Brian J. McCluskey

Prevalence of subclinical hypocalcemia in dairy herds.

The prevalence of subclinical hypocalcemia in the transition cow is unknown. Cows with subclinical hypocalcemia have no clinical signs of hypocalcemia but may be more susceptible to other diseases. The objective of this study was to determine the prevalence of subclinical hypocalcemia in the US dairy herds. As a part of the United States Department of Agricultures National Animal Health Monitoring System 2002 Dairy study, serum samples were collected from 1462 cows within 48 h of parturition. The samples were sorted by lactation number: 1st (n=454), 2nd (n=447), 3rd (n=291), 4th (n=166), 5th (n=72), and 6th (n=32). Subclinical hypocalcemia (<2.0 mM) increased with age and was present in 25%, 41%, 49%, 51%, 54%, and 42% of 1st-6th lactation cows, respectively. Cows with serum calcium concentrations >2.0 mM had significantly lower serum non-esterified fatty acids indicating better energy balance than those with subclinical hypocalcemia. Subclinical hypocalcemia may make cows more susceptible to secondary diseases but more research will be required to determine if this is true.

Comparison of milk and serum enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.

Milk and serum samples from 35 dairy herds in 17 states were evaluated for cow- and herd-level Mycobacterium avium subspecies paratuberculosis (MAP) antibody test agreement. Evaluation of 6,349 samples suggested moderate agreement between milk and serum enzyme-linked immunosorbent assay (ELISA) results, with a kappa value of 0.50. Cow-level sensitivity (Se) for 18 dairy operations with 1,921 animals was evaluated relative to fecal culture results. At the cow level, the milk ELISA relative Se was not significantly different from that of the serum ELISA (21.2 and 23.5%, respectively). Logistic regression models revealed a positive association between lactation number and milk ELISA status. Non-Holstein cows were more likely to test milk ELISA positive than Holstein cows. Cows in the first 2 weeks of lactation and after week 45 of lactation were more likely to test milk ELISA positive than cows between 3 and 12 weeks of lactation. Milk production > 80% of herd average was negatively associated with testing milk ELISA positive. Animals in the West and Midwest regions were less likely than animals in the Southeast region to test ELISA positive by either test. Estimates for herd-level sensitivity for the milk and serum ELISA, relative to fecal culture results, ranged from 56 to 83%. At the cow and herd levels, milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use Dairy Herd Improvement Association testing.

Prevalence of Escherichia coli O157 and other Shiga-toxin-producing E. coli in lambs at slaughter.

Ground beef has been implicated in most outbreaks of Shiga-toxin-producing Escherichia coli O157 infections in humans.1–3 No outbreaks of infection with this organism have been linked to lamb; however, ruminants such as sheep may contribute to transmission of this organism to humans. Shiga-toxin-producing E. coli (STEC) have been reported to be more prevalent in sheep and goats than in cattle and have been isolated from several retail meats, including lamb.4,5 Sheep can be colonized by E. coli O157 experimentally and through natural exposure, and this serotype has been isolated from retail lamb in the United States.6–8 Here, we describe a pilot study conducted to determine the prevalence of E. coli O157 and other STEC serotypes in market lambs just prior to slaughter and to evaluate specific factors that may be associated with a higher or lower prevalence of E. coli O157 in lambs at slaughter. Lambs were sampled at a slaughter facility that processed lambs only located in the midwestern United States. All samples were collected over 3 days in October 1995. Sixty lambs were sampled from lots that exceeded 100 lambs; otherwise, 30 lambs were sampled per lot. Each lot was categorized by source (single farm flock vs. commercial feedlot), distance shipped, and elapsed time from farm to slaughter. Fecal samples were collected by rectal extraction of fecal pellets or, if feces were not in pellet form, by rectal swab with a cotton-tipped swab. For detection of E. coli O157, a single fecal pellet or fecal swab was placed in a culture tube containing 3 ml of tryptic soy brotha to which had been added 40 mg/ml vancomycinb and 50 ng/ml cefiximec (TSBcv). From every 10 lamb in order of sampling, a larger volume of feces was obtained with a gloved hand for culture of 10 g of feces (in addition to the pellet or swab cultures on the same lambs). The larger samples were placed into a polypropylene tube containing 20 ml of TSBcv to which was also added 2.5 mg/ml potassium tellurited (TSBcvt). From each of the same systematically selected lambs, a single fecal pellet was placed into a culture tube containing 10 ml of EC broth containing 20 mg/ml of novobiocind (ECBn). Samples in ECBn were used to determine the presence of STEC

A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturers directions. The S/P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

Comparison of the Serum Neutralization Test and a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Vesicular Stomatitis Virus New Jersey and Vesicular Stomatitis Virus Indiana

A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587–0.9155) and 0.6912 (95% CI: 0.6246–0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.

