Iowa State University Veterinary Diagnostic Research Articles

Survey of Iowa beef veterinarians on Johne's disease in cow-calf operations

Johne's disease has historically been considered a disease of dairy cattle with little impact on beef herds in the United States. Over the past several years, a substantial increase in the number ofJohne's positive submissions from Midwestern beef operations has been observed at the Iowa State University Veterinary Diagnostic Laboratory, suggesting an increase in the prevalence of disease in the cow-calf industry. Thus, there is an urgent need to better understand the current knowledge ofbeefveterinarians related to Johne's disease and assess the impact of the disease in the cow-calf industry.

Retrospective study of lameness cases in growing pigs associated with joint and leg submissions to a veterinary diagnostic laboratory

Objective: The objective of this study was to categorize and quantify the most common causes of joint- or leg-associated lameness by summarizing available information from cases presented to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) between 2010 and 2015. Materials and methods: All cases of lameness or locomotor dysfunction in 7- to 40-week-old pigs submitted to the ISU VDL between May 1, 2010 and April 30, 2015 were retrieved. After removing cases that did not meet the inclusion criteria, the remaining cases were individually reviewed and assigned a primary and secondary diagnosis. Results: Of the 1847 cases retrieved, 464 met the inclusion criteria. The 4 most common primary diagnosis categories were Mycoplasma hyosynoviae (93 cases; 20%), metabolic bone disease (86 cases; 18.5%), infectious arthritis due to non-Mycoplasma bacterial infection (81 cases; 17.5%), and lameness with inconclusive findings (101 cases; 21.8%). There were 23.3% of the cases (108 of 464 cases) that had a secondary diagnosis with metabolic bone disease (28.7%; 31 of 108 cases) identified as the most common secondary diagnosis. Implications: This study reinforces the importance of careful clinical examination, proper sampling, and confirming causes with appropriate diagnostic testing for accurate diagnosis of lameness.

Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine

Porcine parainfluenza virus type 1 (PPIV-1) is a member of the genus Respirovirus in the family Paramyxoviridae. The PPIV-1 was initially detected in 2013 from slaughter pigs in Hong Kong, China although its role in respiratory disease has remained unknown without virus isolates for experimental inoculation in swine. The objective of this study was to determine the relative frequency of PPIV-1 detection in diagnostic samples collected from swine in the United States, describe the cell culture isolation of PPIV-1, and characterize PPIV-1 cell culture isolates in vitro. Among 842 porcine specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 2016–2017, 43.3% were PPIV-1 positive by a real-time, reverse transcriptase PCR suggesting PPIV-1 may be common in swine. Two strains of PPIV-1 were successfully isolated in an LLC-MK2 cell line from a PPIV-1 RT-qPCR positive nasal swab (USA/MN25890NS/2016) and lung (USA/IA84915LG/2017). The PPIV-1 cytopathic effect was demonstrated in tissue culture and enveloped viral particles were observed by electron microscopy. The whole genome, F, and HN gene sequences of both isolates share 98.2%, 98.5%, and 98.2% nucleotide homology, respectively, and phylogenetic analysis indicated they are closely related to other PPIV-1 strains detected in swine from the United States. Whole virus PPIV-1-specific monoclonal antibodies were generated for PPIV-1 detection in infected LLC-MK2 cells by indirect immunofluorescence and immunocytochemistry assays. The virus isolates and monoclonal antibodies obtained in the present study can be used to investigate the pathogenesis of PPIV-1 and develop new diagnostic tests.

Complete Genome Sequences of Two Novel Human-Like H3N2 Influenza A Viruses, A/swine/Oklahoma/65980/2017 (H3N2) and A/Swine/Oklahoma/65260/2017 (H3N2), Detected in Swine in the United States.

Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

General overview of the detection of Mycoplasma hyopneumoniae DNA by quantitative polymerase chain reaction in diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2004 to 2016

Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of enzootic pneumonia and a major causative agent of the porcine respiratory disease complex. This study summarizes and describes the general diagnostic trends on Mhp detection by quantitative polymerase chain reaction (qPCR) in cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2004 to 2016. The following variables were included in the analysis: animal age, geographic location, sample type, season, and submission year. The overall frequency of detection found was 27.04% and ranged from 17.9% to 40.7%. Lung homogenate and bronchial swabs had a greater Mhp qPCR detection rate than other sample types (P < .001) followed by bronchoalveolar lavage (P < .001), while oral fluids had the lowest Mhp detection rate (P < .001). The fall season had a greater percentage of positive Mhp qPCR results when compared to other seasons (P < .001), while spring had the lowest percentage. Finishing-age pigs had a greater percentage of Mhp qPCR detection when compared to other age groups (P < .001), while suckling pigs had the lowest percentage (P < .001).

ISU FLUture: a veterinary diagnostic laboratory web-based platform to monitor the temporal genetic patterns of Influenza A virus in swine

BackgroundInfluenza A Virus (IAV) causes respiratory disease in swine and is a zoonotic pathogen. Uncontrolled IAV in swine herds not only affects animal health, it also impacts production through increased costs associated with treatment and prevention efforts. The Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) diagnoses influenza respiratory disease in swine and provides epidemiological analyses on samples submitted by veterinarians.DescriptionTo assess the incidence of IAV in swine and inform stakeholders, the ISU FLUture website was developed as an interactive visualization tool that allows the exploration of the ISU VDL swine IAV aggregate data in the clinical diagnostic database. The information associated with diagnostic cases has varying levels of completeness and is anonymous, but minimally contains: sample collection date, specimen type, and IAV subtype. Many IAV positive samples are sequenced, and in these cases, the hemagglutinin (HA) sequence and genetic classification are completed. These data are collected and presented on ISU FLUture in near real-time, and more than 6,000 IAV positive diagnostic cases and their epidemiological and evolutionary information since 2003 are presented to date. The database and web interface provides rapid and unique insight into the trends of IAV derived from both large- and small-scale swine farms across the United States of America.ConclusionISU FLUture provides a suite of web-based tools to allow stakeholders to search for trends and correlations in IAV case metadata in swine from the ISU VDL. Since the database infrastructure is updated in near real-time and is integrated within a high-volume veterinary diagnostic laboratory, earlier detection is now possible for emerging IAV in swine that subsequently cause vaccination and control challenges. The access to real-time swine IAV data provides a link with the national USDA swine IAV surveillance system and allows veterinarians to make objective decisions regarding the management and control of IAV in swine. The website is publicly accessible at http://influenza.cvm.iastate.edu.

Comparison of treatment history and antimicrobial sensitivity results of common bovine respiratory disease bacterial pathogens from cases submitted to the ISU-VDL from 2013-2015

Summarized susceptibility results from diagnostic laboratory cases are often used to evaluate trends in antibiotic resistance. The objective of this retrospective study was to evaluate summarized antimicrobial susceptibility data from bovine respiratory disease cases submitted to the Iowa State University Veterinary Diagnostic Laboratory in the context of reported antibiotic treatment history.

Considerations for veterinary practitioners involved in feed-related toxicology cases

The objective of this paper is to identify key considerations for production animal veterinarians involved in legal cases regarding feed-related errors. Veterinarians are trained in the science of management and production, but are often ill-prepared for advising clients on potential litigation that may result when large monetary losses are incurred. The availability of modern analytical equipment and technology can provide practitioners with more information to understand the interactions of toxins with production medicine. This information may further elucidate the source of potential losses. The following considerations were derived from experiences at the Iowa State University Veterinary Diagnostic Laboratory when involved in several legal cases. Since it is likely that production veterinarians will be involved in a legal case at some point in their career, this paper is intended to help practitioners to be better prepared for cases involving feed-related toxicology cases.

Economic impact of university veterinary diagnostic laboratories: A case study

Veterinary diagnostic laboratories (VDLs) play a significant role in the prevention and mitigation of endemic animal diseases and serve an important role in surveillance of, and the response to, outbreaks of transboundary and emerging animal diseases. They also allow for business continuity in livestock operations and help improve human health. Despite these critical societal roles, there is no academic literature on the economic impact of VDLs. We present a case study on the economic impact of the Iowa State University Veterinary Diagnostic Laboratory (ISUVDL). We use economic contribution analysis coupled with a stakeholder survey to estimate the impact. Results suggest that the ISUVDL is responsible for $2,162.46 million in direct output, $2,832.45 million in total output, $1,158.19 million in total value added, and $31.79 million in state taxes in normal years. In an animal health emergency this increases to $8,446.21 million in direct output, $11,063.06 million in total output, $4,523.70 million in total value added, and $124.15 million in state taxes. The ISUVDL receives $4 million annually as a direct state government appropriation for operating purposes. The $31.79 million in state taxes in normal years and the $124.15 million in state taxes in an animal health emergency equates to a 795% and 3104% return on investment, respectively. Estimates of the economic impact of the ISUVDL provide information to scientists, administrators, and policymakers regarding the efficacy and return on investment of VDLs.

