A de novo mutation in CACNA1A is associated with autosomal dominant bovine familial convulsions and ataxia in Angus cattle.

Bovine familial convulsions and ataxia (BFCA) is considered an autosomal dominant syndrome with incomplete penetrance. Nine Angus calves from the same herd were diagnosed with BFCA within days of birth. Necropsy revealed cerebellar and spinal cord lesions associated with the condition. Parentage testing confirmed that all affected calves had a common sire. The sire was then bred to 36 cows across two herds using artificial insemination, producing an additional 14 affected calves. The objective of this investigation was to identify hypothesized dominant genetic variation underlying the condition. Whole-genome sequencing was performed on the sire, six affected and seven unaffected paternal half-sibling calves and combined with data from 135 unrelated controls. The sire and five of the six affected calves were heterozygous for a nonsense variant (Chr7 g.12367906C>T, c.5073C>T, p.Arg1681*) in CACNA1A. The other affected calves (N = 8) were heterozygous for the variant but it was absent in the other unaffected calves (N = 7) and parents of the sire. This variant was also absent in sequence data from over 6500 other cattle obtained via public repositories and collaborator projects. The variant in CACNA1A is expressed in the cerebellum of the ataxic calves as detected in the transcriptome and was not differentially expressed compared with controls. The CACNA1A protein is part of a highly expressed cerebellar calcium voltage gated channel. The nonsense variant is proposed to cause haploinsufficiency, preventing proper transmission of neuronal signals through the channel and resulting in BFCA.

Plasma, urine and tissue concentrations of Flunixin and Meloxicam in Pigs.

BACKGROUND: The objective of this study was to determine the renal clearance of flunixin and meloxicam in pigs and compare plasma and urine concentrations and tissue residues. Urine clearance is important for livestock show animals where urine is routinely tested for these drugs. Fourteen Yorkshire/Landrace cross pigs were housed in individual metabolism cages to facilitate urine collection. This is a unique feature of this study compared to other reports. Animals received either 2.2 mg/kg flunixin or 0.4 mg/kg meloxicam via intramuscular injection and samples analyzed by mass spectrometry. Pigs were euthanized when drugs were no longer detected in urine and liver and kidneys were collected to quantify residues. RESULTS: Drug levels in urine reached peak concentrations between 4 and 8 h post-dose for both flunixin and meloxicam. Flunixin urine concentrations were higher than maximum levels in plasma. Urine concentrations for flunixin and meloxicam were last detected above the limit of quantification at 120 h and 48 h, respectively. The renal clearance of flunixin and meloxicam was 4.72 ± 2.98 mL/h/kg and 0.16 ± 0.04 mL/h/kg, respectively. Mean apparent elimination half-life in plasma was 5.00 ± 1.89 h and 3.22 ± 1.52 h for flunixin and meloxicam, respectively. Six of seven pigs had detectable liver concentrations of flunixin (range 0.0001-0.0012 µg/g) following negative urine samples at 96 and 168 h, however all samples at 168 h were below the FDA tolerance level (0.03 µg/g). Meloxicam was detected in a single liver sample (0.0054 µg/g) at 72 h but was below the EU MRL (0.065 µg/g). CONCLUSIONS: These data suggest that pigs given a single intramuscular dose of meloxicam at 0.4 mg/kg or flunixin at 2.2 mg/kg are likely to have detectable levels of the parent drug in urine up to 2 days and 5 days, respectively, after the first dose, but unlikely to have tissue residues above the US FDA tolerance or EU MRL following negative urine testing. This information will assist veterinarians in the therapeutic use of these drugs prior to livestock shows and also inform livestock show authorities involved in testing for these substances.

Metronidazole for the treatment of Tritrichomonas foetus in bulls.

