Yaxuan Sun

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality

ABSTRACT We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5′ untranslated region (5′-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Alterations of the Ileal and Colonic Mucosal Microbiota in Canine Chronic Enteropathies

Background The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon. Aim To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls. Methods Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis. Results The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05). Conclusion Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota.

Development and validation of an endoscopic activity score for canine inflammatory bowel disease

The aim of this study was to develop and prospectively validate a simple endoscopic score of disease activity for dogs with inflammatory bowel disease (IBD). Archived endoscopic still images and video recordings of gastric, duodenal, and colonic endoscopic examinations were displayed to novice and experienced endoscopists for assessment of inflammatory activity using established descriptions. The mucosal appearances evaluated were normal tissue, erosions, friability, increased granularity, lymphangiectasia (duodenum), and mass (colon). Fleiss and Cohens Kappa statistics were used to estimate the inter-observer agreement of the index. For duodenal assessment, there were statistically significant (P <0.05) differences in inter-observer agreement, with experienced endoscopists performing better than novice endoscopists in the accurate identification of mucosal appearance of the duodenum. In contrast, there was no significant difference between novice and experienced endoscopists in their interpretation of gastric (P = 0.10) and colonic (P = 1.0) mucosal appearances. Validation studies using endoscopic video clips to assess the same endoscopic parameters by quantitative (lesion number and severity) and qualitative (presence of mucosal lesions) methods showed moderate-to-substantial agreement between experienced endoscopists. Based on the observations that the quantitative and qualitative scores of mucosal appearances are virtually identical, and that qualitative assessment was performed more quickly and objectively than quantitative assessment, we propose a simple endoscopic activity score based on qualitative criteria alone in dogs with inflammatory bowel disease.

Sampling guidelines for oral fluid-based surveys of group-housed animals

Formulas and software for calculating sample size for surveys based on individual animal samples are readily available. However, sample size formulas are not available for oral fluids and other aggregate samples that are increasingly used in production settings. Therefore, the objective of this study was to develop sampling guidelines for oral fluid-based porcine reproductive and respiratory syndrome virus (PRRSV) surveys in commercial swine farms. Oral fluid samples were collected in 9 weekly samplings from all pens in 3 barns on one production site beginning shortly after placement of weaned pigs. Samples (n=972) were tested by real-time reverse-transcription PCR (RT-rtPCR) and the binary results analyzed using a piecewise exponential survival model for interval-censored, time-to-event data with misclassification. Thereafter, simulation studies were used to study the barn-level probability of PRRSV detection as a function of sample size, sample allocation (simple random sampling vs fixed spatial sampling), assay diagnostic sensitivity and specificity, and pen-level prevalence. These studies provided estimates of the probability of detection by sample size and within-barn prevalence. Detection using fixed spatial sampling was as good as, or better than, simple random sampling. Sampling multiple barns on a site increased the probability of detection with the number of barns sampled. These results are relevant to PRRSV control or elimination projects at the herd, regional, or national levels, but the results are also broadly applicable to contagious pathogens of swine for which oral fluid tests of equivalent performance are available.

Quantifying the effect of lactogenic antibody on porcine epidemic diarrhea virus infection in neonatal piglets

Abstract The contribution of lactogenic antibody to the protection of piglets against porcine epidemic diarrhea virus (PEDV) was evaluated. Pregnant multiparous sows and their litters were allocated to one of 3 treatment groups: Group 1–6 serum antibody-negative sows and a subset (n=11) of their piglets. Group 2–8 serum antibody-positive sows and their 91 piglets. Piglets were orally inoculated with PEDV at 4 (Group 1) or 2 (Group 2) days of age. Group 3–2 PEDV serum antibody-negative sows and 22 piglets, provided a baseline for piglet survivability and growth rate. Piglets were monitored daily for clinical signs, body weight, and body temperature through day post-inoculation (DPI) 12 (Groups 2 and 3) or 14 (Group 1). Serum and mammary secretions were tested for PEDV IgG, IgA, and virus-neutralizing antibody. Feces were tested by PEDV real-time, reverse transcriptase PCR (rRT-PCR). Piglets on sows without (Group 1) or with (Group 2) anti-PEDV antibody showed significantly different responses to PEDV infection in virus shedding (p < 0.05), thermoregulation (p < 0.05), growth rate (p < 0.05), and survivability (p <0.0001). Specifically, Group 1 piglets shed more virus on DPIs 1 to 5, were hypothermic at all sampling points except DPIs 9, 11, and 12, gained weight more slowly, and exhibited lower survivability than Group 2 piglets. Within Group 2 litters, significant differences were found in virus shedding (p < 0.05), and body temperature (p < 0.05), but not in piglet survival rate. The number of sows and litters in Group 2 was insufficient to derive the relationship between specific levels of lactogenic antibody (FFN, IgA, and IgG) and the amelioration of clinical effects. However, when combined with previous PEDV literature, it can be concluded that the optimal protection to piglets will be provided by dams able to deliver sufficient lactogenic immunity, both humoral and cellular, to their offspring.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions

Abstract Introduction: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs. Material and Methods: Four groups of pigs (six pigs per group) were inoculated with A. pleuropneumoniae serovars 1, 5, 7, or 12. Weekly serum samples and daily oral fluid samples were collected from individual pigs for 56 days post inoculation (DPI) and tested by LPS and ApxIV ELISAs. The ApxIV ELISA was run in three formats to detect immunlgobulins M, G, and A (IgM, IgG and IgA) while the LPS ELISA detected only IgG. Results: All pigs inoculated with A. pleuropneumoniae serovars 1 and 7 were LPS ELISA serum antibody positive from DPI 14 to 56. A transient and weak LPS ELISA antibody response was observed in pigs inoculated with serovar 5 and a single antibody positive pig was observed in serovar 12 at ≥35 DPI. Notably, ApxIV serum and oral fluid antibody responses in pig inoculated with serovars 1 and 7 reflected the patterns observed for LPS antibody, albeit with a 14 to 21 day delay. Conclusion: This work suggests that ELISAs based on ApxIV antibody detection in oral fluid samples could be effective in population monitoring for A. pleuropneumoniae.

SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS)

Abstract:  Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease.