Vickie L. Cooper

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.

Deep Sequencing Analysis Reveals Temporal Microbiota Changes Associated with Development of Bovine Digital Dermatitis

ABSTRACT Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression. Over 175 million sequences were evaluated by utilizing both shotgun and 16S metagenomic techniques. Based on the shotgun sequencing results, there was no evidence of a fungal or DNA viral etiology. The bacterial microbiota of biopsy specimens progresses through a systematic series of changes that correlate with the novel morphological lesion scoring system developed as part of this project. This scoring system was validated, as the microbiota of each stage was statistically significantly different from those of other stages (P < 0.001). The microbiota of control biopsy specimens were the most diverse and became less diverse as lesions developed. Although Treponema spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The consortium of Treponema spp. identified at the onset of disease changes considerably as the lesions progress through the morphological stages identified. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development.

Case–control study of microbiological etiology associated with calf diarrhea

Abstract Calf diarrhea is a major economic burden for the US cattle industry. A variety of infectious agents are implicated in calf diarrhea and co-infection of multiple pathogens is not uncommon in diarrheic calves. A case–control study was conducted to assess infectious etiologies associated with calf diarrhea in Midwest cattle farms. A total of 199 and 245 fecal samples were obtained from diarrheic and healthy calves, respectively, from 165 cattle farms. Samples were tested by a panel of multiplex PCR assays for 11 enteric pathogens: bovine rotavirus group A (BRV-A), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine enterovirus (BEV), bovine norovirus (BNoV), Nebovirus, bovine torovirus (BToV) Salmonella spp. (Salmonella), Escherichia coli (E. coli) K99+, Clostridium perfringens with β toxin gene and Cryptosporidium parvum (C. parvum). The association between diarrhea and detection of each pathogen was analyzed using a multivariate logistic regression model. More than a half of the fecal samples from the diarrheic calves had multiple pathogens. Statistically, BRV-A, BCoV, BNoV, Nebovirus, Salmonella, E. coli K99+, and C. parvum were significantly associated with calf diarrhea (p <0.05). Among them, C. parvum and BRV-A were considered to be the most common enteric pathogens for calf diarrhea with high detection frequency (33.7% and 27.1%) and strong odds ratio (173 and 79.9). Unexpectedly BNoV (OR=2.0) and Nebovirus (OR=16.7) were identified with high frequency in diarrheic calves, suggesting these viruses may have a significant contribution to calf diarrhea.

Significance of genetic variation of PRRSV ORF5 in virus neutralization and molecular determinants corresponding to cross neutralization among PRRS viruses

A high rate of genetic and antigenic variability among porcine reproductive and respiratory syndrome viruses (PRRSVs) hampers effective prevention and control of the disease caused by PRRSV. The major envelope protein (GP5) encoded by the ORF5 of PRRSV has a critical role in inducing virus neutralizing (VN) antibody and cross protection among different strains of PRRSV. This study was conducted to identify sequence elements related to cross neutralization by comparing the ORF5 sequences of 69 field isolates in conjunction with their susceptibility to VN antibody raised against the VR2332 strain in vitro and in vivo. Five common variable sites (amino acid position 32-34, 38-39, 57-59, 137 and 151) were identified between susceptible and resistant viral isolates. Mutants whose ORF5 amino acid sequences were substituted with the sequences corresponding to the 5 identified common variable sites individually or concurrently were generated from a VR2332-backboned infectious clone by site mutagenesis. The change in the susceptibility of the mutants to VN antibodies specific for VR2332 or a heterologous PRRSV was assessed to determine the association of those 5 identified sites with cross neutralization. Among the five sites, the changes of amino acid sequences at three sites (32-34, 38-39, and 57-59) located in the N-terminal ectodomain of ORF5 significantly influenced the susceptibility of the mutant viruses to VN antibody, suggesting that sequence homology at these sites can be utilized as genetic markers to predict the degree of cross neutralization among different PRRSVs.

Naturally occurring influenza infection in a ferret (Mustela putorius furo) colony

Tissue samples from 2 juvenile ferrets (Mustela putorius furo) from a colony that was undergoing an outbreak of respiratory disease were submitted to the Iowa State University Veterinary Diagnostic Laboratory. Microscopic examination of lung samples revealed bronchointerstitial pneumonia with necrotizing bronchiolitis. Influenza A virus was detected in sections of formalin-fixed lung by immunohistochemistry and reverse transcription polymerase chain reaction assay. A field investigation of the premises and analysis of additional samples led to the confirmation and characterization of an influenza virus with high homology to contemporary reassortant H1N1 swine influenza viruses. Although ferrets have been used extensively to research the virulence and transmissibility of avian, human, and swine influenza virus strains, no published information exists on naturally occurring outbreaks of swine influenza in ferrets.

Respiratory disease diagnostics of cattle.

Cattle appear to be more susceptible to respiratory disease based on anatomic and physiologic factors. Diagnostic sampling and tests can provide valuable information when investigating causes of respiratory disease within a group of cattle. Diagnostic tests should be selected based on several criteria including quality of the sample, diagnostic question, producer goals, history, diagnostic laboratory, diagnostician, and economy. Veterinarians and producers should agree on the diagnostic question, testing goals, and use of results before submitting samples.

Novel Senecavirus A in Swine with Vesicular Disease, United States, July 2015.

To the Editor: Senecavirus A (SVA; formerly known as Seneca Valley virus [SVV] belongs to the genus Senecavirus, family Picornaviridae (1,2). SVA was first isolated in 2001 as a contaminant of the PER.C6 cell line and designated as SVV-001 (1,3). Since its discovery, SVA has been infrequently detected in swine with idiopathic vesicular disease (IVD) (4–6), which clinically resembles foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, and vesicular stomatitis. The virus has also been retrospectively detected in previous cases with various clinical conditions in the United States during 1988–2001 (7). However, the clinical significance of SVA in swine could not be determined (7,8).

A Highly Effective Protocol for the Rapid and Consistent Induction of Digital Dermatitis in Holstein Calves.

Bovine Digital Dermatitis (DD) is a leading cause of lameness in dairy cattle. DD is reportedly increasing in prevalence in beef cattle feedlots of the US. The exact etiologic agent(s) responsible for the disease have yet to be determined. Multiple studies have demonstrated the presence of a variety of Treponema spp. within lesions. Attempts to reproduce clinically relevant disease using pure cultures of these organisms has failed to result in lesions that mirror the morphology and severity of naturally occurring lesions. This manuscript details the systematic development of an experimental protocol that reliably induces digital dermatitis lesions on a large enough scale to allow experimental evaluation of treatment and prevention measures. In total, 21 protocols from five experiments were evaluated on their effectiveness in inducing DD lesions in 126 Holstein calves (504 feet). The protocols varied in the type and concentration of inoculum, frequency of inoculation, duration the feet were wrapped, and type of experimental controls need to validate a successful induction. Knowledge gained in the first four experiments resulted in a final protocol capable of inducing DD lesions in 42 of 44 (95%) feet over a 28 day period. All induced lesions were macroscopically and microscopically identified as clinical DD lesions by individuals blinded to protocols. Lesions were also located at the site of inoculation in the palmer aspect of the interdigital space, and induced clinically measurable lameness in a significant portion of the calves. Collectively these results validate the model and provide a rapid and reliable means of inducing DD in large groups of calves.

Rickets case series and diagnostic review of hypovitaminosis D in swine

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.