Kerry A. Rood

Herd-level prevalence of Johne's disease in Utah and adjacent areas of the intermountain west as detected by a bulk-tank milk surveillance project.

The objectives of this study were to estimate the dairy herd-level prevalence of Johnes disease (JD) in Utah and nearby areas of the intermountain west and to estimate the sensitivity of a single bulk-tank milk test for JD detection. Two milk samples from all bulk tanks on the study farms were collected 1 mo apart. Samples were frozen and shipped to a laboratory for JD testing. An ELISA to measure total IgG antibody specific against Mycobacterium avium ssp. paratuberculosis, the etiological agent that causes JD, and a quantitative real-time PCR to detect M. avium ssp. paratuberculosis DNA were used; both tests were designed for bulk milk. Of the dairy farms in the study area, 170/246 (69%) participated. Positive JD results were found in bulk milk from 67/170 (39%) of dairy farms in Utah and adjacent areas. There were 138 JD-positive bulk-tank results from 241 bulk-tank samples from the 67 positive herds. The sensitivity of the bulk milk testing for detection of JD was 138/241(57%). From the 103 JD-negative farms, 235 bulk-tank samples tested negative for JD. The probability of false-negative results on a single bulk-milk sample was (1 - 0.57) = 0.43. For farms with 1 bulk tank, 2 samples collected 1 mo apart, with both samples testing negative (by both ELISA and quantitative real-time PCR) for JD, the true-negative probability was [1 - (0.43)(2)] = (1 - 0.18) = 82%. For farms with at least 2 bulk tanks, at least 4 samples tested, with all results negative for JD, the true-negative probability was at least 97%. Results support other estimates that prevalence of JD has increased over the last 15 to 20 yr. However, the prevalence detected was 3 times that from a recent report where 13% of dairy herds in the western US were positive. The increase in JD suggests that current control programs, at least as applied, are not effective. Bulk milk testing is a practical way to screen dairy herds for presence of JD. Studies are needed regarding the use of individual cow milk tests for accuracy, practicality, and effectiveness in reducing the prevalence of JD in dairy herds.

Abscesses in Captive Elk Associated with Corynebacterium pseudotuberculosis, Utah, USA

We isolated Corynebacterium pseudotuberculosis from an abscess of the head of a captive elk submitted for necropsy and from a similar abscess of a living herd mate. To our knowledge, this is the first documented case in elk and should be considered a potential cause of subcutaneous abscesses in wild elk.

Protection Against Chikungunya Virus Induced Arthralgia Following Prophylactic Treatment with Adenovirus Vectored Interferon (mDEF201)

Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ∼100-fold while spleen virus increased >1000-fold within 1day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 10(7)pfu/ml dose of mDEF201 administered 21days to 24h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease.

Bovine Viral Diarrhea Milk ELISA Test Detecting Anti-p80 Antibody - Association with Milk Handling Methods and Cow Characteristics

A milk ELISA test for Antibody (Ab) against Bovine Viral Diarrhea (BVD) virus was studied in a dairy herd with past diagnoses of calves dying from BVD and Persistently Infected (PI) cows, with culling of all known PI cows. Modified live BVD vaccine was administered to calves 3 months and 4 months old, all cows at dryoff 45 to 60 days before calving, and 15-21 days in milk (DIM). Cows were tested 1 month apart (247 and 258 cows, respectively) using a competitive ELISA for milk Ab binding to p80 BVD non-structural protein. Results are reported as % binding by a second Ab; higher second Ab binding means the milk had less anti-p80 BVD Ab. Cows with 90-100% binding in milk on both tests were classified as low Ab–interpreted as a cow with PI or vaccine failure. Milk handling method was significant; fresh milk mean 49% second Ab binding was higher than for milk preserved 3 other ways. In fresh milk, 15 cows had 90-98% binding on one test, but 14/15 were milking during both herd tests and were below 90% on the other tests. Stage of lactation significantly affected results; anti-BVD Ab was higher from 1-30 DIM and lower from 61-150 DIM than at other stages of lactation. Ear notches were sampled concurrently from all cows for BVD antigen capture ELISA testing. Neither the milk ELISA results (no cows > 90% second Ab binding on both milk tests) nor ear notch testing classified any cows as PI animals. The milk BVD test might be useful to the dairy industry as a practical and convenient test for screening herd replacements, especially when large numbers of lactating cows are purchased and mixed into different pens throughout a dairy herd.

