Veterinary Diagnostic Labs Research Articles

Distribution trends of bovine Anaplasma marginale in Iowa, based upon veterinary diagnostic lab submissions from 2017-2022

Anaplasma marginale is the common etiological agent of bovine anaplasmosis. Infection results in erythrocyte de­struction leading to the clinical signs of anemia, hypoxia, abortion, jaundice and sudden death. Cattle that survive this acute phase or have lower levels of initial bacteremia become chronic, persistent carriers. Carrier animals serve as reser­voirs for the organism and allow infection to spread via me­chanical or biological vectors, to susceptible herdmates. The objective of this study was to report the distribution trends of A. marginale positive test results during a 5-year period in Iowa. This study evaluated Iowa State University Veterinary Diagnostic Lab (ISU VDL) testing results from bovine cases submitted from January 1, 2017 to January 1, 2022, for molecu­lar and serological detection of all stages of anaplasmosis. Cattle residing in Iowa with positive results for PCR or com­petitive ELISA were included in the analysis. The state was divided into 4 geographical districts (1 = northeast, 2 = south­east, 3 = southwest, and 4 = northwest) and the number of pos­itive tests in each district was determined. Total positive tests by district included: northeast 282; southeast 277; southwest 223; northwest 281. Disease spatial analysis mapping, based upon geographic longitude and latitude location of herds with positive diagnostic tests, illustrate progressive disease expan­sion from the southern counties of Iowa, near the Missouri border, into the northern parts of Iowa. These results suggest that A. marginale has developed a widespread distribution across the state of Iowa.

Calf diarrhea diagnostics

The decision to submit calf diarrhea samples to a veterinary diagnostic lab can often be challenging. Balancing the value of the information with the ability to promote positive manage­ment changes is the conundrum the veterinarian often finds themself in. It is important for veterinarians to understand the available tests, interpret results properly, and convey those re­sults in a manner that promotes improvements in calf health.

PSIV-A-6 Effects of a yeast cell wall additive on the growth performance and fecal Eimeria load of finishing lambs

Abstract Yeast-based additives have been shown to alter the rumen environment and exhibit immunomodulatory properties that can lead to enhancements in health status and subsequent performance. The objectives of this study were to evaluate the impacts of supplementing a yeast cell wall additive (Safwallâ, Phileo by Lesaffre, Milwaukee, WI) on finishing lamb growth performance and fecal Eimeria load. A total of 44 Polypay lambs were used in a 35-dexperiment at the Kansas State University Sheep and Meat Goat Center. On d 0, a fresh fecal sample was collected from each lamb and submitted to the Kansas State University Veterinary Diagnostic Lab to determine a baseline Eimeria count via quantitative fecal float. Lambs were then randomly assigned to either a control diet or diet supplemented with 1.5 g Safwallâ. Diets were fed for 35-d and contained no coccidiostat. Lambs had ad libitum access to both feed and water for the duration of the experiment. A basal lamb finisher diet was manufactured as a control diet and Safwallâ was hand-mixed into rations daily to make the treatment diet. Lambs and feeders were individually weighed weekly to track dry matter intake (DMI) and changes in body weight (BW). Data were analyzed using the GLIMMIX procedure of SAS (v.9.4, SAS Inst., Cary, NC) with an alpha set at (P < 0.05). From d 0 to 7 there was no evidence of a difference (P = 0.19) in DMI between treatment groups; however, during the second week of the study (d 7 to 14) lambs fed the control diet had significantly greater (P = 0.02) DMI compared with those fed a diet supplemented with yeast cell wall (Table 1). There was no evidence of differences in DMI between treatments for the remainder of the study (P > 0.05). Likewise, BW did not differ significantly (P > 0.05) between treatments on d 0 or by d 35. There was no evidence that fecal Eimeria load differed significantly (P > 0.05) on d 0 or 21; however, on d 35, lambs fed the control diet had a decreased Eimeria load compared with those fed a diet with yeast cell wall, but neither group had biologically relevant levels of Eimeria present in the feces. In summary, feeding a diet containing a yeast cell wall additive did not alter finishing lamb growth performance. While lambs fed the control diet had reduced fecal Eimeria at the end of the study, there was a reduction in this variable across both treatments. Further research should be conducted with younger animals with a known Eimeria challenge.

Changes in thyroid hormone concentrations over time in dogs with autoimmune thyroiditis.

The objective of this study was to follow long-term changes in the concentration of thyroid hormones in dogs with subclinical thyroiditis. Samples were obtained from 125 dogs with subclinical thyroiditis. The study population included 70 female and 55 male dogs. The mean testing interval was 3.9 years from initial testing (SD, 2.3 years; range, 1 to 9 years). Dogs with subclinical thyroiditis were identified retrospectively using results from the Orthopedic Foundation for Animals Canine Thyroid Profile performed by the Endocrinology Section of the Michigan State University Veterinary Diagnostic Lab. Owners were invited to submit follow-up serum samples with their veterinarian along with a medical history form, including subsequent treatments. At the time of retesting, 30% of the dogs had progressed to hypothyroidism and/or were treated with thyroxine. Fifty percent maintained positive or equivocal thyroglobulin autoantibody (TgAA) results while remaining euthyroid. Fourteen percent of the dogs became TgAA negative and remained euthyroid. In 6% of the cases tested, proper medical histories were not available, and a final classification could not be determined. These results indicate that most dogs with elevated thyroglobulin autoantibodies either exhibit persistent autoimmune thyroiditis with continued risk of hypothyroidism or progress to hypothyroidism when monitored for more than 1 year. Thyroid function in dogs with subclinical thyroiditis should be monitored every 12 months or if there is change in the clinical presentation.

Evaluating Trends in Strangles Outbreaks Using Temperature and Precipitation Data in the United States of America for 2018-2022.

Strangles is a highly contagious upper respiratory infection of equids that is globally distributed. The causative agent of strangles, Streptococcus equi subspecies equi, can be spread through indirect contact with infected fomites, and studies have shown this microbe to live well in varying environmental conditions. The purpose of this study was to analyze strangles case numbers across the United States of America from 2018 to 2022 to investigate potential temporal or weather patterns associated with outbreaks. Diagnosed case records were obtained from the Equine Disease Communication Center, university databases, government agencies, or veterinary diagnostic labs, and geographic information systems (GISs) were used to map cases and to acquire relevant meteorological data from outbreak areas. These data were analyzed using logistic regression to explore trends that occur between outbreaks and changes in temperature and precipitation. Initial review of weather data suggested monthly changes in strangles case numbers corresponded with changing seasons. Logistic regression indicated that changes in monthly average temperature and minimum temperature were significantly associated with increased or decreased odds of strangles outbreaks, respectively. Future analyses should focus on weather data isolated within a smaller region or state to better resolve trends in strangles outbreaks throughout the continental USA.

92 Seasonal variation in plasma vitamin A and E concentrations in horses with or without pasture access

Vitamins A and E are essential nutrients for horses and often provided in the diet by fresh forage but become depleted when forage is cut and dried for hay and can lead to potentially fatal health problems in horses. This study evaluated seasonal changes in plasma vitamin A and E for a mature horse herd in the Upper Midwest to determine the impact of pasture access on the prevalence of vitamin deficiency. Researchers hypothesized horses on dry lot would be deficient all year while horses with pasture access would have increased vitamin A and E concentrations during the grazing season. The study took place between February and November 2022 in River Falls, Wisconsin. Twenty-seven mature horses were divided into 2 treatments based on management practices including 1) horses consuming a hay-only diet in a dry lot (n = 13) and 2) horses with pasture access in the summer (n = 14). All horses had ad libitum access to water and a vitamin and mineral mix (200 IU/kg Vitamin E; 176,320 IU/kg Vitamin A) for the duration of the study and received ad libitum cool-season grass hay round bales from September through May. Hay-only horses continued this diet from June through August while pastured horses continuously grazed cool-season grass pasture. Jugular venous blood samples were taken in February (winter), May (spring), August (summer), and November (fall) and plasma samples were sent theMichigan State Veterinary Diagnostic Lab for analysis of plasma vitamin A and E concentrations. Data were analyzed using PROC MIXED in SAS with significance set at P ≤ 0.05. Adequate vitamin A was classified as 175 to 300 ng/ml, and adequate vitamin E was classified as 2.0 to 4.0 µg/ml. Vitamin A deficiency was observed in 81% and 85% of horses in winter and spring, respectively, with an average concentration of 145 ng/ml. Pastured horses exhibited an increase in plasma vitamin A in the summer compared with dry lot horses (P ≤ 0.05), however 64% of pastured horses remained deficient. In the fall, vitamin A deficiency increased to 97% for all horses and plasma vitamin A averaged 139 ng/ml. When evaluating vitamin E, 70 to 74% of horses were deficient in the winter and spring, respectively, with an average concentration of 1.83 µg/ml. Plasma vitamin E concentrations did not change over time for dry lot horses and remained inadequate in the summer at 1.43 µg/ml (P > 0.05) while concentrations in pastured horses rose to 2.4 µg/ml (P ≤ 0.05). These results confirm recommendations to provide additional vitamin A and E to horses on a hay-only diet and demonstrates the short-lived increases in vitamin A and E for horses provided limited pasture access.

88 Factors affecting serum vitamin E status in horses

Vitamin E (Vit E) is commonly supplemented to horses to meet NRC requirements, support performance, and treat deficiencies that may appear via blood work. Some veterinarians report horses test deficient in serum Vit E (<2.0 ug/mL), despite adequate Vit E in the diet. The objective of this study was to test the hypothesis that several factors could affect serum Vit E status in horses, including intake, access to pasture, and lab variability. Thirty horses (540.2 kg BW ± 51.5, mean ± SD) were utilized and housed at the Purina Animal Nutrition Center, Gray Summit, MO. HAY horses (n = 12) each received 2.7 kg of pelleted feed (Strategy, Vit E 334.9 IU/kg) per day plus 2.0% BW of forage (mature grass hay, Vit E 1.6 IU/kg), with no access to pasture. PASTURE horses (n = 18) each received 2.7 kg of the same pelleted feed, with free-choice access to pasture (estimated 2.0% BW/day intake, Vit E 5.1 IU/kg). All blood samples for Vit E concentration were taken at 0730 – 0800 via jugular venipuncture into serum collection tubes. Except where noted, samples were processed immediately via centrifugation with serum sent to 2 labs (Michigan State University Veterinary Diagnostic Lab, VDL, and Cornell University Animal Health Diagnostic Center, AHDC) for Vit E analysis (LC-HPLC). For 2 additional samples, blood was processed after 8 h at room temperature and after 8 h of refrigeration to see if results varied fromimmediately processed samples. Data were analyzed with GLIMMix ANOVA (SAS 9.4) for Vit E status, as well as lab and diet comparisons. All horses tested within the normal reference range (>2.0 ug/mL) for Vit E at both labs. There was a trend (P = 0.07) for a difference in Vit E status based on lab, with VDL values slightly higher than AHDC. There was a difference in HAY vs PASTURE at VDL (P = 0.004), with higher Vit E in PASTURE. There was a trend for a difference in HAY vs PASTURE at AHDC (P = 0.06), with higher Vit E in PASTURE. These differences could be explained by the higher estimated intake in Vit E intake in PASTURE, which was significant at P < 0.001. Conversely, it could be related to the form of Vit E in the diet, especially as provided by fresh grass. Statistics were not applied to the samples that underwent different handling procedures due to low numbers, but processing did not appear to greatly alter Vit E results in these 2 samples. In conclusion, horses with adequate Vit E intake all tested within normal reference range, while dietary source and/or intake, along with lab location appeared to play a small role. More work is necessary to determine why certain subsets of horses have low serum Vit E, despite adequate levels provided in the diet.

Placentitis and abortion in domestic pigs (Sus scrofa domesticus) associated with Trueperella abortisuis on US swine farms

We document a case series of abortions and placentitis in domestic pigs from the Midwest United States where aerobic bacterial cultures consistently isolated Trueperella abortisuis. Cases were submitted between 2017-2020 to the Kansas State Veterinary Diagnostic Lab. Microscopically, there was suppurative placentitis with necrosis and intralesional, gram-positive coccobacilli. In all cases, molecular diagnostics were negative for major causes of abortion in pigs. This is the first known report of T abortisuis isolated from swine abortions or placentitis in the United States.

Returning from a COVID detour.

“This is only temporary,” I told myself as I switched from pursuing my Ph.D. research to being a COVID-19 diagnostic technician. During the summer, I had been volunteering in a diagnostic laboratory, assaying samples for the presence of SARS-CoV-2. Now, the lab was offering to take over paying my stipend, tuition, and fees so I could commit more time to that work—and so my Ph.D. adviser wouldn't have to support me while I did so. My research progress and graduation were my priority. At the same time, I wanted to continue to do my part to protect my community from the pandemic, as well as help out the lab and ease my adviser's financial burden. I was torn between the needs of my community and my personal needs. > “I am grateful my skills were useful during the pandemic.” Prior to starting my Ph.D., I had spent several years working at state veterinary diagnostic laboratories. I enjoyed the diagnostic work, but I wanted to pursue Ph.D.-level research. Four years in, everything was going well as I learned how to combine the precision and speed from my diagnostic training with the flexible thinking and creativity needed for research. Then, of course, the pandemic hit. My research was mostly stalled as my lab was closed, and I was feeling helpless and frustrated at home. I was interested to learn about a public health effort to track the virus in our community, a collaboration between my university and the county health department—and that veterinary diagnostic labs were pitching in. I realized I had the knowledge and training to help with the project, called TRACE COVID-19. Despite having left diagnostics, now was not the time to focus on my personal career goals. I was done watching from the sidelines. Initially, I volunteered in the diagnostic lab 8 to 10 hours a day, a few times a week. It seemed like a rewarding use of my excess time. But my adviser didn't approve. He was paying me, after all, and didn't think I should be dedicating time to diagnostics. Still, I continued to volunteer through the summer months, using the time when I would otherwise be quarantining at home. When my research lab opened at limited capacity, volunteering did not hinder my research progress. But when the diagnostic laboratory offered to fund me, I faced a dilemma. I worried that if the diagnostic lab took over my funding, it might be logistically challenging to fully return to my Ph.D. research when the time came. At the same time, I didn't want to abandon my work with the diagnostic lab. My adviser also encouraged me to keep working for TRACE, saying it would augment my CV. So, I naively forged ahead, thinking I could work on my research in my spare time. Instead, TRACE consumed my life. The number of samples delivered daily was overwhelming, and in the evenings, I faced the onslaught alone. I skipped meals and neglected exercise, and my mental health suffered. But I kept telling myself it was only temporary. By the end of the fall term, I was burned out and ready to transition back to research. To my dismay, my adviser was running low on funding. My “temporary” decision was looking more and more permanent, and what had begun as rewarding work was becoming a ball and chain. Extricating myself took flexibility, creativity, and patience. I reached out to faculty members for advice and support, and everyone was willing to help—something I had forgotten during my isolation in the diagnostic lab. One of them encouraged me to apply for a position at a local biotech company. I was thrilled when the company offered me the job, as well as the funding I needed to complete my degree. My way forward was now clear. I don't regret my time with the TRACE project, despite the sacrifices to my research progress and mental health. I am grateful my skills were useful during the pandemic. And I've learned that, when I encounter detours and setbacks on my career path, I should look to the bigger picture and remind myself that it's only temporary.