Treatment history and antimicrobial susceptibility results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolates from bovine respiratory disease cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015.

Bovine respiratory disease is the most costly disease facing the cattle industry. Increasing resistance to antimicrobial treatment has been presented as a significant contributing factor, often through summarized susceptibility testing data. We assessed the relationship between previous antimicrobial treatment and antimicrobial susceptibility results from isolates of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni cultured from bovine respiratory cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015. Antimicrobial susceptibility data from 1,251 bacterial isolates were included for analysis. More bacterial isolates from cattle that received antimicrobial treatment showed resistance compared to isolates from untreated cattle, and the percentage of resistant isolates increased as the number of antimicrobial treatments increased. Resistance to enrofloxacin, spectinomycin, tilmicosin, and tulathromycin was present in >75% of M. haemolytica isolates from cattle that had received 3 or more antimicrobial treatments; resistance to each of those 4 antimicrobials was present in ≤10% of M. haemolytica isolates from untreated cattle. Similar but less dramatic trends were apparent for isolates of P. multocida and H. somni. The percentage of multi-drug resistant bacterial isolates also increased with the number of treatments. Results of our study suggest that previous antimicrobial treatment may have a profound effect on antimicrobial susceptibility testing. Summarized susceptibility results from diagnostic laboratories should not be used to make generalized statements regarding trends in antimicrobial resistance without providing context regarding antimicrobial treatment history.

Development of Polioencephalomyelitis in Cesarean-Derived Colostrum-Deprived Pigs Following Experimental Inoculation with Either Teschovirus A Serotype 2 or Serotype 11

Teschovirus encephalomyelitis is a sporadic disease associated with Teschovirus A (PTV) serotype 1 and, less frequently, other serotypes. In recent years, the number of cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of posterior paresis has increased. Submission histories from various regions of the United States suggest a trend for clinical disease to persist in herds and affect a wider age-range of pigs than historically reported. Polioencephalitis and/or myelitis was consistently present and PTV was detected in affected neural tissue by PCR in a portion of cases. Sequencing from two clinical cases identified PTV-2 and PTV-11. To assess neuropathogenicity of these isolates, 5-week-old cesarean derived and colostrum-deprived pigs were assigned to three groups: negative control (n = 4), PTV-2-inoculated (n = 7), and PTV-11-inoculated (n = 7). Three PTV-2-inoculated pigs developed mild incoordination of the hind limbs, one of which progressed to posterior ataxia. While all PTV-11-inoculated pigs showed severe neurological signs consistent with Teschovirus encephalomyelitis, no evidences of neurological signs were observed in sham-inoculated animals. All PTV-2- and PTV-11-inoculated pigs had microscopic lesions consistent with Teschovirus encephalomyelitis. To our knowledge, this is the first description of PTV-11 and experimental study demonstrating the neuropathogenicity of PTV-11 in the United States.

PCV2d-2 is the predominant type of PCV2 DNA in pig samples collected in the U.S. during 2014–2016