Dermatopathy in juvenile Angus cattle due to vitamin A deficiency

In juvenile cattle, vitamin A deficiency is reported most commonly as a neurological condition; only rarely are there dermatologic manifestations. In the current study, alopecia, severe epidermal and follicular orthokeratosis, and acanthosis due to hypovitaminosis A are reported in 2 of 32 Angus calves, with a third animal suspected. Affected animals responded to vitamin A supplementation, and no additional calves displayed signs. Vitamin A acts on skin by regulating DNA transcription in keratinocytes, reducing the number of tonofilaments and desmosomes, both involved in cell-to-cell adhesion. Hence, adequate levels of dietary vitamin A are necessary for normal keratinocyte turnover, and deficiencies result in retention of keratinized cells (orthokeratosis). The present report reminds diagnosticians to consider vitamin A deficiency in cases of orthokeratotic dermatopathy in cattle.

Gene expression and lymphocyte population at the fetal-maternal interface in sheep pregnancies established by somatic cell nuclear transfer

The hypothesis of this study was that the leukocyte populations and expression levels of genes related to immune response, growth factors and apoptosis would be altered at the fetal-maternal interface in somatic cell nuclear transfer (SCNT)-generated sheep pregnancies. Placental and endometrial samples from sheep pregnancies established by SCNT and natural breeding (control) were collected at 45 days and at term. Expression of genes related to growth factors, apoptosis and immune response was examined using quantitative reverse transcription polymerase chain reaction. Endometrial leukocyte populations and major histocompatibility class I (MHC-I) protein expression were examined by immunohistochemistry. At term we observed altered expression of genes related to apoptosis, growth factors and immune response in placental and endometrial tissue of SCNT pregnancies. In Day-45 pregnancies there was less-pronounced abnormal expression and only genes related to apoptosis and growth factors were abnormal in the placenta. Endometrial gene expression profiles were similar to age-matched controls. Placental MHC-I protein expression was similar in SCNT and controls at 45 days but increased in the SCNT at term. The altered gene expression at the fetal-maternal interface likely contributes to the placental dysfunction and overgrowth observed in sheep SCNT pregnancies.

A genome-wide association study for mastitis resistance in phenotypically well-characterized Holstein dairy cattle using a selective genotyping approach

A decrease in the incidence of bovine mastitis, the costliest disease in the dairy industry, can be facilitated through genetic marker-assisted selective breeding programs. Identification of genomic variants associated with mastitis resistance is an ongoing endeavor for which genome-wide association studies (GWAS) using high-density arrays provide a valuable tool. We identified single nucleotide polymorphisms (SNPs) in Holstein dairy cattle associated with mastitis resistance in a GWAS by using a high-density SNP array. Mastitis-resistant (15) and mastitis-susceptible (28) phenotypic extremes were identified from 224 lactating dairy cows on commercial dairy farm located in Utah based on multiple criteria of mastitis resistance over an 8-month period. Twenty-seven quantitative trait loci (QTLs) for mastitis resistance were identified based on 117 SNPs suggestive of genome-wide significance for mastitis resistance (p ≤ 1 × 10−4), including 10 novel QTLs. Seventeen QTLs overlapped previously reported QTLs of traits relevant to mastitis, including four QTLs for teat length. One QTL includes the RAS guanyl-releasing protein 1 gene (RASGRP1), a candidate gene for mastitis resistance. This GWAS identifies 117 candidate SNPs and 27 QTLs for mastitis resistance using a selective genotyping approach, including 10 novel QTLs. Based on overlap with previously identified QTLs, teat length appears to be an important trait in mastitis resistance. RASGRP1, overlapped by one QTL, is a candidate gene for mastitis resistance.

Estimation of the Trichomonad Concentration in Beef Bull Preputial Samples and Resultant Population Effect of Pooling on Detection of Tritrichomonas foetus Using qPCR

The primary objective was assessment of effect of sample pooling on Tritrichomonas foetus detection using qPCR in beef bulls within 5 logs of trichomonad concentration in spiked preputial samples. The secondary objective was to use qPCR Ct values to estimate the percentage of beef bulls in the field population within each log of trichomonad concentration. Solutions containing none, 1,10,100,1000 and 10,000 trichomonads (T)/ml were made and tested by qPCR. Test sensitivities for single positive samples were: 1 T/ml, 20/40 (50%); 10 T/ml, 40/40 (100%); 100 T/ml, 40/40 (100%); 1000 T/ml, 40/40 (100%); 10,000 T/ml, 40/40 (100%). For 5-sample pools (1 positive and 4 negative), sensitivities were: 1 T/ml, 6/40 (15%), 10 T/ml, 26/40 (65%); 100 T/ml 40/40 (100%); 1000 T/ml, 40/40 (100%); 10,000 T/ml, 40/40 (100%). Specificity for both single and pooled negative samples was 40/40 negative (100%). Diagnostic field samples (n = 130) in which T. foetus was detected by qPCR were classified by Ct to estimate the proportion of positive bulls within each trichomonad concentration: 1 T/ml 10 (7.7%); 10 T/ml 17 (13.1%); ≥ 100 T/ml 103 (79.2%). Accounting for the above sensitivities and proportions of bulls, sensitivity for trichomoniasis detection in the bull population was 96.2% using single preputial samples and 88.9% using 5-sample pools. Therefore 7.3% of all T. foetus-positive bulls were not detected using 5-sample pools compared with using single samples. Detection of bovine trichomoniasis by qPCR is minimally affected by using 5-sample pools, considering the trichomonad concentration of most positive bulls.

