State Veterinary Diagnostic Laboratory Research Articles

Comparison of In Vitro Bacterial Susceptibility to Common and Novel Equine Wound Care Dressings.

Antimicrobial resistance is becoming a problem of concern in the veterinary field, necessitating the use of effective topical treatments to aid the healing of wounds. Honey has been used for thousands of years for its medicinal properties, but in recent years medical-grade Manuka honey has been used to treat infected wounds. The goal of this study was to determine the relative susceptibility of four common equine wound pathogens to ten different types of antimicrobial agents based on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The pathogens studied include ATCC lab-acclimated Pseudomonas aeruginosa, Escherichia coli, and methicillin-resistant Staphylococcus aureus and one from an equine sample submitted to the Colorado State Veterinary Diagnostic Laboratory (Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus)). An additional goal of the study was to describe the comparison of bactericidal activity of medical-grade Manuka honey, local honey, and commercial, food-grade honey to other commonly used wound dressings (20% hypertonic saline, silver sulfadiazine cream, PHMB gauze, and PHMB foam). The objective is to provide veterinary practitioners with comparative data on the use of a variety of antimicrobial dressings for inhibiting the growth of common wound bacteria. MIC and MBC for Manuka, store, and local honeys were comparable to those of sterile gauze, sugar, and hypertonic saline. Across bacterial species, local honey proved to have more bactericidal activity when compared to Manuka honey and commercial, food-grade honey. The MIC and MBC for PHMB gauze and foam was consistently at a higher dilution compared to the other antimicrobials. The majority of antimicrobials exhibited stronger inhibitory and bactericidal activity against a Streptococcus zooepidemicus isolate obtained from a wound compared to other bacteria that were ATCC lab-acclimated. Additional research for in vivo applications needs to be done to see whether differences exist in effective wound management.

46 The Distribution of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 Inoculated Feed After Chemical Mitigation to Flush a Feed Mill

Abstract Contaminated feed is a route of virus transmission between feed mills and swine farms. To reduce the risk of transmission, an understanding of the virus distribution and mitigation strategies are needed. The objective was to evaluate the distribution in the feed and environment of a feed mill of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca Valley virus-1 (SVV1) inoculated feed before and after the use of chemical mitigants. A 22.7 kg batch of feed was inoculated with PEDV, PRRSV, and SVV1 and ran through a mixer, bucket elevator, conditioner, pellet die, and cooler. Following the virus-inoculated batch, a flush treatment of either 1) ground corn (GC), 2) GC + 1.5% liquid formaldehyde (LF; SalCURB LF Liquid, Kemin, Des Moines, IA), 3) GC + 1.5% LF + 25% abrasive material (LF-BB; SalCURB; Shell & Bone Builder, Iowa Limestone Company, Urbandale, IA) 4) double flush – GC + 25% abrasive material follow by GC +1.5% LF (2F; Shell & Bone Builder; SalCURB) or 5) dry formaldehyde (DF; SalCURB F2 Dry, Kemin, Des Moines, IA) was manufactured, followed by 3 virus-free batches. Feed and environmental samples were taken from each piece of equipment. Dust samples were taken from the inoculated, flush, and final batches from non-feed contact surfaces. Samples were analyzed via a triplex PCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold and proportion PCR positive were analyzed using SAS GLIMMIXv9.4 (SAS, Inc., Cary, NC). A treatment×batch×location interaction was not observed (P > 0.05) in feed for any of the viruses. A thermal processing×treatment×batch interaction was observed (P < 0.05) for PEDV, PRRSV, and SVV1. The implementation of a flush, either chemical or physical, in combination with thermal processing led to reduced quantities (P < 0.05) of viral RNA for PEDV and PRRSV as compared with the positive pre-thermal feed. Chemically mitigated flushes reduced the quantity of SVV1 RNA in the flush and first post-flush batch more than GC pre-thermal processing (P < 0.05) with similar quantities of viral RNA post-thermal processing. In the post-thermal environmental samples, a batch×treatment interaction was observed for PEDV and SVV1, driven by increased quantities of viral RNA (P < 0.05) in the positive batch with no detectable RNA in the flush and post-flush batches. Decreasing quantities of viral RNA (P < 0.05) in dust were observed between the positive and last batch for all viruses; however, SVV1 RNA was still detectable in the dust following the last batch in all treatments. The use of chemical mitigants and thermal processing reduced the quantity of viral RNA for PEDV, PRRSV, and SVV1; however, viral presence was still observed in dust on non-feed contact surfaces which could pose as a source of contamination if re-introduced into finished feed.

