J.S. Van Kessel

A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case-control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map-negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance (P < 5 x 10(-5)). Two regions, one on chromosome 3 (near EDN2) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively (P < 5 x 10(-7), genome-wide Bonferonni P < 0.05).

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.

The importance of culling in Johne's disease control

Johnes disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection and results in economic losses in the dairy industry. To control MAP transmission in herds, test-based culling has been recommended and immediate culling of high shedding animals is typically implemented. In this study, we quantified the effects of MAP control in US dairy herds, using the basic reproduction ratio R(0). The effectiveness of culling strategies was evaluated for good and poor herd management (low- and high-transmission rates, respectively) by a phase diagram approach. To establish a quantitative relationship between culling rates and test properties, we defined the average detection times for low and high shedding animals. The effects of various culling strategies and test characteristics, such as test sensitivity, test turnaround time, and testing interval, were analyzed. To understand the overall effect of model parameters on R(0), we performed global uncertainty and sensitivity analyses. We also evaluated the effectiveness of culling only high shedding animals by comparing three test methods (fecal culture, fecal polymerase chain reaction, PCR, and enzyme-linked immunosorbent assay, ELISA). Our study shows that, in the case of good herd management, culling of only high shedding animals may be effective in controlling MAP transmission. However, in the case of poor management, in addition to immediate culling of high shedding animals, culling of low shedding animals (based on the fecal culture test) will be necessary. Culling of low shedding animals may be delayed 6-12 months, however, if a shorter testing interval is applied. This study suggests that if farmers prefer culling only high shedding animals, faster MAP detection tests (such as the fecal PCR and ELISA) of higher sensitivity should be applied with high testing frequency, particularly on farms with poor management. Culling of infectious animals with a longer testing interval is generally not effective to control MAP.

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.

Survival of Escherichia coli in cowpats in pasture and in laboratory conditions

Aims:  To compare survival of Escherichia coli and faecal coliforms (FC) in bovine faeces deposited in a pasture or incubated in a controlled laboratory environment at temperatures within the same range.

Using a Portable Real-Time PCR Assay To Detect Salmonella in Raw Milk †

The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.

Identification of loci associated with tolerance to Johne’s disease in Holstein cattle

Johnes disease, caused by Mycobacterium avium subspecies paratuberculosis (Map), is a fatal disease in cattle. The objective of this study was to identify loci associated with tolerance in cows infected with Map. Tolerance was defined as a cows fitness at a given level of Map infection intensity. Fitness was measured by Map faecal cultures, and Map infection intensity was measured by culturing four gut tissues. The quantitative phenotype of tolerance was defined by numerical indexes of cultures of peak (peak tolerance, PT) and average (average tolerance, AT) faecal and tissue Map from 245 Holstein cows. The categorical phenotype was defined as: ≥ 100 cfu Map tissue infection, and faecal shedding ≥ 75 cfu (intolerant) or <10 cfu (tolerant cows). In 94 cows, Map was identified in ≥ 1 tissue, including 44 cows with ≥ 100 Map tissue cfu and 36 with ≥ 1 faecal cfu. A genome-wide association analysis was performed after filtering, leaving genotypes for 45,789 SNPs in 90 animals for the quantitative phenotype and 16 cases and 25 controls for the categorical analysis of tolerance. rs41748405:A>C (BTA15) was associated with PT (P = 1.12 × 10(-7)) and AT (P = 2.17 × 10(-6)). Associations were identified with PT and adjacent SNPs ss61512613:A>G and ss61530518:A>G (BTA6) (P < 3.0 × 10(-5)), and with AT for ss61469568:A>G (BTA 2) (P = 3.3 × 10(-5)) and ss86284768:A>G (BTA1) (P = 3.31 × 10(-5)). For the categorical phenotype, an association was found with ss8632653:A>G (BTA6) (P < 5.0 × 10(-5)). This is the first study to identify loci associated with tolerance to Johnes disease.

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.

Comparison of release and transport of manure‐borne Escherichia coli and enterococci under grass buffer conditions

Aim:  To test the hypothesis that Escherichia coli and enterococci bacteria have similar release rates and transport characteristics after being released from land‐applied manure.