin administered for dry cow therapy, and pirlimycin administered for clinical mastitis treatment. Herd size and ADUR of systemically administered ceftiofur were also positively associated. In conclusion, ß-lactams were most commonly used on Canadian dairy farms. Among antimicrobials of very high importance in human medicine, the use of fluoroquinolones was rare, whereas third-generation cephalosporins and penicillin combinations containing colistin were used very frequently on Canadian dairy farms.

Manageable risk factors associated with the lactational incidence, elimination, and prevalence of Staphylococcus aureus intramammary infections in dairy cows.

Staphylococcus aureus intramammary infections (IMI) are a major cause of mastitis on farms worldwide. Incidence and elimination rates are the key determinants of prevalence of Staph. aureus, and risk factors associated with these rates must be identified, prioritized, and controlled to obtain long-term reduction in prevalence. The objectives of this study were to identify manageable risk factors associated with the lactational incidence, elimination, and prevalence of Staph. aureus IMI. A cohort of 90 Canadian dairy farms was recruited and followed in 2007 and 2008. Quarter milk samples were collected repeatedly from a selection of cows, and bacteriological culture was realized to assess incidence, elimination, and prevalence of Staph. aureus IMI. Practices used on farms were measured using direct observations and a validated questionnaire. A linear regression model was used to explore the relationship between herd IMI prevalence and incidence and elimination rates. Multilevel logistic regression models were used to compute measures of associations between practices used on farms and IMI incidence, elimination, and prevalence. The herd incidence rate was the most important predictor of herd IMI prevalence: a reduction of the incidence rate equivalent to its interquartile range (0.011 new IMI/quarter-month) was associated with a prevalence reduction of 2.2 percentage points; in comparison, an equivalent increase of the elimination rate by its interquartile range (0.36 eliminated IMI/quarter-month) resulted in a prevalence reduction of 0.4 percentage points. Postmilking teat disinfection and blanket dry-cow therapy were already implemented by most herds. Most of the practices associated with Staph. aureus IMI incidence were related to milking procedures. Among these, wearing gloves during milking showed desirable associations with IMI incidence, elimination, and prevalence. Similarly, adequate teat-end condition and use of premilking teat disinfection were associated with lower IMI incidence and prevalence. The initial herd prevalence of Staph. aureus IMI was positively associated with subsequent IMI incidence. This indicates that, in some situations, an initial reduction of the pool of infected quarters could be justified. Some housing practices were associated with IMI incidence, elimination, or prevalence. The effects of these latter practices, however, were often influenced by specific cow characteristics such as parity or days in milk. These results highlight the importance of good milking practices to prevent Staph. aureus IMI acquisition and, therefore, reduce their prevalence.

The National Cohort of Dairy Farms--a data collection platform for mastitis research in Canada.

Costs and feasibility of extensive sample collection and processing are major obstacles to mastitis epidemiology research. Studies are often consequentially limited, and fundamental mastitis researchers rarely have the opportunity to conduct their work in epidemiologically valid populations. To mitigate these limitations, the Canadian Bovine Mastitis Research Network has optimized research funds by creating a data collection platform to provide epidemiologically meaningful data for several simultaneous research endeavors. This platform consists of a National Cohort of Dairy Farms (NCDF), Mastitis Laboratory Network, and Mastitis Pathogen Culture Collection. This paper describes the implementation and operation of the NCDF, explains its sampling protocols and data collection, and documents characteristics, strengths and limitations of these data for current and potential users. The NCDF comprises 91 commercial dairy farms in 6 provinces sampled over a 2-yr period. Primarily Holstein-Friesian herds participating in Dairy Herd Improvement milk recording were selected in order to achieve a uniform distribution among 3 strata of bulk tank somatic cell counts and to reflect regional proportions of freestall housing systems. Standardized protocols were implemented for repeated milk samplings on clinical mastitis cases, fresh and randomly selected lactating cows, and cows at dry-off and after calving. Just fewer than 133,000 milk samples were collected. Demographic and production data were recorded at individual cow and farm levels. Health management data are documented and extensive questionnaire data detailing farm management and cleanliness information are also captured. The Laboratory Network represents coordinated regional mastitis bacteriology laboratories using standardized procedures. The Culture Collection archives isolates recovered from intramammary infections of cows in the NCDF and contains over 16,500 isolates, all epidemiologically cross-referenced between linked databases. The NCDF is similar to Canadian dairies in relation to mean herd size, average production, and freestall percentages. Pathogen recovery was greater than anticipated, particularly for coagulase-negative staphylococci and Corynebacterium spp. International scientists are encouraged to use this extensive archive of data and material to enhance their own mastitis research.

Invited review: effect of udder health management practices on herd somatic cell count.

