Seroprevalence of Brucella canis antibodies in dogs entering a Minnesota humane society, Minnesota, 2016-2017.

BACKGROUND: Canine brucellosis, caused by the bacterium Brucella canis, is a zoonotic and largely reproductive disease of dogs. The disease is a recognized problem in canine breeding populations, and the risk to individuals assisting with birthing is well described. Prior to 2015, all cases of canine brucellosis reported to the Minnesota Board of Animal Health were in dogs used for breeding. In 2015, canine brucellosis was identified in eight Minnesota rescue dogs, all originating from specific geographic areas in South Dakota. Our objective was to measure the seroprevalence of B. canis in stray and previously owned dogs entering a large Minnesota animal rescue organization to determine if our observations represented a localized or generalized disease issue among rescue dogs. METHODS: A stratified random sample of stray and previously owned dogs entering the largest Minnesota animal rescue organization between November 1, 2016 and November 7, 2017, was tested for B. canis antibodies by the 2-Mercaptoethanol Rapid Slide Agglutination Test (2ME-RSAT) (Zoetis d-TEC® CB kit). Sample sizes for each strata were calculated using previously published seroprevalence estimates. Blood from selected dogs was collected, serum harvested, and transported to the Minnesota Veterinary Diagnostic Laboratory for testing. Positive samples in the 2ME-RSAT were shipped to Cornell University for confirmation by Agarose Gel Immunodiffusion (AGID) testing. Demographics, state and setting of origin, and health status were collected on study-dogs. RESULTS: Of the 10,654 dogs accepted by AHS during the study period, 943 (8.9%) were selected for testing. Most study dogs arrived from Oklahoma (28%), Alabama (18%), and Minnesota (12%). The median age of study dogs was 1.5 years; 303 (32%) were intact males and 294 (31%) were intact females. Most study dogs were strays (n = 716, 76%). Of the total, 22 (3.1%) stray and eight (3.5%) owner-surrendered dogs were presumptively positive by RSAT; one (0.11%) of the stray dogs was positive by 2ME-RSAT and confirmed by AGID. The positive dog was a healthy-appearing 1 year-old neutered male beagle from Texas. CONCLUSIONS: The seroprevalence of canine brucellosis in dogs entering Minnesota for adoption from multiple states was low. Never-the-less, care must to be taken to consider all potential risks and outcomes of interstate and international dog trade, including the spread of infectious diseases such as canine brucellosis.

The emergence and evolution of influenza A (H1α) viruses in swine in Canada and the United States.

Swine are a key reservoir host for influenza A viruses (IAVs), with the potential to cause global pandemics in humans. Gaps in surveillance in many of the world's largest swine populations impede our understanding of how novel viruses emerge and expand their spatial range in pigs. Although US swine are intensively sampled, little is known about IAV diversity in Canada's population of ~12 million pigs. By sequencing 168 viruses from multiple regions of Canada, our study reveals that IAV diversity has been underestimated in Canadian pigs for many years. Critically, a new H1 clade has emerged in Canada (H1α-3), with a two-amino acid deletion at H1 positions 146-147, that experienced rapid growth in Manitoba's swine herds during 2014-2015. H1α-3 viruses also exhibit a higher capacity to invade US swine herds, resulting in multiple recent introductions of the virus into the US Heartland following large-scale movements of pigs in this direction. From the Heartland, H1α-3 viruses have disseminated onward to both the east and west coasts of the United States, and may become established in Appalachia. These findings demonstrate how long-distance trading of live pigs facilitates the spread of IAVs, increasing viral genetic diversity and complicating pathogen control. The proliferation of novel H1α-3 viruses also highlights the need for expanded surveillance in a Canadian swine population that has long been overlooked, and may have implications for vaccine design.

Outbreak of highly pathogenic avian influenza in Minnesota in 2015.

The incursion of highly pathogenic avian influenza (HPAI) into the United States during 2014 resulted in an unprecedented foreign animal disease (FAD) event; 232 outbreaks were reported from 21 states. The disease affected 49.6 million birds and resulted in economic losses of $950 million. Minnesota is the largest turkey-producing state, accounting for 18% of U.S. turkey production. Areas with concentrated numbers of turkeys in Minnesota were the epicenter of the outbreak. The first case was presumptively diagnosed in the last week of February 2015 at the Minnesota Veterinary Diagnostic Laboratory (MVDL) and confirmed as HPAI H5N2 at the National Veterinary Services Laboratories on March 4, 2015. A total of 110 farms were affected in Minnesota, and the MVDL tested >17,000 samples from March to July 2015. Normal service was maintained to other clients of the laboratory during this major FAD event, but challenges were encountered with communications, staff burnout and fatigue, training requirements of volunteer technical staff, test kit validation, and management of specific pathogen-free egg requirements.