Porcine circovirus type 2 (PCV2) vaccination was introduced in the US in 2006 and since has been adopted by most pig producers. While porcine circovirus associated disease (PCVAD) outbreaks are now relatively uncommon in the US, PCV2 remains a concern which is emphasized by increasing numbers of PCR and sequencing requests for PCV2. In the present study, randomly selected lung tissues from 586 pigs submitted in 2015 were tested for presence of PCV2 DNA. Positive samples were further characterized by sequencing and combined with available PCV2 open-reading-frame (ORF) 2 sequences from the client data base of the Iowa State University Veterinary Diagnostic Laboratory. The prevalence of PCV2 in the randomly selected lung tissues was 23% (135/586) with 11.3% PCV2a, 29% PCV2b and 71.8% for PCV2d subgroup PCV2d-2. A total of 455 ORF2 sequences obtained from 2014 through 2016 were analyzed and PCV2d accounted for 66.7% of the 2014 sequences, 71.8% of the 2015 sequences, and 72% of the 2016 sequences. Interestingly, only 1.9% (9/455) of the sequences belonged to the recently identified PCV2e genotype. The present data indicates that despite an almost 100% PCV2 vaccine coverage in the US, PCV2 DNA can still be detected in almost 1 of 4 randomly selected pig tissues. PCV2d-2 is now the predominant genotype in the USA suggesting that PCV2d-2 may have some advantage over PCV2a and PCV2b in its ability to replicate in pigs under vaccination pressure.

Outbreak of H5N2 highly pathogenic avian Influenza A virus infection in two commercial layer facilities: lesions and viral antigen distribution.

The largest outbreak of highly pathogenic avian Influenza A virus (HPAIV) infection in U.S. history began in December 2014 resulting in the euthanasia of millions of birds and collateral economic consequences to the U.S. poultry industry. We describe 2 cases of H5N2 HPAIV infection in laying hens in Iowa. Following a sharp increase in mortality with minimal clinical signs, 15 dead birds, from 2 unrelated farms, were submitted to the Iowa State University Veterinary Diagnostic Laboratory. Common lesions included diffuse edema and multifocal hemorrhage of the comb, catarrhal exudate in the oropharynx, and multifocal tracheal hemorrhage. Less common lesions included epicardial petechiae, splenic hemorrhage, and pancreatic necrosis. Influenza A virus nucleoprotein was detected by immunohistochemistry in multiple cell types including ependymal cells, the choroid plexus, neurons, respiratory epithelium and macrophages in the lung, cardiac myocytes, endothelial cells, necrotic foci in the spleen, Kupffer cells in the liver, and necrotic acinar cells in the pancreas. Real-time polymerase chain reaction and sequencing confirmed H5N2 HPAIV with molecular characteristics similar to other contemporary U.S. H5N2 HPAIVs in both cases.

Whole-Genome Sequences of Novel Porcine Circovirus Type 2 Viruses Detected in Swine from Mexico and the United States

A unique porcine circovirus type 2 capsid protein (ORF2) sequence was detected in swine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. The complete genome sequences of four viruses, one from Mexico and three from the United States, were determined to further characterize this novel PCV2 genotype.

PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in a regional surveillance study for Coronaviruses in 2012 and was detected for the first time in United States (US) swine in February 2014. However, it remains unknown if PDCoV had been introduced into the US prior to that time period. In the present study, 1734 clinical samples (903 cases) submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) for enteric disease diagnosis between October 2012 and December 2013 were tested retrospectively for PDCoV using a virus-specific real-time reverse transcription (RT) PCR targeting conserved region of the membrane gene. PDCoV genome was first detected in a fecal sample collected on August 19th 2013 from Minnesota. Subsequently, PDCoV was observed in samples collected on August 20th and August 27th from Iowa and on August 29th from Illinois. Therefore, with available samples submitted to the ISU VDL, it can be inferred that PDCoV has been present in US swine at least since August 2013.

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90-95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6-100% identity among the PCR amplicons from the 4 farms and 97-99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6-99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011-2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6-100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Prevalence of Campylobacter spp. relative to other enteric pathogens in grow-finish pigs with diarrhea