A systematic review of the scientific literature on relationships between management practices used on dairy farms and herd somatic cell count (SCC) was undertaken to distinguish those management practices that have been consistently shown to be associated with herd SCC from those lacking evidence of association. Relevant literature was identified using a combination of database searches (PubMed, Medline, CAB, Agricola, and Web of Science) and iterative screening of references. To be included in the review, a manuscript had to be published after 1979 in French, English, or Dutch; study design had to be other than case report or case series; herds studied had to be composed of ≥ 40 milking cows producing on average ≥ 7,000kg of milk in 305 d; interventions studied had to be management practices applied at the herd level and used as udder health control strategies; and SCC had to be measured using electronic cell counting methods. The 36 manuscripts selected were mainly observational cross-sectional studies; 8 manuscripts dealt exclusively with automatic milking systems and 4 with management of calves and heifers and its effect on SCC in early lactation heifers. Most practices having consistent associations with SCC were related to milking procedures: wearing gloves during milking, using automatic take-offs, using postmilking teat dipping, milking problem cows last, yearly inspection of the milking system, and use of a technique to keep cows standing following milking; all were consistently associated with lower herd SCC. Other practices associated with lower SCC were the use of a freestall system, sand bedding, cleaning the calving pen after each calving, surveillance of dry-cow udders for mastitis, use of blanket dry-cow therapy, parenteral selenium supplementation, udder hair management, and frequent use of the California Mastitis Test. Regarding SCC of heifers, most of the consistent associations reported were related to interventions made during the peripartum period. Studies on automatic milking systems have frequently reported elevation of the herd SCC following transition to the new system. These elevations seemed to be mediated both by the lack of monitoring of chronically infected cows and by an elevated incidence of intramammary infections. By assembling the results reported in many different studies, this review generates a more comprehensive understanding of the management practices influencing SCC and highlights areas of SCC control knowledge that lack evidence of effectiveness.

Effect of nonclinical Staphylococcus aureus or coagulase-negative staphylococci intramammary infection during the first month of lactation on somatic cell count and milk yield in heifers.

Coagulase-negative staphylococci (CNS) are the most prevalent cause of intramammary infections in heifers around calving, but Staphylococcus aureus should not be ignored because it is also prevalent, contagious, and more likely to persist into lactation. The objective of this study was to determine the effect of a subclinical infection caused by S. aureus or CNS diagnosed during the first month of lactation in heifers on SCC, milk production, and culling risk during the entire first lactation. Data were obtained from a cohort of 50 farms following a mastitis monitoring and control program and subscribing to the animal health record system (DS@HR) through the ambulatory clinic of the Faculté de médecine vétérinaire of the Université de Montréal (St-Hyacinthe, Québec, Canada). This program included routinely collecting a composite milk sample at each farm visit from all recently freshened heifers. A total of 2,273 Holstein heifers were examined. Among the 1,691 heifers meeting the full selection criteria, 90 (5%) were diagnosed with S. aureus, 168 (10%) were diagnosed with CNS, and 153 (9%) were negative (no pathogen isolated). Test-day natural logarithm somatic cell count (lnSCC) was modeled in a repeated measures linear regression model with herd as random effect. The model-adjusted mean lnSCC in S. aureus and CNS groups were significantly higher than in the culture-negative group from 40 to 300 d in milk. At the test-day level, lnSCC in S. aureus and CNS groups were on average 1.2 and 0.6 higher, respectively, than the culture-negative group. A similar model for milk yield showed that mean milk yield was not statistically different between culture groups from 40 to 300 d in milk. The presence of a S. aureus or CNS intramammary infections in the first month of lactation in heifers correlates with future increased SCC over the entire first lactation.

Relationship between feeding strategy, lying behavior patterns, and incidence of intramammary infection in dairy cows.

The objectives of this study were to investigate whether feeding strategy influences post-milking standing time in dairy cows and to determine whether this time relates to incidence of intramammary infection (IMI). Fifteen lactating dairy cows (5 most recently fresh, 10 randomly chosen) from each of 6 tie stall dairy farms were enrolled for a total of 90 cows. Quarter samples of milk were taken from each cow once every 3 wk for a total of 3 samplings, and routine bacteriological culture and identification procedures were conducted. National Mastitis Council guidelines were used to define IMI, and occurrence of a new IMI was defined as a positive culture sample following a negative culture sample. Data on lying behavior patterns were collected using data loggers for every cow for 7 d before each milk sampling. For these 7 d, individual milking and feeding times of the cows were also recorded. Our results demonstrated that the provision of feed around milking time (between 30 min before and 60 min after) resulted in the longest post-milking standing times. The shortest post-milking standing times were seen in those cows that were fed >30 min before milking. Feeding cows >60 min after milking resulted in only slightly shorter post-milking standing times than those fed between 30 min before and 60 min after milking. Cows lying down for the first time 40 to 60 min after milking had 1.4 times lower odds of acquiring a new environmental IMI than cows lying down within 40 min after milking. As post-milking standing time increased past 60 min, the odds of acquiring a new environmental IMI increased as well. The cows lying down for the first time 60 to 90, 90 to 120, and >120 min after milking showed 3.2, 5.8, and 7.4 times higher odds, respectively, of acquiring a new environmental IMI compared with cows lying down for the first time within 40 min of milking. Our results suggest that despite being able to manage post-milking standing times by providing fresh feed at different times around milking, the use of such a feeding strategy in tie stall systems is an unrealistic IMI prevention strategy.