Serum biochemical changes in cats with naturally acquired feline cytauxzoonosis

The geographical distribution of feline cytauxzoonosis is expanding in the US. Clinical signs of feline cytauxzoonosis, including lethargy, anorexia, and icterus, are similar to hepatic lipidosis and cholangiohepatitis. Hematologic and serum biochemical abnormality patterns may assist practitioners in prioritizing feline cytauxzoonosis as a differential diagnosis over hepatic lipidosis and cholangiohepatitis. Hematology and serum biochemical profiles of cats with naturally acquired feline cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. Retrospective search and analysis of the Kansas State Veterinary Diagnostic Laboratory or Kansas State University Veterinary Health Center records between January 2007 and June 2018 for cats with cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. Patients with acute feline cytauxzoonosis presented with frequent nonregenerative anemia (20/28 [71%]), leukopenia (23/28 [82%]), thrombocytopenia (23/23 [100%]), hyperbilirubinemia (27/28 [97%]), hypoalbuminemia (26/28 [93%]), reduced (18/28 [64%]) or low normal (10/28 [36%]) serum ALP activity, and hyponatremia (23/28 [82%]). Reduced ALP activity was unique to cats with feline cytauxzoonosis relative to hepatic lipidosis and cholangiohepatitis. No correlation between the severity of anemia and the magnitude of hyperbilirubinemia was identified in feline cytauxzoonosis patients. The combination of nonregenerative anemia, leukopenia, thrombocytopenia, hyperbilirubinemia, and reduced serum ALP activity in icteric cats may increase the clinical suspicion, but is not pathognomonic, for acute feline cytauxzoonosis. Hematologic and serum biochemical abnormalities of naturally acquired feline cytauxzoonosis are like those reported with feline bacterial sepsis. Blood smear evaluation for intraerythrocytic Cytauxzoon felis piroplasms, tissue aspirates for schizont-laden macrophages, and/or molecular testing are required to diagnose feline cytauxzoonosis.

Diagnosis of Anaplasma marginale in cattle at the Iowa State University veterinary diagnostic laboratory 2003–2021

Anaplasma marginale is a blood-borne rickettsia-like organism that infects cattle erythrocytes and causes anaplasmosis. This study reviews diagnostic data of all A. marginale diagnostics performed from 2003 to August 2021 in the Iowa State Veterinary Diagnostic Laboratory. Typically, the referring veterinarian's initial tentative diagnosis was based on presenting clinical signs or necropsy findings. Confirmatory testing at the ISU-VDL consisted of light microscopy evaluation of stained blood smears or molecular diagnostic procedures. A total of 94 cases were submitted with tissue samples from deceased animals, of which 79 were from Iowa and 15 were from other states. The most typical gross lesions were widespread yellow adipose tissue and splenomegaly. Typical histopathological lesions included marked bile stasis and hemosiderin-laden macrophages in the liver and spleen, respectively. Starting in 2013, when PCR was implemented to confirm cases of anaplasmosis, 315/1125 (28%) were positive to A. marginale, and 810 were negative, using a cut-off of 35.0 Ct. The average (±SD) of the positive PCR Ct was 19.5 (±6.0), and the first and third quartiles were 14.9 and 23.4. Most cases occurred between August and November, peaking in September, whether from necropsies or positive blood samples by PCR. The most common tick observed in Iowa, Dermacentor variabilis, is likely the main vector for transmission. Further surveys should be conducted to estimate seroprevalence by geographical location, the density of cattle populations, distribution of known vectors according to season, and strains of A. marginale.

Evaluating dry vs. wet disinfection in boot baths on detection of porcine epidemic diarrhea virus and porcine reproductive and respiratory virus RNA.