Salmonella spp., Lawsonia intracellularis, and Brachyspira spp. are pathogens commonly associated with diarrhea in growing and finishing pigs. Brachyspira spp. infection has recently reemerged as a significant concern due to an increase in the incidence of swine dysentery; however, the mechanisms underlying this increase in dysentery remain largely unknown. Pigs are also well-recognized as potential carriers of Campylobacter spp., particularly Campylobacter coli, yet enteric disease in swine associated with infection by these bacteria is considered uncommon and diagnosis has historically been based upon exclusion of other causes. Accordingly, Campylobacter culture is often excluded in routine diagnostic testing of cases of porcine enterocolitis and the incidence of infection is therefore largely unknown. In this study, feces from 155 cases of clinical diarrhea in grow-finish pigs submitted to the Iowa State University Veterinary Diagnostic Laboratory were cultured for Campylobacter spp. in addition to other testing as indicated for routine diagnostic investigation. Campylobacter culture was positive from 82.6% (128/155) of samples with C. coli accounting for 75% of isolates and Campylobacter jejuni for the remaining 25%. In 14.8% (23/155) of cases a Campylobacter spp. was the sole infectious agent detected; however, there was no association with any particular Campylobacter spp. Interestingly, for those cases with a laboratory diagnosis of Brachyspira-associated disease, 100% (15/15) were also culture positive for Campylobacter spp. suggesting a possible interrelationship between these bacteria in the pig gut. No association was noted between Campylobacter culture results and infection with either Salmonella spp. or L. intracellularis.

Rickets

Rickets can be attributed to nutritional, genetic, hormonal, or toxic disturbances and is classified as a metabolic bone disease. Rickets is most often associated with inappropriate dietary levels of calcium, phosphorus, and/or vitamin D. During a 27-month period (January 2010 through March 2012), the Iowa State University Veterinary Diagnostic Laboratory investigated causes of sudden, unexpected death and lameness in growing pigs throughout the Midwestern United States. Clinical observations from 17 growing pig cases included weakness, lameness, reluctance to move, muscle fasciculations and/or tremors, tetany, and death. Ribs were weak, soft, and bent prior to breaking; rachitic lesions were apparent at costochondral junctions in multiple cases. Acute and/or chronic bone fractures were also noted in multiple bones. Failure of endochondral ossification, expanded physes, infractions, thin trabeculae, and increased osteoclasts were noted microscopically. Decreased bone ash and serum 25(OH)D(3), combined with clinical and microscopic evaluation, confirmed a diagnosis of vitamin D-dependent rickets in all cases. In 3 cases, disease was linked to a specific nutrient supplier that ultimately resulted in a voluntary feed recall; however, most cases in the current investigation were not associated with a particular feed company. The present report describes vitamin D-associated rickets and its importance as a potential cause of weakness, lameness, muscle fasciculations, recumbency or sudden unexpected death in swine, and describes appropriate samples and tests for disease diagnosis.

Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces

Recently a commercial antigen-capture enzyme-linked immunosorbent assay kit in the form of a dipstick (Bovine Enterichek, Biovet Inc.) was made available to bovine practitioners and producers for the rapid detection of Betacoronavirus 1 (BCV-1), Rotavirus A (RV-A), Escherichia coli K99(+), and Cryptosporidium parvum in feces from diarrheic calves. The diagnostic performance of Bovine Enterichek was evaluated in comparison with a multiplex real-time polymerase chain reaction assay (mrtPCR). One hundred fecal samples were procured from diagnostic submissions to Iowa State University Veterinary Diagnostic Laboratory and were used for the assessment. The agreement quotient (kappa) in results for each pathogen between Bovine Enterichek and mrtPCR were 0.095 (BCV-1), 0.521 (RV-A), 0.823 (E. coli K99( + )), and 0.840 (C. parvum). In comparison to mrtPCR, the diagnostic sensitivity of Bovine Enterichek was 60.0%, 42.3%, 71.4%, and 81.5%, and the diagnostic specificity was 51.4%, 100%, 100%, and 98.6% for BCV-1, RV-A, E. coli K99( + ), and C. parvum, respectively. The current study suggested that Bovine Enterichek can be a rapid test tool in the field for detection of RV-A, C. parvum, or E. coli K99(+) in feces from calves at acute stage of clinical disease. However, test results for BCV-1 by the kit should be interpreted with caution due to low specificity and sensitivity of the kit.

Mycoplasma-associated arthritis: Critical points for diagnosis

Mycoplasma hyosynoviae and Mycoplasma hyorhinis are known causative agents of arthritis in postweaned swine. Data from the Iowa State University Veterinary Diagnostic Laboratory shows that diagnosis of M hyosynoviae- and M hyorhinis-associated arthritis has increased in swine in the Midwest United States. This diagnostic note summarizes disease characteristics of both pathogens and describes appropriate procedures to diagnose Mycoplasma-associated arthritis. An accurate diagnosis is critical to establishing effective treatment and prevention measures in affected herds.