D.R. Wolfgang

Bulk-tank milk analysis: A useful tool for improving milk quality and herd udder health

Bulk-tank milk (BTM) analysis is now widely accepted as a useful tool for evaluating milk quality and monitoring udder-health status in a herd. Bacterial and somatic cell count (SCC) estimation of BTM, when done repeatedly over a period of time, can become a significant knowledge base. When interpreted within the context of the farms management practices, this information provides a basis for evaluating current and potential milk quality and mastitis problems in a herd. This article describes the process of using BTM analysis to make decisions on improving milk quality and herd udder health. It should be kept in mind that although individual cow samples for milk culture and SCC are more definitive for diagnosis and monitoring of udder health, BTM analysis is less expensive, more convenient, and faster than testing milk samples from individual animals or groups of cows.

A longitudinal study on the impact of Johne's disease status on milk production in individual cows

Longitudinal data from 3 commercial dairy herds in the northeast United States were collected from 2004 to 2007. Johnes disease status, as indicated by Mycobacterium avium ssp. paratuberculosis infection levels, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues at slaughter. Milk production data were collected from the Dairy Herd Improvement Association. The effect of Johnes disease status on milk production was analyzed using a mixed linear model with an autocorrelation random effect structure. Infected animals produced more milk than uninfected cows before they began shedding M. avium ssp. paratuberculosis. Cows infected with M. avium ssp. paratuberculosis had monthly decreases of 0.05 to 1 kg in daily milk production relative to uninfected animals, with greater decreases in progressive disease categories. Animals with fecal culture results of >30 cfu/g produced approximately 4 kg less milk per day compared with uninfected cows. These results will be valuable in calculating the economic effect of Johnes disease.

Dynamics of endemic infectious diseases of animal and human importance on three dairy herds in the northeastern United States.

Endemic infectious diseases in dairy cattle are of significant concern to the industry as well as for public health because of their potential impact on animal and human health, milk and meat production, food safety, and economics. We sought to provide insight into the dynamics of important endemic infectious diseases in 3 northeastern US dairy herds. Fecal samples from individual cows and various environmental samples from these farms were tested for the presence of major zoonotic pathogens (i.e., Salmonella, Campylobacter, and Listeria) as well as commensal bacteria Escherichia coli and enterococci. Additionally, the presence of Mycobacterium avium ssp. paratuberculosis was tested in fecal and serum samples from individual cows. Test results and health and reproductive records were maintained in a database, and fecal, plasma, DNA, and tissue samples were kept in a biobank. All bacteria of interest were detected on these farms and their presence was variable both within and between farms. The prevalence of Listeria spp. and L. monocytogenes in individual fecal samples within farm A ranged from 0 to 68.2% and 0 to 25.5%, respectively, over a period of 3 yr. Within farm B, continuous fecal shedding of Salmonella spp. was observed with a prevalence ranging from 8 to 88%; Salmonella Cerro was the predominant serotype. Farm C appeared less contaminated with Salmonella and Listeria, although in the summer of 2005, 50 and 19.2% of fecal samples were positive for Listeria and L. monocytogenes, respectively. The high prevalence of E. coli (89 to 100%), Enterococcus (75 to 100%), and Campylobacter (0 to 81%) in feces suggested they were ubiquitous throughout the farm environment. Fecal culture and ELISA results indicated a low prevalence of Mycobacterium avium ssp. paratuberculosis infection in these farms (0 to 13.6% and 0 to 4.9% for culture-positive and ELISA-positive, respectively), although the occasional presence of high shedders was observed. Results have major implications for food safety and epidemiology by providing a better understanding of infectious disease dynamics on dairy farms. Comprehensive understanding of these infections may lead to better farm management practices and pathogen reduction programs to control and reduce the on-farm contamination of these pathogens and to prevent their further entry into the food-chain.

Effect of Johne's disease status on reproduction and culling in dairy cattle

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johnes disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johnes disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and high-shedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. High-shedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals.

Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies Paratuberculosis infection in cattle using quarter milk and bulk tank milk samples.

A study was conducted to evaluate sensitivity, specificity, and predictive value of IS900-PCR assay for detection of Mycobacterium avium subspecies paratuberculosis in pooled quarter milk and bulk tank milk. Feces, blood and pooled quarter milk from 1493 lactating cattle on 29 herds were analyzed. Bulk tank milk (n = 29 bulk tanks) samples were also examined. Culture analysis revealed that 10.9%, 2.8%, and 20.6% of fecal, pooled quarter milk samples and bulk tanks were positive for M. avium subsp. paratuberculosis, respectively. While 13.5% and 27.5% of pooled quarter milk samples and bulk tanks were positive by IS900 PCR assay, respectively. Moderate to high antibody titers for M. avium subsp. paratuberculosis were detected in 223 of 1493 (14.4%) cows. Cows positive on fecal culture were taken as true positives relative to which the IS900 PCR assay was evaluated. The sensitivity and predictive value of KELA, pooled quarter milk culture, and IS900 PCR assay increased with lactation age. While the specificity of the tests decreased with increase in lactation age. Overall, the IS900 PCR assay using pooled quarter milk samples had a sensitivity, specificity and predictive value of 0.87, 0.95 and 0.71, respectively. The IS900 PCR assay using bulk tank milk had poor sensitivity (0.21), specificity (0.5) and predictive value (0.6). Pooled quarter milk culture analysis had a very low sensitivity (0.17). The kinetics ELISA had lower sensitivity (0.59), specificity (0.90) and predictive value (0.43) as compared to the IS900 PCR assay using pooled quarter milk samples. Results from our study suggest that IS900 PCR assay using bulk tank milk may not be useful for screening herds with M. avium subsp. paratuberculosis infected animals. In conclusion, use of IS900 PCR assay for cows in 2(nd) lactation and higher, using aseptically collected pooled quarter milk samples, can be a useful tool for screening and monitoring lactating cattle in herds with M. avium subsp. paratuberculosis infection.

