Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds

OBJECTIVE: To evaluate sensitivity of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis (MAP) in large dairy herds and assess the use of the method for estimation of MAP prevalence. ANIMALS: 1,740 lactating cows from 29 dairy herds in California. PROCEDURE: Serum from each cow was tested by use of a commercial ELISA kit. Individual fecal samples were cultured and used to create pooled fecal samples (10 randomly selected fecal samples/pool; 6 pooled samples/herd). Sensitivity of MAP detection was compared between Herrold's egg yolk (HEY) agar and a new liquid culture method. Bayesian methods were used to estimate true prevalence of MAP-infected cows and herd sensitivity. RESULTS: Estimated sensitivity for pooled fecal samples among all herds was 0.69 (25 culture-positive pools/36 pools that were MAP positive). Sensitivity increased as the number of culture-positive samples in a pool increased. The HEY agar method detected more infected cows than the liquid culture method but had lower sensitivity for pooled fecal samples. Prevalence of MAP-infected cows was estimated to be 4% (95% probability interval, 2% to 6%) on the basis of culture of pooled fecal samples. Herd-level sensitivity estimate ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity for culture of pooled fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Use of pooled fecal samples from 10 cows was a cost-effective tool for herd screening and may provide a good estimate of the percentage of MAP-infected cows in dairy herds with a low prevalence of MAP.     

Risk of postnatal exposure to Sarcocystis neurona and Neospora hughesi in horses

OBJECTIVE: To estimate risk of exposure and age at first exposure to Sarcocystis neurona and Neospora hughesi and time to maternal antibody decay in foals. ANIMALS: 484 Thoroughbred and Warmblood foals from 4 farms in California. PROCEDURE: Serum was collected before and after colostrum ingestion and at 3-month intervals thereafter. Samples were tested by use of the indirect fluorescent antibody test; cutoff titers were > or = 40 and > or = 160 for S neurona and N hughesi, respectively. RESULTS: Risk of exposure to S neurona and N hughesi during the study were 8.2% and 3.1%, respectively. Annual rate of exposure was 3.1% for S neurona and 1.7% for N hughesi. There was a significant difference in the risk of exposure to S neurona among farms but not in the risk of exposure to N hughesi. Median age at first exposure was 1.2 years for S neurona and 0.8 years for N hughesi. Highest prevalence of antibodies against S neurona and N hughesi was 6% and 2.1 %, respectively, at a mean age of 1.7 and 1.4 years, respectively. Median time to maternal antibody decay was 96 days for S neurona and 91 days for N hughesi. There were no clinical cases of equine protozoal myeloenchaphlitis (EPM). CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to S neurona and N hughesi was low in foals between birth and 2.5 years of age. Maternally acquired antibodies may cause false-positive results for 3 or 4 months after birth, and EPM was a rare clinical disease in horses < or = 2.5 years of age.     

The Kendall Oration--Veterinarians in society--some future directions

Every four years a distinguished veterinarian is invited to provide the profession with a contemporary perspective on our craft. This year Australian Chief Veterinary officer Gardner Murray delivered a truly international viewpoint through the Kendall Oration at AVA's National Conference in Canberra.     

Fell Pony syndrome in a pony in North America

A 5-week-old Fell Pony colt was examined for fever, lethargy, and anemia. The colt had been lethargic for 1 week before examination, had continued to nurse, had a temperature of 104°F (40°C), and was treated with ceftiofur (5 mg/kg IM q12h). Approximately 36 hours before examination, the colt developed watery diarrhea. Blood work performed by the referring veterinarian on the day of admission revealed a PCV of 10%.     

Validation of real-time polymerase chain reaction tests for diagnosing feline immunodeficiency virus infection in domestic cats using Bayesian latent class models

The objectives of the current study were to estimate the sensitivity and specificity of three real-time polymerase chain reaction (PCR) tests for diagnosis of feline immunodeficiency virus (FIV) infection in domestic cats, both individually and when interpreted in series with one of two serological tests, separately in populations of cats at low and high risk of being infected with FIV. One PCR test targeted the pol gene and two targeted the gag gene of FIV. For comparison, sensitivities and specificities of the individual serological tests (IDEXX SNAP(?) test and AGEN Simplify(?) test) were also estimated. The study populations consisted of domestic cats thought to be not vaccinated against FIV. Low-risk (males aged 4 years or less and females; n=128) and high-risk (males over 4 years; n=128) cats were selected from those where blood samples were submitted to a commercial clinical pathology service. Bayesian latent class models were used to obtain posterior probability distributions for sensitivity and specificity for each test, based on prior distributions obtained from three experts. Medians of the posterior sensitivity distributions for the PCR tests based on the pol gene and two regions of the gag gene tests ranged from 0.85 to 0.89, compared to 0.89-0.97 for the two serological tests. The medians of posterior specificity distributions for these PCR tests were 0.94-0.96, and 0.95-0.97 for the serological tests. In contrast, the PCR based on one region of the gag gene had lower median sensitivity. Sensitivities of combinations of these serological and PCR tests interpreted in series were low; medians of posterior sensitivity distributions ranged from 0.75 to 0.83. Relative to the low-risk population, median sensitivities in the high-risk population were lower for all tests other than the AGEN Simplify(?) test; specificities were similar in both populations. We conclude that the sensitivities of the two PCR tests based on the pol gene and two regions of the gag gene, respectively, in non-vaccinated cats are probably lower than the sensitivities of the two serological tests we assessed. We do not recommend screening cats whose FIV vaccination status is uncertain with one of these serological tests and then testing positives with one of these PCR tests because in non-vaccinates, the sensitivities of combinations of these serological and PCR tests interpreted in series are low. Assessment of the validity of these PCR assays in FIV-vaccinated cats is required.     

Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples

ABSTRACT: BACKGROUND: The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis. RESULTS: We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2--10 mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods. CONCLUSIONS: The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA.     

Haematological and biochemical findings in bovine post-parturient haemoglobinuria and the accompanying Heinz-body anaemia

Sarcocysts morphologically similar to Sarcocystis muris were observed in the skeletal muscles of mice dosed with free isosporan sporocysts recovered from the faeces of naturally infected cats. Kittens fed these mice shed similar sporocysts measuring 8.7 ±0.1 x 10.8 ±0.1 μm in their faeces five to six days later. Feeding these sporocysts to mice and rats resulted in the formation of identical sarcocysts in mice, but not rats. Attempts to induce similar infections in mice using naturally acquired isolates of I. felis were unsuccessful.     

Soil and Atmospheric Inputs to PAH Concentrations in Salt Marsh Plants

Polycyclic aromatic hydrocarbons (PAHs) were measured in two wetland plant species grown outdoors in pots of sediment contaminated with up to 730,000 μg/kg total PAHs. After approximately 3 months, the plants were harvested, cleaned, and analyzed for an expanded suite of PAHs which included both the 16 priority PAHs and 22 alkyl PAH homologs. Sediment and air samples were also collected and analyzed. PAH compounds were detected in all of the samples, with the highest concentrations in the contaminated sediment. The root sample concentrations were generally about one order of magnitude lower than that of the sediment, and were strongly correlated with the concentration in the sediment in which they were grown. The concentrations in foliage were much lower and did not correlate with sediment concentration. Concentrations of low molecular weight PAH compounds detected in the foliage were not significantly lower in plants grown in control sediments, suggesting that the sediment is not the primary source of these PAHs. Several high molecular weight PAHs were detected only in plants grown in contaminated sediment. Plants of both species grown in control sediment were larger than plants grown in contaminated sediment.