BACKGROUND: Tritrichomonas foetus is a sexually transmitted protozoon that causes reproductive failure, among cattle, so disruptive that many western US states have initiated control programs. Current control programs are based on the testing and exclusion of individual bulls. Unfortunately, these programs are utilizing screening tests that are lacking in sensitivity. Blanket treatment of all the exposed bulls and adequate sexual rest for the exposed cows could provide a more viable disease control option. The objectives of this study were twofold. The first objective was to demonstrate effectiveness for metronidazole treatment of a bull under ideal conditions and with an optimized treatment regime. This type of study with a single subject is often referred to as an n-of-1 or single subject clinical trial. The second objective of the current study was to review the scientific basis for the banning of metronidazole for use in Food Animals by the Animal Medicinal Drug Use Clarification Act of 1994 (AMDUCA). RESULTS: Results from an antimicrobial assay indicated that metronidazole at a concentration of 0.5 µg/mL successfully eliminated in vitro protozoal growth of bovine Tritrichomonas foetus. The estimated effective intravenous dose was two treatments with 60 mg/kg metronidazole, 24 h apart. A bull that had tested positive for Tritrichomonas foetus culture at weekly intervals for 5 weeks prior to treatment was negative for Tritrichomonas foetus culture at weekly intervals for five consecutive weeks following this treatment regimen. An objective evaluation of the published evidence on the potential public health significance of using metronidazole to treat Tritrichomonas foetus in bulls provides encouragement for veterinarians and regulators to consider approaches that might lead to permitting the legal use of metronidazole in bulls. CONCLUSION: The study demonstrated successful inhibition of Tritrichomonas foetus both in vitro and in vivo with metronidazole. The current status of metronidazole is that the Animal Medicinal Drug Use Clarification Act of 1994 prohibits its extra-label use in food-producing animals. Veterinarians and regulators should consider approaches that might lead to permitting the legal use of metronidazole in bulls.

Transboundary Tick and Tick-Borne Pathogen Threats to Cattle.

Transboundary incursions of ticks and tick-borne pathogens are ever present concerns for US cattle industries. Global trade in livestock and wildlife, historic and emerging transboundary issues with endemic tick populations and pathogens, and migratory bird flyways are pathways of concern. Transboundary challenges are presented for the Asian long-horned tick and Theileria orientalis Ikeda, for 2 cattle fever tick species [Rhipicephalus (Boophilus) annulatus and R (B) microplus] and Babesia bigemina and B bovis, and for the tropical bont tick and Ehrlichia ruminantium.

Effects of delivery via pressure-adjustable pneumatic gas-powered dart gun of three antimicrobial drugs (ceftiofur crystalline free acid, tildopirosin, and tulathromycin) on drug disposition and meat quality in cattle.

BACKGROUND: Although Beef Quality Assurance guidelines do not recommend use of darting methods to deliver drugs, cattle in the US may be raised on farms and ranches without restraint facilities, and reports from the field suggest that dart guns are being used to deliver antimicrobial drugs. Few studies report whether this route of administration results in altered drug disposition or carcass quality. METHODS: Forty steers were blocked by sire and then randomly assigned to treatment with saline, ceftiofur crystalline free acid, tildipirosin, or tulathromycin delivered via dart gun. To assess drug disposition, eight ceftiofur, six tulathromycin, and six tildipirosin-treated calves were selected to measure plasma concentrations of drugs up to 10 days after drug administration. Steers were then fed a balanced ration for approximately 6.5 months and slaughtered. To evaluate carcass quality, tenderness of steaks from darted-side and non-darted sides was evaluated via Warner-Bratzler shear force testing. Due to the prohibition of extralabel routes of administration for ceftiofur in the U.S., animals treated with this drug did not enter the food supply. RESULTS: Ceftiofur disposition differed from published reports with lower mean Cmax but similar mean apparent elimination half-life. Tildipirosin disposition differed from published reports with lower Cmax and shorter apparent elimination half-life. Tulathromycin was similar to previous published reports but Cmax and apparent elimination half-life was highly variable. All steaks (from darted and non-darted sides) from cattle treated with ceftiofur and saline were more tender than from cattle treated with tulathromycin or tildipirosin (P = 0.003). There was a trend toward more tenderness in steaks from the non-darted compared to the darted side. Steaks from the darted side for one treatment, tildipirosin, were less tender than the non-darted side. CONCLUSIONS: Pharmacokinetic parameters of ceftiofur crystalline free acid, tildipirosin, and tulathromycin to cattle using pressure-adjustable pneumatic gas-powered dart gun were estimated in this study. Delivery of tildipirosin and tulathromycin to cattle with dart gun may also result in detectable decreases in tenderness of harvested steaks.

Technical Note: The comparison of pH and redox potential in different locations in the reticulo-rumen of growing beef steers supplemented with different levels of quebracho extract.

Rumen acidosis is a common metabolic disorder occurring when organic acid production exceeds clearance capacity, reducing ruminal pH. The occurrence of acidosis has been directly correlated to the ratio of concentrate to forage in the diet. However, rates of substrate fermentation and acid absorption vary at different locations in the reticulo-rumen. The objective of this study was to determine the pH and redox potential (Eh) in different locations of the reticulo-rumen using 16 ruminally cannulated steers (309 ± 43 kg) receiving different supplementation levels of quebracho extract (QT; Schinopsis balansae) within a grower type diet (CP: 13.4%; total digestible nutrients [TDN]: 70.4%; and ME: 2.55 Mcal/kg, dry matter [DM] basis). Animals were randomly assigned to one of four dietary treatments: QT at 0%, 1%, 2%, and 3% of DM (QT0, QT1, QT2, and QT3, respectively), containing about 0%, 0.7%, 1.4%, and 2.1% of condensed tannins (CT), DM basis, respectively. Animals were adapted to the basal diet for 12 d before being introduced to predetermined treatments for 4 weeks (wk), with diets provided twice daily to allow ad libitum intake. Weekly measurements of ruminal fluid pH and Eh were taken 4 h post-feeding using a portable pH meter with two probes (pH and redox) in four locations of the reticulo-rumen (reticulum, cranial sac, dorsal sac, and ventral sac). Data were analyzed using a random coefficients model with the pen as a random effect and wk as repeated measures, with DM intake included as a covariate. There was no interaction among treatments, location, and wk (P ≥ 0.882) on reticulo-ruminal pH. Overall, ruminal pH was lower for QT0 and QT1 compared to QT3 (P < 0.001). The pH in the reticulum was greater than those of the ventral and dorsal sacs (6.05 vs. 5.94, 5.89, respectively; P ≤ 0.001) but similar to cranial sac (6.00). Reticular pH was positively correlated with the ruminal locations (≥0.78; P < 0.001). The linear equation to estimate ruminal mean pH using reticulum pH had an intercept and slope different from zero (P ≤ 0.04), but CT (% DM) was not different from zero (P = 0.15), root mean square error of 0.15, and R2 of 0.778: 0.723 (±0.36) + 0.857 (±0.059) × reticulum pH + 0.033 (±0.023) × CT. The Eh was lower for QT0 in week 1 than all other treatments (P < 0.001). We concluded that reticulo-ruminal pH differs among locations in the rumen regardless of QT supplementation level and days on feed, with reticular pH being the highest.

Detection of flunixin in the urine of untreated pigs housed with pigs treated with flunixin meglumine at labeled doses.

The objective of this study was to determine the likelihood that swine treated with flunixin meglumine could contaminate their environment, which could cause untreated swine housed in the same pen to ingest or absorb enough drug to be detected in their urine. Currently, any detectable level of flunixin found in the urine of pigs exhibited at livestock shows in Texas can disqualify the exhibitor. We conducted 2 trials in this study. The first, a pilot trial, placed pigs in 2 pens, with each pen housing a pig that did not receive a drug and a treated pig that received 2.2 mg/kg of flunixin intramuscularly. This trial demonstrated that transfer of the drug from treated to untreated pigs housed in close proximity was possible. The second trial was conducted using 10 pens, with a treated and untreated pig in each pen. Each pig receiving treatment was randomly selected and administered 2.2 mg/kg of flunixin intramuscularly; then, urine and plasma were collected from all swine for 10 d. Flunixin was detected at or above the limit of detection of 0.1 ng/mL in the urine of all treated and untreated pigs throughout the 10-d trial. Treated pigs had higher urine levels of flunixin than their untreated pen mates for 4 d post-treatment (P < 0.0001), but there was no statistical difference between pen mates during the last 5 d of the trial, making it impossible to differentiate treated from untreated pigs.

Improvements in Tritrichomonas foetus molecular testing.

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

Evaluation of Commercial Prototype Bacteriophage Intervention Designed for Reducing O157 and Non-O157 Shiga-Toxigenic Escherichia coli (STEC) on Beef Cattle Hide.

Microbiological safety of beef products can be protected by application of antimicrobial interventions throughout the beef chain. This study evaluated a commercial prototype antimicrobial intervention comprised of lytic bacteriophages formulated to reduce O157 and non-O157 Shiga-toxigenic Escherichia coli (STEC) on beef cattle hide pieces, simulating commercial pre-harvest hide decontamination. STEC reduction in vitro by individual and cocktailed phages was determined by efficiency of plating (EOP). Following STEC inoculation onto hide pieces, the phage intervention was applied and hide pieces were analyzed to quantify reductions in STEC counts. Phage intervention treatment resulted in 0.4 to 0.7 log10 CFU/cm² (p < 0.01) E. coli O157, O121, and O103 reduction. Conversely, E. coli O111 and O45 did not show any significant reduction after application of bacteriophage intervention (p > 0.05). Multiplicity of infection (MOI) evaluation indicated E. coli O157 and O121 isolates required the fewest numbers of phages per host cell to produce host lysis. STEC-attacking phages may be applied to assist in preventing STEC transmission to beef products.