Construction of covalently coupled, concatameric dimers of 7TM receptors.

7TM receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM receptors. Such 15 TM concatameric homo- and heterodimers of the beta(2)-adrenergic and the NK(1) receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two receptors. It is concluded that covalently joined 7TM receptor dimers with surprisingly normal receptor properties can be constructed with use of an artificial transmembrane connector, which perhaps can be used to fuse other membrane proteins.

Efficacy of benznidazol treatment for asymptomatic chagasic patients from state of Rio Grande do Sul evaluated during a three years follow-up.

The efficacy of benznidazol on the treatment of chagasic patients from the state of Rio Grande do Sul was evaluated during a three-year follow-up. A cohort of 80 asymptomatic chronic chagasic patients or blood bank donors (49 male and 31 female) was studied. Their ages varied from 17-42 years, with a mean and a median of 30 and 35 years, respectively. The 80 patients presented positive serology, hemoculture and polymerase chain reaction (PCR). They were treated with 5 mg/Kg benznidazol twice a day for 60 days. Serological, parasitological and PCR methods were used to evaluate response. Serology was performed using commercial ELISA and indirect immunofluorescence (IFI) tests, parasitemia was monitored by hemoculture in LIT medium and PCR with primers S35/S36 was used to amplify a Trypanosoma cruzi 330 bp kDNA repetitive sequence. PCR positivity of 240 seropositive individuals was compared using DNA preparations from whole blood/guanidine EDTA (GE), buffy-coat/GE and frozen buffy-coat. Fifty non-chagasic individuals were used as negative controls. PCR positivity was 86.7% for the frozen buffy-coat, 71.7% for the GE/buffy-coat and 69.2% for the GE/whole blood. The hemocultures became negative just after treatment and remained negative during the three years of follow-up. In the third year after treatment, 9/80 (11.3%) patients presented negative PCR and, from those, four also presented negative serological tests. Furthermore, a reduction in three serological titers was observed in 27/80 (33.8%) of the patients treated. Taken together, the results show that four of the 80 (5.0%) chronic chagasic patients from the state of Rio Grande do Sul were cured after treatment with benznidazol.

Efficacy of benznidazol treatment for asymptomatic chagasic patients from state of Rio Grande do Sul evaluated during a three years follow-up

The efficacy of benznidazol on the treatment of chagasic patients from the state of Rio Grande do Sul was evaluated during a three-year follow-up. A cohort of 80 asymptomatic chronic chagasic patients or blood bank donors (49 male and 31 female) was studied. Their ages varied from 17-42 years, with a mean and a median of 30 and 35 years, respectively. The 80 patients presented positive serology, hemoculture and polymerase chain reaction (PCR). They were treated with 5 mg/Kg benznidazol twice a day for 60 days. Serological, parasitological and PCR methods were used to evaluate response. Serology was performed using commercial ELISA and indirect immunofluorescence (IFI) tests, parasitemia was monitored by hemoculture in LIT medium and PCR with primers S35/S36 was used to amplify a Trypanosoma cruzi 330 bp kDNA repetitive sequence. PCR positivity of 240 seropositive individuals was compared using DNA preparations from whole blood/guanidine EDTA (GE), buffy-coat/GE and frozen buffy-coat. Fifty non-chagasic individuals were used as negative controls. PCR positivity was 86.7 percent for the frozen buffy-coat, 71.7 percent for the GE/buffy-coat and 69.2 percent for the GE/whole blood. The hemocultures became negative just after treatment and remained negative during the three years of follow-up. In the third year after treatment, 9/80 (11.3 percent) patients presented negative PCR and, from those, four also presented negative serological tests. Furthermore, a reduction in three serological titers was observed in 27/80 (33.8 percent) of the patients treated. Taken together, the results show that four of the 80 (5.0 percent) chronic chagasic patients from the state of Rio Grande do Sul were cured after treatment with benznidazol.