Validation of a competitive ELISA and a virus neutralization test for the detection and confirmation of antibodies to Senecavirus A in swine sera.

Senecavirus A (SVA; family Picornaviridae) is a nonenveloped, single-stranded RNA virus associated with idiopathic vesicular disease (IVD) in swine. SVA was detected in pigs with IVD in Brazil, United States, Canada, and China in 2015, triggering the need to develop and/or validate serologic assays for SVA. Our objective was to fully validate a previously developed competitive enzyme-linked immunosorbent assay (cELISA) as a screening test for antibodies to SVA. Additional objectives included the development and validation of a virus neutralization test (VNT) as a confirmatory test for SVA antibody detection, and the comparison of the cELISA, VNT, and an existing immunofluorescent antibody test (IFAT) for the detection of SVA antibodies in serial bleeds from SVA outbreaks. The diagnostic specificity and sensitivity were 98.2% (97.2-98.9%) and 96.9% (94.5-98.4%) for the cELISA, and 99.6% (99.0-99.9%) and 98.2% (95.8-99.4%) for the VNT, respectively. There was strong agreement among cELISA, VNT, and IFAT when compared based on kappa coefficient. Based on these performance characteristics, these tests are considered suitable for serologic detection of SVA in pigs.

Investigation into the Airborne Dissemination of H5N2 Highly Pathogenic Avian Influenza Virus During the 2015 Spring Outbreaks in the Midwestern United States.

We investigated the plausibility of aerosol transmission of H5N2 highly pathogenic avian influenza (HPAI) virus during the 2015 spring outbreaks that occurred in the U.S. midwest. Air samples were collected inside and outside of infected turkey and layer facilities. Samples were tested to assess HPAI virus concentration (RNA copies/m(3) of air), virus viability, and virus distribution by particle size. HPAI virus RNA was detected inside and up to 1000 m from infected facilities. HPAI virus was isolated from air samples collected inside, immediately outside, up to 70 m from infected facilities, and in aerosol particles larger than 2.1 µm. Direct exposure to exhausted aerosols proved to be a significant source of environmental contamination. These findings demonstrate HPAI virus aerosolization from infected flocks, and that both the transport of infectious aerosolized particles and the deposition of particles on surfaces around infected premises represent a potential risk for the spread of HPAI.

Molecular characterization of quail bronchitis virus isolated from bobwhite quail in Minnesota.

From 2008 to 2012, 4 separate cases of quail bronchitis virus infection were seen in bobwhite quail (Colinus virginianus) raised in Minnesota. The quail chicks ranged in age from 5 d to 8 wk and suffered from respiratory distress and elevated mortality. On necropsy, gross lesions consisted of mucus in trachea, congested lungs, caseous air sacculitis, accumulation of chalky white urates on internal organs, necrotic foci in liver, and enlarged spleen. Histologic examination revealed fibrinoheterophilic rhinitis, heterophilic bronchitis, heterophilic tracheitis, and interstitial pneumonia in addition to deciliation, desquamation, and necrosis of bronchial respiratory epithelium. Karyomegaly with basophilic intranuclear inclusions was also seen in affected epithelium. Severe epicarditis, pericarditis, myocarditis, multifocal necrotizing hepatitis, and splenitis were additional pathological findings. Quail bronchitis virus (QBV) was isolated from all four samples when inoculated in specific-pathogen-free (SPF) embryonated chicken eggs. The virus was confirmed by electron microscopy and polymerase chain reaction using fowl adenovirus (FAdV) hexon gene-specific primers. Nucleotide sequences of the four isolates showed 99.0% identity with CELO strain of fowl adenovirus A. Nine nucleotide substitutions were observed; 3 of these were nonsynonymous (A281G, C314T and G565C), leading to changes in deduced amino acid sequences (S94G, T105M and A189P, respectively). Based on partial sequence of the hexon gene, QBV isolates of this study clustered closely with fowl adenovirus A and were different from FAdV groups B through E and from adenoviruses of goose, duck, turkey, and pigeon. Further studies are indicated to determine the impact of nonsynonymous substitutions on host specific pathogenicity of these viruses.

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.