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control, 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL), and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of a co-inoculants of PRRSV (1 × 105 TCID50 per mL) and PEDV (1 × 105 TCID50 per mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one minute, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon was greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Pseudomonas aeruginosa susceptibility, antibiogram and clinical interpretation, and antimicrobial prescribing behaviors for dogs with otitis in the Midwestern United States.

Pseudomonas aeruginosa (P.aeruginosa) can cause otitis in dogs that is nonresponsive to empirical therapy. This study evaluated P.aeruginosa isolates (N=216) from canine ear swabs submitted to the Kansas State Veterinary Diagnostic Laboratory from 2018-2020 to create an antibiogram and minimum inhibitory concentration distributions using Clinical Laboratory Standards Institutes breakpoints. Multidrug resistance was defined as non-susceptibility to ≥1 drug from ≥3 antimicrobial classes. Submitting veterinarians (N=83) were invited to complete a survey about antimicrobial use and otitis management. Susceptibility was higher for aminoglycosides [gentamicin (82%, 177/216) and amikacin (81%, 175/216)] than fluoroquinolones [marbofloxacin (67%, 145/216), enrofloxacin (32%, 70/216), and orbifloxacin (18%, 39/216)]. Most responding veterinarians (54%, 15/28) prescribe topical aminoglycosides as first-line therapy for canine otitis, but 71% (15/21) prescribe fluoroquinolones if rods are seen cytologically. Ceftazidime, imipenem, and piperacillin-tazobactam showed high susceptibility and are used rarely. Multidrug resistance was present in 13% (28/216) of isolates. Based on in vitro susceptibility, topical aminoglycosides might be more effective than fluoroquinolones for P.aeruginosa otitis, but efficacy studies are required. Susceptibility testing is encouraged for cases not responding to empirical therapy but has limitations because topical preparations have high concentrations and otic breakpoints are not available.

53 Evaluating the Efficacy of Boot Baths with Wet and Dry Disinfectants for Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus

Abstract Maintaining biosecurity between barns is challenging. Boot baths, either wet or dry, can be implemented to limit pathogen spread. The objective was to evaluate the efficacy of boot baths using wet or dry disinfectants for porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). Treatments included 1) control, 2) wet disinfectant (Synergize, Neogen, Lexington, KY), and 3) dry disinfectant (Traffic C.O.P., PSP, LLC, Rainsville, AL). Prior to disinfection, 0.5 mL of both PRRSV (~1×105 TCID50/mL) and PEDV (~1×105 TCID50/mL) was placed onto a new boot with a layer of autoclaved corn dust and allowed to dry for 15 minutes. After the mixture dried, the boot was put on and stepped into its respective boot bath. After 3 seconds, the boot was lifted out of the bath and stepped onto a stainless-steel coupon to simulate walking through a facility. Both boot and coupon were allowed to dry for 1 minute before swabs were taken from both surfaces. Samples were analyzed in a duplex PCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a treatment×surface×virus interaction (P &amp;gt; 0.10). The interaction between treatment×surface impacted (P &amp;lt; 0.05) the quantity of detectable RNA. The control had greater concentration of virus on the coupon than the boot. The reverse was true for boots treated with wet disinfectant, where the boot had a greater concentration of virus than the coupon. Treatment×virus also impacted detectable RNA (P &amp;lt; 0.05), where wet and control boots had greater quantities of PEDV RNA than PRRSV. There was no detectable virus when dry disinfectant was used. For this trial, dry disinfectant was the most efficacious in reducing the viral RNA on both boots and subsequent surfaces; however, further research in commercial settings is warranted.

PSIII-4 Characterization of Digital Overgrowth in Sow Breeding Herds: A Case Study

Abstract Digital overgrowth has been documented as one of the most common foot abnormalities observed in U.S. commercial sow breeding herds and often leads to lameness and culling. Previous research has reported that 8.2% of lactating sows had digital overgrowth. When examining a 3,500 gestating sows, at least 32% were scored with moderate or severe digital overgrowth. Digital overgrowth negatively impacts piglet pre-wean mortality, lower NBA, and lower weaning weights. In addition to the digital overgrowth, sows have increased risk for cracked hooves and other hoof lesions. The objective of this study was to characterize a true standard (mm) for digital overgrowth and to determine if varying toe cell structure contributes to toe overgrowth. Initially, 2 crossbred sows that visually exhibited digital overgrowth were evaluated into this case study. Sows were euthanized at the Iowa State Veterinary Diagnostic Laboratory. Sow toe measurements were collected from the tip of the toe to the coronary band that is anatomically located at the sagittal line. Medial and lateral toe measurements were recorded for each limb. The mean toe measurements were front right limb: lateral digit (LD; 40 mm), medial digit (MD; 40 mm); front left limb: LD (36.5 mm), MD (38.5 mm); hind right limb: LD (73.5 mm), MD (49 mm); hind left limb: LD (50.5 mm), MD (50 mm). Dew claw and histopathological evaluations are in progress.