Prevalence of Clostridium difficile Toxin Genes in the Feces of Veal Calves and Incidence of Ground Veal Contamination

A study was conducted in two parts to determine the prevalence of toxigenic Clostridium difficile in veal calves and retail meat. The first part of the study focused on the veal production continuum (farm to abattoir). Fifty calves from 4 veal herds (n=200) were followed for 18-22 weeks from the time of arrival on the veal farm to the time of slaughter. Fecal samples were collected from calves every 4-6 weeks. Half of the calves included in the study (n=100) were followed to the abattoir where carcass swabs were collected post slaughter. Fecal samples and carcass swabs were screened for genes encoding C. difficile toxins TcdA, TcdB, and CDT by using real-time polymerase chain reaction (PCR). Carcass swabs were also screened for toxigenic C. difficile by using traditional culture methods. In the second part of the study, ground veal products (n=50 samples) purchased from local grocery stores were examined for toxigenic C. difficile by using real-time PCR and traditional culture methods. Fecal samples from 56 of 200 (28%) calves tested positive for C. difficile toxin genes at least once over the course of the study. Calf age (p=0.011) influenced prevalence of C. difficile toxin genes in calf feces. Toxin genes of C. difficile were detected in one carcass swab by multiplex real-time PCR only. Toxigenic C. difficile was detected by PCR and culture in four (8%) and three (6%) ground veal samples, respectively. The findings of the study reveal that toxigenic C. difficile was most prevalent in veal calves (12%) just before slaughter, although viable toxigenic C. difficile was not recovered from veal carcasses. On the contrary, viable toxigenic C. difficle was recovered from 6% retail meat, thus suggesting that contamination occurs either during or after veal fabrication.

Environmental Sampling To Predict Fecal Prevalence of Salmonella in an Intensively Monitored Dairy Herd

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas-were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone.

In vitro antimicrobial inhibition of Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory using flow cytometry and a broth microdilution method

Mycoplasma bovis is an important pathogen of cattle, causing mastitis, pneumonia, conjunctivitis, otitis, and arthritis. Currently there are only a few reports of sensitivity levels for M. bovis isolates from the United States. Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory between December 2007 and December 2008 (n = 192) were tested for antimicrobial susceptibility to enrofloxacin, erythromycin, florfenicol, spectinomycin, ceftiofur, tetracycline, and oxytetracycline using a broth microdilution method. The most effective antimicrobials against M. bovis determined by using the broth microdilution method were florfenicol, enrofloxacin, and tetracycline with minimum inhibitory concentration (MIC) ranges of 2–32 µg/ml, 0.1–3.2 µg/ml, and 0.05 to >12.8 µg/ml, respectively. Spectinomycin, oxytetracycline, and tetracycline showed a wide-ranging level of efficacy in isolate inhibition with broth microdilution with MIC ranges of 4 to >256 µg/ml, 0.05 to >12.8 µg/ml, and 0.05 to >12.8 µg/ml, respectively. A significant difference in the susceptibility levels between quarter milk and lung isolates was found for spectinomycin. When MIC values of a subset of the M. bovis isolates (n = 12) were tested using a flow cytometric technique, the MIC ranges of enrofloxacin, spectinomycin, ceftiofur, erythromycin, tetracycline, oxytetracycline, and florfenicol ranges were 0.1–0.4 µg/ml, 4 to >256 µg/ml, >125 µg/ml, >3.2 µg/ml, <0.025 to >6.4 µg/ml, 0.8 to >12.8 µg/ml, and <2–4 µg/ml, respectively. Flow cytometry offers potential in clinical applications due to high-throughput capability, quick turnaround time, and the objective nature of interpreting results.

Correlation between Herrold egg yolk medium culture and real-time quantitative polymerase chain reaction results for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental samples

Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearmans rank correlation between Ct and CFU was good (–0.66) in fresh and thawed pooled fecal samples, and excellent (–0.76) and good (–0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (–0.53) to excellent (–0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

A mathematical model of the dynamics of Salmonella Cerro infection in a US dairy herd

We developed a mathematical model of the transmission dynamics of salmonella to describe an outbreak of S. Cerro infection that occurred in a Pennsylvania dairy herd. The data were collected as part of a cooperative research project between the Regional Dairy Quality Management Alliance and the Agricultural Research Service. After the initial detection of a high prevalence of S. Cerro infection in the herd, a frequent and intensive sampling was conducted and the outbreak was followed for 1 year. The data showed a persistent presence of S. Cerro with a high prevalence of infection in the herd. The dynamics of host and pathogen were modelled using a set of nonlinear differential equations. A more realistically distributed (gamma-distributed) infectious period using multiple stages of infection was considered. The basic reproduction number was calculated and relevance to the intervention strategies is discussed.