Feed Intake and Weight Changes in Bos indicus-Bos taurus Crossbred Steers Following Bovine Viral Diarrhea Virus Type 1b Challenge Under Production Conditions.

Bovine viral diarrhea virus (BVDV) has major impacts on beef cattle production worldwide, but the understanding of host animal genetic influence on illness is limited. This study evaluated rectal temperature, weight change and feed intake in Bos indicus crossbred steers (n = 366) that were challenged with BVDV Type 1b, and where family lines were stratified across three vaccine treatments of modified live (MLV), killed, (KV) or no vaccine (NON). Pyrexia classification based on 40.0 °C threshold following challenge and vaccine treatment were investigated for potential interactions with sire for weight change and feed intake following challenge. Pyrexia classification affected daily feed intake (ADFI, p = 0.05), and interacted with day (p < 0.001) for ADFI. Although low incidence of clinical signs was observed, there were marked reductions in average daily gain (ADG) and cumulative feed intake during the first 14 day post-challenge; ADG (CV of 104%) and feed efficiency were highly variable in the 14-day period immediately post-challenge as compared to the subsequent 14-day periods. A sire × vaccine strategy interaction affected ADFI (p < 0.001), and a sire by time period interaction affected ADG (p = 0.03) and total feed intake (p = 0.03). This study demonstrates that different coping responses may exist across genetic lines to the same pathogen, and that subclinical BVDV infection has a measurable impact on cattle production measures.

Prevalence of and risk factors for paratuberculosis in purebred beef cattle.

OBJECTIVE: To estimate the prevalence of paratuberculosis in purebred beef cattle in Texas and identify risk factors for seropositivity. DESIGN: Epidemiologic survey. ANIMALS: 4,579 purebred cattle from 115 beef ranches in Texas. PROCEDURE: Blood was collected, and serum was analyzed for antibodies with a commercial ELISA. Fecal samples were collected and frozen at -80 degrees C until results of the ELISA were obtained, and feces from seropositive cattle were submitted for mycobacterial culture. Herd owners completed a survey form on management factors. RESULTS: Results of the ELISA were positive for 137 of the 4,579 (3.0%) cattle, and 50 of the 115 (43.8%) herds had at least 1 seropositive animal. Results of mycobacterial culture were positive for 10 of the 137 (7.3%) seropositive cattle, and 9 of the 50 (18%) seropositive herds had at least 1 animal for which results of mycobacterial culture were positive. Risk factors for seropositivity included water source, use of dairy-type nurse cows, previous clinical signs of paratuberculosis, species of cattle (Bos taurus vs Bos indicus), and location. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that seroprevalence of paratuberculosis among purebred beef cattle in Texas may be greater than seroprevalence among beef cattle in the United States as a whole; however, this difference could be attributable to breed or regional differences in infection rates or interference by cross-reacting organisms. Veterinarians should be aware of risk factors for paratuberculosis as well as the possibility that unexpected serologic results may be found in some herds.

Diagnostic Tools for Bluetongue and Epizootic Hemorrhagic Disease Viruses Applicable to North American Veterinary Diagnosticians.

This review provides an overview of current and potential new diagnostic tests for bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses compiled from international participants of the Orbivirus Gap Analysis Workshop, Diagnostic Group. The emphasis of this review is on diagnostic tools available to North American veterinary diagnosticians. Standard diagnostic tests are readily available for BT/EHD viruses, and there are described tests that are published in the World Organization for Animal Health (OIE) Terrestrial Manual. There is however considerable variation in the diagnostic approach to these viruses. Serological assays are well established, and many laboratories are experienced in running these assays. Numerous nucleic acid amplification assays are also available for BT virus (BTV) and EHD virus (EHDV). Although there is considerable experience with BTV reverse-transcriptase PCR (RT-PCR), there are no standards or comparisons of the protocols used by various state and federal veterinary diagnostic laboratories. Methods for genotyping BTV and EHDV isolates are available and are valuable tools for monitoring and analyzing circulating viruses. These methods include RT-PCR panels or arrays, RT-PCR and sequencing of specific genome segments, or the use of next-generation sequencing. In addition to enabling virus characterization, use of advanced molecular detection methods, including DNA microarrays and next-generation sequencing, significantly enhance the ability to detect unique virus strains that may arise through genetic drift, recombination, or viral genome segment reassortment, as well as incursions of new virus strains from other geographical areas.