Pax5 and CD3 immunophenotyping of lymphoma in 2 central bearded dragons.

Two central bearded dragons (Pogona vitticeps), a 3-y-old male and a 5-y-old female, were diagnosed with different manifestations of lymphoma at the Kansas State Veterinary Diagnostic Laboratory between 2019 and 2020. The 3-y-old male was presented for postmortem evaluation and was in poor body condition. Microscopically, nearly all examined organs contained variable numbers of neoplastic round cells. Neoplastic cells in the stomach and liver had moderate immunoreactivity to CD3 consistent with multicentric T-cell lymphoma, and non-neoplastic lymphocytes infiltrating the stomach mass had strong immunoreactivity to Pax5. The 5-y-old female had an ulcerated oral mass located in the right lingual gingiva submitted as an excisional biopsy. Microscopically, the mass was composed of large numbers of neoplastic round cells in the epithelium and connective tissue that were strongly and diffusely positive for CD3 and frequently positive for Pax5, consistent with a dual-positive, localized, epitheliotropic T-cell lymphoma. Neoplastic and non-neoplastic lymphocytes did not stain with CD20 or CD79a. Neoplasms are increasingly reported as a cause of morbidity and mortality in reptiles. Our 2 cases illustrate various presentations of T-cell lymphoma and the effectiveness of CD3 and Pax5 immunohistochemistry in bearded dragons.

Marijuana toxicosis in 2 donkeys.

Marijuana toxicosis is typically seen by companion animal veterinarians. However, with increased marijuana availability, there is a greater potential for toxicosis in other species. Herein we describe a case of suspected marijuana toxicosis in a female and a male American Mammoth donkey, aged 8 y and 20 y, respectively, fed cannabis buds. Both cases were presented because of depression and lethargy. However, the jenny had ataxia, mild colic, tachycardia, tachypnea, and decreased tongue tone. Plasma samples from the jenny on presentation and 3 d following hospitalization were submitted to the Kansas State Veterinary Diagnostic Laboratory to be screened for cannabinoids using high-pressure liquid chromatography coupled with tandem mass spectroscopy (HPLC-MS/MS). A single serum sample from the jack was taken on presentation and submitted to the Animal Health Diagnostic Center at Cornell University for Δ9-tetrahydrocannabinol (THC) and cannabidiol analysis using HPLC-MS/MS. THC was detected in all samples. Clinical signs were noted 24-36 h after ingestion, which included mild-to-moderate neurologic deficits, mild colic, tachycardia, tachypnea, and decreased tongue tone. Both donkeys recovered uneventfully within 24 h of peak effects. Utilizing a cannabinoid screening assay in collaboration with a veterinary diagnostic laboratory may be useful when an equine practitioner suspects marijuana toxicosis in a patient.

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Dog in Connecticut in February 2021.

We report the first detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 3-month-old dog in Connecticut that died suddenly and was submitted to the state veterinary diagnostic laboratory for postmortem examination. Viral RNA was detected in multiple organs of the dog by reverse transcription real time-PCR (RT-qPCR). Negative and positive sense strands of viral RNA were visualized by in situ hybridization using RNAscope technology. Complete genome sequencing and phylogenetic analysis of the hCoV-19/USA/CT-CVMDL-Dog-1/2021 (CT_Dog/2021) virus were conducted to identify the origin and lineage of the virus. The CT_Dog/2021 virus belonged to the GH/B1.2. genetic lineage and was genetically similar to SARS-CoV-2 identified in humans in the U.S. during the winter of 2020–2021. However, it was not related to other SARS-CoV-2 variants identified from companion animals in the U.S. It contained both the D614G in spike and P323L in nsp12 substitutions, which have become the dominant mutations in the United States. The continued sporadic detections of SARS-CoV-2 in companion animals warrant public health concerns about the zoonotic potential of SARS-CoV-2 and enhance our collective understanding of the epidemiology of the virus.