Porcine epidemic diarrhea virus introduction into the United States: Root cause investigation.

Abstract Porcine epidemic diarrhea (PED) was identified in the United States in the spring of 2013, and professionals from many parts of the U.S. swine industry responded rapidly to understand and control the newly emerging disease. In less than two months, the disease had spread to more than 200 herds in thirteen states. Experts from the US Department of Agriculture (USDA) engaged in laboratory diagnostics, analytic support, epidemiology expertise, and data management to facilitate the effort. By 2014, a great deal had been learned about the disease; however, the question of how it entered the United States remained unanswered. In 2014, USDA formed an investigative group to address the question and leverage current knowledge with resources and partnerships not readily available to non-federal investigators. The group formed collaborations with other government and non-government organizations and individuals, and followed many avenues of inquiry; ultimately arriving at a small number of scenarios that describe possible mechanisms for PED introduction. For a scenario to be plausible, it had to explain: contamination of a person or product in the source country, its transit and entry to the United States, rapid dispersal across a wide geographic area, and exposure/infection of pigs. It had to be compatible with findings of swine herd investigations and research studies. Potential products had to have been imported legally during the time prior to the beginning of the epidemic, or delivered to the United States through prohibited channels. Follow-up studies were initiated to gather more evidence for the most plausible scenarios. Of the scenarios, flexible intermediate bulk containers (“feed totes”) used to transport bulk feed serving as fomites for movement of PED virus provided the simplest explanation for the accumulated findings of the investigation.

Factors Associated with Highly Pathogenic Avian Influenza H5N2 Infection on Table-Egg Layer Farms in the Midwestern United States, 2015.

SUMMARY A case-control study was conducted among commercial table-egg layer and pullet operations in Iowa and Nebraska, United States, to investigate potential risk factors for infection with highly pathogenic avian influenza (HPAI) H5N2. A questionnaire was developed and administered to 28 case farms and 31 control farms. Data were collected at the farm and barn levels, enabling two separate analyses to be performed—the first a farm-level comparison of case farms vs. control farms, and the second a barn-level comparison between case barns on case farms and control barns on control farms. Multivariable logistic regression models were fit using a forward-selection procedure. Key risk factors identified were farm location in an existing control zone, rendering and garbage trucks coming near barns, dead-bird disposal located near barns, and visits by a company service person. Variables associated with a decreased risk of infection included visitors changing clothing, cleaning and disinfecting a hard-surface barn entryway, and ceiling/eaves ventilation in barns.

Surveillance for highly pathogenic H5 avian influenza virus in synanthropic wildlife associated with poultry farms during an acute outbreak

In November 2014, a Eurasian strain H5N8 highly pathogenic avian influenza virus was detected in poultry in Canada. Introduced viruses were soon detected in the United States and within six months had spread to 21 states with more than 48 million poultry affected. In an effort to study potential mechanisms of spread of the Eurasian H5 virus, the United States Department of Agriculture coordinated several epidemiologic investigations at poultry farms. As part of those efforts, we sampled synanthropic birds and mammals at five infected and five uninfected poultry farms in northwest Iowa for exposure to avian influenza viruses. Across all farms, we collected 2,627 samples from 648 individual birds and mammals. House mice were the most common mammal species captured while house sparrows, European starlings, rock pigeons, swallows, and American robins were the most commonly captured birds. A single European starling was positive for Eurasian H5 viral RNA and seropositive for antibodies reactive to the Eurasian H5 virus. Two American robins were also seropositive. No mammal species showed evidence of infection. These results indicate synanthropic species merit further scrutiny to better understand potential biosecurity risks. We propose a set of management practices aimed at reducing wildlife incursions.

A single-tube multiplex reverse transcription-polymerase chain reaction for detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses in insects

A multiplex single-tube reverse transcription—polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10—30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.

Use of Sentinel Herds to Study the Epidemiology of Vesicular Stomatitis in the State of Colorado

Abstract: Approximately 20 sentinel premises in Colorado were visited quarterly during a 3‐year prospective study to investigate the persistence of VS viruses in horses. A survey to assess management practices, health events, animal movements and environmental data was completed at each visit. Collection of serum samples and oral swabs along with a clinical examination of sentinel horses were performed at each visit. Serum samples were tested by 2 or more of 4 available serological tests. The data collected for two years (August 1998 to August 2000) are reported here. During this period there was seroconversion in 1 and 8 horses based on capture IgM tests for seroytpes New Jersey and Indiana, respectively. Kaplan‐Meier curves were generated for those premises with horses that seroconverted and the mean survival time was 4.17 quarters (range 1.85‐7.0). The occurrence of seroconversions during periods when no clinical disease was observed suggests the persistence of vesicular stomatitis viruses in the environment of the sentinel premises.