289 Serum Trace Minerals in Late Gestation Sows at Variable Risk for Pelvic Organ Prolapse

Abstract Within the last decade, pelvic organ prolapse (POP) resulting in sow mortality has become an increasing concern for the U.S. swine industry, contributing approximately 21% of all sow deaths. While little is known regarding the etiology preceding POP in sows, many have proposed an association with vitamin and trace minerals abundance to POP incidence. We tested the hypothesis that sows differing in POP risk would have differences in serum trace minerals and vitamins. A perineal scoring (PS) system (PS1 - presumed low risk; PS2 - presumed moderate risk; and PS3 - presumed high risk) to assess risk for POP during late gestation was used to score 213 individual sows. Blood was collected from sows of two different farms during late gestation (days 105–115) that scored a PS3 (n = 20) and a parity matched sow scored as PS 1 (n = 16). Subsequently, 1.5, 0.8, and 23.1% of sows scored as PS1, PS2, or PS3, respectively, experienced POP. Serum was analyzed at the Iowa State University Veterinary Diagnostic Lab for trace mineral content (Calcium, Copper, Iron, Potassium, Magnesium, Manganese, Molybdenum, Phosphorus, Selenium, and Zinc). Additionally, vitamin E was evaluated in serum via GC-MS. Differences (P &amp;lt; 0.05) in copper, potassium, molybdenum, phosphorus, and selenium was observed between farms. Further a PS x Farm interaction (P = 0.06) was observed for serum copper abundance which across all farms was 12% less (P &amp;lt; 0.04) in PS3 compared to PS1 sows. No impact on serum vitamin E was observed between PS sows. These data demonstrate sows with greater POP risk may have potential differences in serum factors although these data also underscore the importance of measuring vitamin and mineral quantities in a tissue specific manner. This project was supported by the National Pork Board and the Foundation for Food and Agriculture Research.

The role of the veterinary diagnostic lab in the management of BRD.

Veterinary diagnostic labs (VDLs) are important service agencies providing essential diagnostic testing for a wide variety of domestic animal species as well as wildlife. They serve key roles in disease monitoring and diagnosis as well as surveillance for diseases of consequence. Of the many roles VDLs serve, one is being a member of the larger team of professionals dealing with the management of the bovine respiratory disease (BRD) complex. VDLs provide a number of services related to the management of BRD. These include disease outbreak investigation, abnormal morbidity characterization, routine monitoring, and biosecurity screening for a variety of infectious agents via methods such as necropsy and histopathology, bacterial culture, antimicrobial sensitivity testing, virus isolation, and serological assays. VDLs continue to look for better methods and assays as instrumentation technology also grows and improves. This is reflected in the growing proliferation of molecular-based assays that provide a high degree of sensitivity and specificity. Professional staff in VDLs work in collaboration with those in academia and private industry to conduct basic research focusing on a different aspect of the BRD complex. VDLs remain a primary source of the varied field-origin infectious agents associated with BRD that are used for research purposes.

Development and application of molecular diagnostics and proteomics to bovine respiratory disease (BRD)

Advances in molecular and proteomic technologies and methods have enabled new diagnostic tools for bovine respiratory pathogens that are high-throughput, rapid, and extremely sensitive. Classically, diagnostic testing for these pathogens required culture-based approaches that required days to weeks and highly trained technical staff to conduct. However, new advances such as multiplex hydrolysis probe-based real-time PCR technology have enabled enhanced and rapid detection of bovine respiratory disease (BRD) pathogens in a variety of clinical specimens. These tools provide many advantages and have shown superiority over culture for co-infections/co-detections where multiple pathogens are present. Additionally, the integration of matrix-assisted laser desorption ionization time of flight mass spectrometry (MS) into veterinary diagnostic labs has revolutionized the ability to rapidly identify bacterial pathogens associated with BRD. Recent applications of this technology include the ability to type these opportunistic pathogens to the sub-species level (specifically Mannheimia haemolytica) using MS-based biomarkers, to allow for the identification of bacterial genotypes associated with BRD versus genotypes that are more likely to be commensal in nature.

External quality assessment of Rift Valley fever diagnosis in 17 veterinary laboratories of the Mediterranean and Black Sea regions.

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.

Update on Trichomoniasis

In recent years diagnostic surveillance for bovine trichomoniasis has increased since most states require testing prior to interstate movement of non-virgin bulls. However, bovine trichomoniasis continues to be an economically significant disease, particularly in beef cattle, in much of the United States. The disease produces no clinical signs in bulls but can have devastating reproductive consequences on females resulting in reduced calf crops and extended calving seasons. The organism is efficiently transmitted via the venereal route. Although prevalence may be highest in older bulls it has been documented to occur in non-virgin bulls of any age. Bulls are believed to be persistently infected but females develop short lived immunity and naturally clear the infection within 3-22 months. Diagnosis is made by culture in selective media and/or by PCR identification of the organism in smegma samples collected by preputial scraping. Samples of reproductive or fetal fluids obtained from females can also be tested. Readers should consult their state veterinary diagnostic labs for instructions on sample preparation and shipment.&#x0D; Control of trichomoniasis is primarily through testing and culling of positive bulls. There are no approved, effective treatments available. However, vaccination of females will, in most cases, improve reproductive indices in infected herds.

Haloxyfop determination by gas chromatography/tandem mass spectrometry in eggs.

Haloxyfop is a pre/post-emergence herbicide with known organ toxicities and teratogenic effects in mammals. The European Union Commission on Food Safety has an established maximum residue limit of 10 μg/kg in all agricultural products including eggs. A sensitive highly specific method would be of value in determination of haloxyfop residues in foodstuffs such as eggs. The Michigan State University Veterinary Diagnostic Lab (MSU VDL) developed a method for the extraction of haloxyfop from eggs based on popular QuEChERS (quick, easy, cheap, effective, rugged, and safe) methodologies, essentially providing acetonitrile extracts following treatment with high ionic strength additives. Extracts derivatized with trimethylsilyl (TMS) groups were examined by gas chromatography/tandem mass spectrometry using developed multiple reaction monitoring (MRM) methodology. The MSU VDL received eggs from chickens exposed to 760 μg/kg haloxyfop in flaxseed. Haloxyfop-TMS m/z 374→73 MRM setting enabled quantitation across the 1-50 ppb range in comparison with an ibuprofen MRM transition as internal standard. The determined limit of quantitation was 2.5 ng/g, and the method successfully identified haloxyfop residues in five of six batches of the chicken eggs, with nonzero values ranging from 2.7 to 14.5 ng/g. These values were consistent with flaxseed incorporation into the diet at 4-7% and known excretion into eggs at 2-3% of daily haloxyfop exposure, and establish the utility of the method in identifying regulatory noncompliance and adulteration of food sources.

Veterinary utility of dried blood spots for detailed analysis of chlorinated pesticides and polychlorinated biphenyls by gas chromatography tandem mass spectrometry

Persistent organic pollutants (POPs) are organic compounds of anthropogenic origin that resist atmospheric and microbial degradation and thus persist in the environment and in food chains for exceptionally long periods of time. Veterinarians and wildlife researchers need simple methodologies for monitoring and measuring such compounds including two large and diverse categories, organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs), compounds that have been largely banned from production and use except for specific exceptions. We present development of methodologies for detection and quantitation of 22 OCs and 10 PCB congeners by tandem quadrupole gas chromatography-mass spectrometric analysis of Dried Blood Spots (DBS). Development was enabled by (1) optimization of suspension and extraction methodologies for DBS; (2) strategic streamlining and condensation of Multiple Reaction Monitoring (MRM) settings on GC/MS/MS; and (3) improvement of GC settings to accommodate all 32 compounds in a single chromatographic run per sample. The method was validated for parameters of linearity, limits of detection and quantitation, recovery and precision, and results from blood were shown to correlate well with those from DBS despite both being only 50 uL in volume. The method was applied successfully to blood samples from nine avian specimens submitted to the MSU Veterinary Diagnostic Lab, and all were shown to bear the burden of varying levels of OCs and/or PCB compounds.

Development of a tick-borne pathogen QPCR panel for detection of Anaplasma, Ehrlichia, Rickettsia, and Lyme disease Borrelia in animals

Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals.

Species characterization and minimum inhibitory concentration patterns of Brachyspira species isolates from swine with clinical disease