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.

PSI-6 Effects of rest period prior to processing on vaccine response and blood metabolites in feedlot heifers

Abstract It is common for feedyards to rest cattle after transport and prior to administration of their receiving vaccinations in an attempt to minimize transport stress and optimize vaccine efficacy and subsequent growth and performance. This study aimed to test how time of rest after feedlot heifer arrival impacts vaccine titer and blood metabolites indicating cattle health. Eighty mixed-breed heifers (250 ± 4.2 kg BW) were purchased at live auction in Oklahoma City, OK and transported to the Kansas State University in Manhattan. Heifers were allotted in a completely randomized design to one of four treatments processed at 0, 6, 24, or 48 hours after arrival. At the time of processing (d 0) and again on d 35, serum samples were collected and analyzed for infectious bovine rhinotrachitis (IBR) titer, large animal chemistry panel, and hepatic profile by the Kansas State Veterinary Diagnostic Laboratory. There were time × treatment interactions (P &amp;lt; 0.02) for serum IBR titer, glucose, urea nitrogen, and bicarbonate. Specifically, heifers had greater (P &amp;lt; 0.05) IBR titer on d 35 than d 0, as expected because they were vaccinated upon arrival. Upon arrival, cattle held for 48 hours prior to processing had significantly greater (P &amp;lt; 0.05) glucose than those held for 24 hours (108 vs. 68 mg/dL, respectively), with no differences in processing treatment by d 35. Urea nitrogen levels in cattle held for 6, 24, or 48 hr prior to processing were greater (P &amp;lt; 0.05) than those held for 0 hours on d 0 or for any length of time by d 35. Finally, cattle held for 0 or 48 hr prior to processing had lower (P &amp;lt; 0.05) bicarbonate levels on d 0 than those held for 6 or 24 hours by d 35. These data show that while initial rest may be helpful to normalize blood metabolites, cattle should be placed within 24 hours of arrival. However, most blood metabolite levels normalize within 35 days.

Development of a nested PCR assay for detection of Streptococcus equi subspecies equi in clinical equine specimens and comparison with a qPCR assay

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.

Emergence of Salmonella enterica serovar 4,[5],12:i:- as the primary serovar identified from swine clinical samples and development of a multiplex real-time PCR for improved Salmonella serovar-level identification.

Rapid identification of the infecting Salmonella serovar from porcine diagnostic samples is vital to allow implementation of appropriate on-farm treatment and management decisions. Although identification at the serogroup level can be rapidly achieved at most veterinary diagnostic laboratories, final Salmonella serovar identification often takes several weeks because of the limited number of reference laboratories performing the complex task of serotyping. Salmonella serogroup B, currently the dominant serogroup identified from swine clinical samples in the United States, contains serovars that vary from highly pathogenic to minimally pathogenic in swine. We determined the frequency of detection of individual group B serovars at the Iowa State Veterinary Diagnostic Laboratory from 2008 to 2017, and validated a multiplex real-time PCR (rtPCR) to distinguish pathogenic serogroup B serovars from those of lesser pathogenicity. Our results indicate that, since 2014, Salmonella enterica ssp. enterica serovar 4,[5],12:i:- has been the dominant serovar identified from swine clinical samples at the ISU-VDL, with S. Typhimurium now the second most common serovar identified. We developed a rtPCR to allow rapid differentiation of samples containing S. 4,[5],12:i:- and S. Typhimurium from samples containing serovars believed to be of less pathogenicity, such as S. Agona and S. Derby. When combined with enrichment culture, this rtPCR has the ability to significantly improve the time to final serovar identification of the 2 most commonly identified pathogenic Salmonella serovars in swine, and allows rapid implementation of serovar-specific intervention strategies.

Epidemiological Review of Francisella Tularensis: A Case Study in the Complications of Dual Diagnoses.