Molecular and serological in-herd prevalence of Anaplasma marginale infection in Texas cattle.

Bovine anaplasmosis is an infectious, non-contagious disease caused by the rickettsial pathogen Anaplasma marginale (A. marginale). The organism has a global distribution and infects erythrocytes, resulting in anemia, jaundice, fever, abortions and death. Once infected, animals remain carriers for life. The carrier status provides immunity to clinical disease, but is problematic if infected and naïve cattle are comingled. Knowledge of infection prevalence and spatial distribution is important in disease management. The objective of this study was to assess A. marginale infection in-herd prevalence in Texas cattle using both molecular and serological methods. Blood samples from 11 cattle herds within Texas were collected and analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) and a commercial competitive enzyme-linked immunosorbent assay (cELISA). Samples from experimentally infected animals were also analyzed and RT-qPCR detected A. marginale infection up to 15 days before cELISA, providing empirical data to support the interpretation of herd prevalence results. Herds with high prevalence were located in the north Texas Rolling Plains and west Trans-Pecos Desert, with RT-qPCR prevalence as high as 82% and cELISA prevalence as high as 88%. Overall prevalence was significantly higher in cattle in north and west Texas compared to cattle in east Texas (p<0.0001 for prevalence based on both RT-qPCR and cELISA). The overall RT-qPCR and cELISA results exhibited 90% agreement (kappa=0.79) and provide the first A. marginale infection prevalence study for Texas cattle using two diagnostic methods. Since cattle are the most important reservoir host for A. marginale and can serve as a source of infection for tick and mechanical transmission, information on infection prevalence is beneficial in the development of prevention and control strategies.

Seroprevalence of Anaplasma marginale in Texas cattle.

To our knowledge the seroprevalence of Anaplasma marginale in Texas has not been reported. The objective of this study was to estimate the point seroprevalence and spatial distribution of Texas cattle persistently infected with A. marginale. This was a cross-sectional observational study examining serum collected from 12,000 adult cattle marketed in 23 selected Texas auction markets during the second week of July 2011. A random subset of those cattle comprising 1835 individuals was evaluated for persistent infection with A. marginale using a commercial cELISA for antibody detection. The pooled apparent seroprevalence for cattle tested at auction markets across the state was 15.02% (95% CI: 11.02-19.53%), with markets in the western portion of the state demonstrating prevalence ⇒ 30%. The winter tick, Dermacentor albipictus is involved in the biological transfer of A. marginale and is prevalent in west Texas. Producers in endemic and non-endemic areas should be encouraged to determine the infection status of replacement cattle in order to implement effective management strategies for the control bovine anaplasmosis.

Change in predominance of Bovine viral diarrhea virus subgenotypes among samples submitted to a diagnostic laboratory over a 20-year time span.

Although the causative agent of bovine viral diarrhea was initially categorized as 1 species, phylogenetic analysis revealed that these viruses belong to 2 different species, Bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, with 2-11 subgenotypes within each species. Distribution of species and subgenotypes has been shown to vary with geographic region. Whether distribution shifts over time is not known. Surveys conducted between 1994 and 2008 reported 3 subgenotypes circulating among cattle in the United States: BVDV-1a, BVDV-1b, and BVDV-2a. The average percent prevalence of BVDV-1a, BVDV-1b, and BVDV-2a strains reported in surveys before 2001 were 21%, 43%, and 36%, respectively. Surveys conducted on viruses isolated after 2001 reported decreasing percentages of BVDV-1a and BVDV-2a strains, with BVDV-1b strains accounting for 75-100% of samples. Comparison of these surveys is confounded by differences in geographic location, collection methods, and sample type used in the survey. The purpose of the present study was to determine whether the prevalence of BVDV subgenotypes shifted in samples collected from the same geographic region and by the same laboratory over time. BVDV strains isolated in years 1988, 1998, and 2008, at the Texas Veterinary Medical Diagnostic Laboratory, Amarillo, Texas, were genotyped, and the prevalence of BVDV-1a, BVDV-1b, and BVDV-2a strains were determined. Typing, on the basis of phylogenetic analysis, was done on 148 samples. The strongest trend detected among these samples was a pronounced decrease in the number of BVDV-1a strains over time.