Dairy herd - level prevalence of Johne's disease and BVD in the Intermountain West of the U.S.A. and farm management practices and characteristics for test-positive herds

Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis (MAP), causative agent of Johne’s disease (JD) and Bovine Viral Diarrhea (BVD) virus were estimated on dairy farms in Utah. Duplicate milks were collected at 3-4 day intervals on 5 dates from each bulk tank on participating farms. Samples were tested at separate laboratories for BVD (real-time, RT-PCR) and for JD/MAP (ELISA and qPCR). 151/209 (72%) eligible dairy farms participated. Farms detected positive were: 58 JD (38%) and 14 BVD (9%); 5 farms had both diseases. Follow up visited farms’ (n=22) means, medians: 778,420 milking cows; 20,052 lbs, 20,311 lbs 305d milk; 175,545/ml, 178,000/ml bulk milk SCC; stalls visibly soiled in rear one-third 37%, 32%, range 5% to 90%. Seventeen of 21 (81%) farms with JD had observed adult cows becoming thin while retaining appetite, 52% had seen adult cows contract diarrhea and subsequently die. Both BVD-positive farms had observed abortions. Free stalls housed milking cows on 91% of farms; dry lots housed dry cows on 55%. Nine farms (41%) had purchased animals within the past year: 27% pregnant heifers, 18% bulls, 9% calves, 14% cows; 9% had purchased only bulls. Whenever animals were last purchased, 14 farms (64%) had performed no disease testing or segregation; 8 farms (36%) utilized at least one biosecurity practice for replacements. Most common were 9-way vaccine including BVD on arrival (27%), 14% segregated replacements for any time, 11% tested for any diseases (none for JD). Fourteen (67%) farms with JD would identify known positive cows; none would segregate positives. Most producers (57%) allowed known JDpositive cows to calve again, farms with BVD were equivocal. No producers would have a separate calving area for JD or BVD-positive cows. Six farms (27%) fed calves only individual cow colostrum and pasteurized milk. All 22 farms vaccinated against BVD.

Johne's disease, Mycoplasma and BVD in Utah-Bulk Tank Milk Testing and Comparison to Previous Regional Prevalence and Individual Herd Results over Time

Dairy herd-level prevalence of Mycobacterium avium subsp. paratuberculosis (MAP), causative agent of Johne’s disease (JD), Mycoplasma spp., and Bovine Viral Diarrhea (BVD) virus were estimated in Utah and surrounding states and compared to previous surveillance results. Milk was collected at 3-4 day intervals on 5 dates (duplicate samples) from each bulk tank on participating farms, samples analyzed separately. One frozen sample was shipped to a laboratory for JD/MAP testing with ELISA and real-time PCR, the other paired sample was shipped to another laboratory for mycoplasma and BVD testing. Mycoplasma was cultured on modified Hayflick medium, standard methods; BVD testing was real-time, RT-PCR. 151/209 (72%) eligible dairy farms participated. Farms detected positive (some had multiple diseases) were: 58 JD (38%); 4 mycoplasma (3%); 14 BVD (9%). Sensitivity of testing was: 284/528 milks = 54% for JD, 17/61 = 28% for mycoplasma (lower than previous reports), 41/117 = 35% for BVD. Of 67 herds positive for JD in 2009, 28 (42%) remained positive, 14 (21%) became test-negative, and 25 (37%) were lost to follow up. Of 16 herds positive for mycoplasma in 2007, one (6%) remained positive, 8 (50%) became testnegative, and 7 (44%) were lost to follow up. Bulk milk remains a practical way to screen dairy herds for presence of JD, mycoplasma and BVD, provided that repeated sampling is used. Mycoplasma-positive herds were more likely to become test-negative in bulk milk in subsequent years than were JD-positive herds. Prevalence of BVD was similar to but slightly lower than previously reported.