La probabilité de détecter le virus de l'influenza A (VIA) dans des échantillons de fluide oral (FO) a été calculée pour chacune des 13 épreuves basées sur une réaction d'amplification en chaine en temps réel utilisant la polymérase réverse (rRT-PCR) et 7 épreuves basées sur l'isolement viral (IV). Les échantillons de FO ont été inoculés avec du VIA H1N1 ou H3N2 et dilués en série par facteur de 10 (10−1 à 10−8). Huit laboratoires participants ont reçu 180 échantillons randomisés de FO (10 réplicats × 8 dilutions × 2 sous-types de VIA plus 20 échantillons témoins négatifs sans VIA) et ont réalisé la méthode de rRT-PCR et d'IV de leur choix. L'analyse des résultats à l'aide d'un modèle de régression logistique pour les effets mélangés a identifié la dilution et l'épreuve comme étant des variables significatives (P < 0,0001) pour la détection de VIA dans du FO par rRT-PCR ou IV. Le sous-type de virus n'était pas significatif pour la détection de VIA soit par rRT-PCR (P = 0,457) ou par IV (P = 0,101). Pour les épreuves rRT-PCR les valeurs seuils de cycle (Ct) augmentaient de manière constante avec la dilution mais variaient énormément. Ainsi, il n'était pas possible de prédire le succès de l'IV sur la base des valeurs de Ct. Le succès de l'IV était inversement relié à la dilution de l'échantillon; l'épreuve était généralement négative aux faibles concentrations de virus. Pour avoir du succès dans la surveillance des maladies et de la santé des porcs il est nécessaire d'avoir des épreuves avec des performances constantes, mais des différences significatives dans la reproductibilité ont été observées parmi les épreuves évaluées.(Traduit par Docteur Serge Messier).

Detection and molecular characterization of astroviruses in turkeys.

This study was conducted to determine the prevalence and molecular characteristics of turkey astrovirus 1 (TAstV-1) and avian nephritis virus (ANV) in turkeys with light turkey syndrome (LTS), which is characterized by lower body weight in market-age turkeys than their standard breed character. We collected pools of fecal samples from four LTS and two non-LTS turkey flocks in Minnesota at 2, 3, 5 and 8 weeks of age. Of the 80 LTS pools tested, 16 (20.0 %) and 11 (13.8 %) were positive for TAstV-1 and ANV, respectively. For non-LTS flocks, these numbers were 8 (20.0 %) and 5 (12.5 %), respectively. The maximum number of birds was positive at five weeks of age. We also tested 130 fecal samples of poult enteritis syndrome (PES) cases submitted to the Minnesota Veterinary Diagnostic Laboratory and found 19 and 11 positive for TAstV-1 and ANV, respectively. RdRp gene sequences were determined for a total of 29 TAstV-1 and 22 ANV samples. Phylogenetic analysis of the RdRp gene revealed 92-100 % and 88-100 % nucleotide sequence identity among TAstV-1 and ANV sequences, respectively. A large number of nucleotide and amino acid substitutions were observed in LTS and PES flocks than in non-LTS flocks. One of the PES sequences grouped with ANV-like sequences detected in chickens, indicating that regular screening of birds should be continued. Further, complete genome analysis should be conducted to determine whether this virus is a novel divergent strain or a recombinant of chicken and turkey ANV-like viruses. The detection of TAstV-1 and ANV in a considerable number of non-LTS cases emphasizes the need for further studies on the transmission pattern and pathogenesis of these viruses to determine their role as pathogens of turkeys.

Environmental Factors Influencing White-Tailed Deer (Odocoileus virginianus) Exposure to Livestock Pathogens in Wisconsin.

White-tailed deer (Odocoileus virginianus) are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland) in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo), bovine viral diarrhea virus (BVDV-1 and BVDV-2), infectious bovine rhinotracheitis virus (IBR), and parainfluenza 3 virus (PI3). We used logistic regression to model potential intrinsic (e.g., age, sex) and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens) variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010-2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%), IBR (7.9%), Leptospira interrogans serovar pomona (11.7%), L. i. bratislava (1.0%), L. i. grippotyphosa (2.5%) and L. i. hardjo (0.3%). Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119) were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens.

Molecular characterization of turkey enteric reovirus S3 gene.

The molecular diversity in S3 gene sequences of turkey reovirus (TRV) was determined in poult enteritis syndrome (PES)-affected and apparently healthy turkey poults. Twenty-nine TRV-positive samples (15 from PES-affected flocks and 14 from apparently healthy flocks) were tested using self-designed primers for the S3 gene. Phylogenetic analysis revealed that the TRV S3 sequences of this study clustered in clade III and formed two different groups in this clade. The avian reoviruses from duck and goose formed clade I and those from chickens formed clade II. The clade III TRV sequences had a nucleotide percent identity of 88.9 to 100% among themselves but only of 59.5 to 63.5% and 69.2 to 72.6% with clades I and II, respectively. More amino acid substitutions were present in TRVs from PES-affected flocks than in those from apparently healthy flocks using ATCC VR-818 (AY444912) as a benchmark. All TRVs of this study showed substitutions at positions 244 and 285. The impact of these changes on the virulence of the virus, if any, needs to be studied.