Introduction:Tularemia is a rare but potentially fatal disease that develops in numerous wild and domestic animals, including lagomorphs, rodents, cats, and humans. Francisella tularensis bacterium, the causative agent of tularemia, was identified by veterinary personnel at Fort Riley, Kansas during a routine post-mortum evaluation of a domestic feline. However, before formal diagnosis was confirmed, the sample was sent and prepared for rabies testing at the Department of Defense (DoD) U.S. Army Public Health Command Central (PHC-C), Food Analysis and Diagnostic Laboratory (FADL). This case report provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. The epidemiologic characteristics of tularemia, F. tularensis as an organism of military interest, potential laboratory management of F. tularensis, and clinical findings on a case of feline tularemia are discussed. It further raises questions as to whether or not dead animals should be treated as sentinels and be pre-screened for select agents, especially in instances of dual diagnoses.Methods:A necropsy was performed on the cat by the Fort Riley veterinarian, DNA extraction and PCR analyses were conducted by FADL microbiologists, histology and immunohistology analyses were conducted by the Kansas State Veterinary Diagnostic Laboratory, and feline tissue and blood were sent to the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) for confirmatory testing and strain identification of tularemia.Results:Tularemia was identified in the spleen of the cat by the Fort Riley veterinarian and during the histological sampling of the spleen by the Kansas State Veterinary Diagnostic Laboratory. A specific subsequent real-time polymerase chain reaction (RT-PCR) in vitro diagnostic detection of target DNA sequences of F. tularensis was conducted by the FADL microbiologists using a Joint Biological Agent Identification and Diagnostic System (JBAIDS) Tularemia Detection Kit to detect a presumptive qualitative result to detect tularemia in feline and blood samples. USAMRIID also performed RT-PCR and identified genomic DNA from F. tularensis Type A, (SPL15.013.02), thus confirming the FADL’s initial presumptive result of F. tularensis. USAMRIID attempted to culture F. tularensis from three samples (swab, feline tissue, and transfer pipette tip), but no growth consistent with F. tularensis was observed on the cysteine heart agar with sheep blood and antibiotics (CHAB) and chocolate (CHOC) plates. Discussions:Our case study of a dual diagnosis of presumptive F. tularensis and possible rabies exposure transmission from a pet cat to its owner provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. Our case study also demonstrates the obligation for cooperation between animal health, human health, and public health professionals in the management of zoonotic diseases.

Bayesian Space-Time Patterns and Climatic Determinants of Bovine Anaplasmosis.

The space-time pattern and environmental drivers (land cover, climate) of bovine anaplasmosis in the Midwestern state of Kansas was retrospectively evaluated using Bayesian hierarchical spatio-temporal models and publicly available, remotely-sensed environmental covariate information. Cases of bovine anaplasmosis positively diagnosed at Kansas State Veterinary Diagnostic Laboratory (n = 478) between years 2005–2013 were used to construct the models, which included random effects for space, time and space-time interaction effects with defined priors, and fixed-effect covariates selected a priori using an univariate screening procedure. The Bayesian posterior median and 95% credible intervals for the space-time interaction term in the best-fitting covariate model indicated a steady progression of bovine anaplasmosis over time and geographic area in the state. Posterior median estimates and 95% credible intervals derived for covariates in the final covariate model indicated land surface temperature (minimum), relative humidity and diurnal temperature range to be important risk factors for bovine anaplasmosis in the study. The model performance measured using the Area Under the Curve (AUC) value indicated a good performance for the covariate model (> 0.7). The relevance of climatological factors for bovine anaplasmosis is discussed.

First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus.

BackgroundCurrently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 (porcine bocavirus 3, 4–1 and 4–2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure.MethodsPorcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples.ResultsSeven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6–99.5 % identity with the North American strains) to be the North American strains nearest relative.ConclusionsThese results are the first to report the presence of PPV6 in North America and demonstrate that the virus is found in multiple geographic areas in the United States and in Mexico. The overall prevalence of PPV6 in PRRSv viremic animals is relatively low. Further, all of the PPV6 genomes found in North America are most closely related to a PPV6 strain first identified in 2014 in healthy pigs from the Tianjin province of China.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0401-6) contains supplementary material, which is available to authorized users.