Typhlocolitis and dysentery due to Brachyspira hyodysenteriae infection represent an economically important disease syndrome in growing pigs. Largely disappearing from U.S. swine herds in the late 1990 s and early 2000s, Brachyspira-associated disease and bacterial isolation from swine with clinical disease has increased in the last several years, and non-B. hyodysenteriae isolates are commonly identified. Antimicrobial resistance has been demonstrated in Brachyspira spp. isolates from Europe and Asia, and may be the reason for the resurgence in U.S. herds. Seventy-nine clinical isolates identified at the Iowa State University Veterinary Diagnostic Lab were tested with multiple polymerase chain reaction assays to establish species identity, and evaluated for minimum inhibitory concentrations (MICs) using an agar dilution method against lincomycin, gentamicin, valnemulin, tiamulin, salinomycin, and carbadox. Only 38.0% of isolates could be confirmed as the known pathogens B. hyodysenteriae (30.4%) or Brachyspira pilosicoli (7.6%). Twenty of the 79 isolates (25.3%) were identified as Brachyspira murdochii, and 13.9% could not be identified to species. The MIC values were consistently high against lincomycin and moderately high against gentamicin. The remaining antimicrobials had MICs that were at the low end of the test ranges. Brachyspira murdochii and Brachyspira spp. had significantly greater MIC values against several of these drugs than other Brachyspira spp. examined. The increased incidence of these less definitively characterized Brachyspira species with increased MIC values to commonly prescribed antimicrobials may, at least in part, explain the increased prevalence and severity of this disease complex in recent years. Further research is necessary to understand these changes.

Differences in capabilities of different enzyme immunoassays to detect anti-hepatitis E virus immunoglobulin G in pigs infected experimentally with hepatitis E virus genotype 3 or 4 and in pigs with unknown exposure

Hepatitis E virus (HEV), a major cause of acute viral hepatitis in humans in many developing countries, is highly prevalent in the pig population worldwide. The objective of this study was to assess the capability of three porcine prototypes of a human enzyme-linked immunosorbent assay (ELISA), an in-house ELISA and a line-immunoassay (LIA) to detect anti-HEV antibodies in pigs infected experimentally with HEV ( n = 57), known to be negative for HEV infection ( n = 27), or with unknown exposure to HEV infection (field samples, n = 90). All 27 samples from non-infected pigs were negative with all five assays. The earliest detection of anti-HEV antibodies occurred at 14 days post-inoculation (dpi) with four of five assays. From 42 dpi, all samples from infected pigs were detected correctly as anti-HEV positive. Kappa analysis demonstrated substantial agreement among tests (0.62–1.00) at 14 dpi and complete agreement (1.00) at 56 dpi. The overall area under the curve for all quantitative tests as determined by receiver operator characteristic analysis ranged from 0.794 to 0.831 indicating moderate accuracy. The results showed that all five assays can detect anti-HEV IgG antibodies accurately in pigs infected experimentally with HEV. In field samples, a higher prevalence of anti-HEV IgG was found in breeding herds than in growing pigs (100% versus 66.7–93.9%). These serological assays should be very useful in veterinary diagnostic labs for HEV diagnosis in swine.

Serum pepsinogen levels to monitor gastrointestinal nematode infections in cattle revisited

The serum pepsinogen level is widely accepted as a useful parameter for monitoring gastrointestinal nematode infections in first-season grazing calves. However, several aspects, with possible implications for its practicality and the way to use the pepsinogen test, have received little attention to date. The objectives of this study were to evaluate (1) the reproducibility of a pepsinogen assay; (2) the required sample size for a reliable diagnosis and (3) the compliance to and effect of advice based on pepsinogen levels determined in year one on the nematode infection levels in the next generation of calves. Despite a high repeatability of the pepsinogen assay within the reference lab, the reproducibility between different labs was poor. There was more variation in pepsinogen levels between herds than between animals within a herd, suggesting that it is most useful to make a herd level diagnosis. Sample size calculation indicated that sampling seven animals sufficed to obtain a reliable indication of calf groups of up to 40 animals. Eighty-two herds were followed-up over two consecutive years and 39 (48%) had followed the advice that was formulated based on pepsinogen levels. Thirty-nine percent of the herds were advised to reduce the intensity of chemoprophylaxis. Samples from animals of those herds showed a higher pepsinogen level in the second year, but none of them had a mean pepsinogen level that is considered excessive. It is concluded that pepsinogen determination at housing from a limited number of animals can contribute to the design of the anthelmintic control strategy in the next year and a more targeted use of anthelmintics. However, more efforts are needed to harmonize the assay between veterinary diagnostic labs.