Survival of porcine reproductive and respiratory syndrome virus in fresh pork.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically important diseases of pigs. Transmission of PRRS virus has been reported through many routes, with aerosol route being the most predominant. There may also be a potential risk of transmission through contami-nated pork, but this has never been investigated. The purpose of this study was to experimentally contaminate fresh pork with three different concentrations of PRRSV and to study virus survival at ambient (25 °C), refrigerated (4 °C), and frozen (-20 °C) temperatures. Concentrations of virus representing natural infectivity level and 'worst case scenario' were studied. The virus was detected in fresh pork at all three virus concentrations for up to 48 h at ambient temperature. At 4 °C, the virus survived for 6 days in pork inoculated with the higher virus concentration and for 3 days in pork inoculated at the lower concentration. At frozen temperature, PRRSV was detected for up to 60 days in pork inoculated at the higher concentration and for 7 days in pork inoculated at the lower concentration. These results suggest that fresh pork has the potential to be a vehicle for virus dissemination depending upon temperature and time of storage.

The occurrence of enteric viruses in Light Turkey Syndrome.

Two studies were conducted to determine the role of enteric viruses in Light Turkey Syndrome (LTS), which is characterized by lower weight in market age turkeys than their standard breed character. In the surveillance study, we selected four LTS and two non-LTS turkey flocks in Minnesota and collected faecal samples at 2, 3, 5 and 8-weeks of age. Astrovirus, rotavirus, and reovirus were detected alone or in various combinations in both LTS and non-LTS flocks. No coronavirus was detected in LTS flocks and no corona- or reovirus was detected in non-LTS flocks. In the second study, 2-week-old turkey poults were divided into two groups; Group A (challenged) was inoculated orally with 10% pooled faecal suspension from LTS flocks and group B (control) was inoculated with phosphate buffered saline (PBS). Clinical signs of depression, huddling, and lack of uniform size were observed in the challenged group but not in the control group. diarrhoea was observed in both groups but was more severe in the challenged group than in the control group. Birds in the challenged group shed astrovirus, rotavirus and reovirus, while the control group shed only astrovirus. Virus shedding in both groups was observed for up to nine weeks of age. Significantly lower body weights were seen in the challenged group starting at seven weeks of age and lasting until 20 weeks of age. These findings suggest that viral enteritis at an early age may set up conditions for the development of LTS in adult turkeys.

Survival of Porcine Reproductive and Respiratory Syndrome Virus in Pork Products.

There is a risk of virus transmission through contaminated pork, and many viruses are considered potential hazards for both humans and livestock. The risk of transmission may be elevated with importation/exportation of meat between countries globally. Survival of porcine reproductive and respiratory syndrome virus (PRRSV) in different pork products has not been studied. The present study evaluated PRRSV survival in four different products: fresh sausage, ham, bacon, and acidified sausage prepared with experimentally contaminated pork. These products were prepared according to standard methods used by the manufacturers of pork products, and then stored at room temperature, 4 °C and -20 °C. PRRSV was detected only in fresh sausage for up to 15 days at 4 °C and for 30 days at -20 °C. No PRRSV was detected at any temperature in any of the other three products. These preliminary data provide valuable information for the pork processing industry, as well as in planning for import/export of these products among different countries.

Isolation and characterization of a turkey arthritis reovirus.

During the spring and summer of 2011, the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota received 14 submissions of 15-to-18-week-old tom turkeys that were recumbent with wing tip bruises ("wing walkers") and uni- or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in specific-pathogen-free embryonated chicken eggs and QT-35 cells. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction, and gene sequence analysis. BLAST analysis on the basis of a 880 bp nucleotide sequence of the S4 gene confirmed all isolates as a reovirus. Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, -2, -3, and -5, and the other subgroup containing TARV-MN4. Isolates in subgroup I had a similarity of 97%-100% with each other, while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90%-93% similarity with turkey enteric reoviruses in the United States, while the other four isolates in subgroup I had 89%-97.6% similarity. These results indicate divergence within TARVs as well as from enteric viruses, which needs to be confirmed by complete genome sequence analysis. Further experimental studies are planned to determine the role of these isolates in turkey arthritis and to compare them with classical chicken reovirus.

Effect of temperature on the survival of porcine circovirus type 2b in fresh pork.

Porcine circovirus type 2b (PCV2b) causes PCV-associated disease in pigs. This study was conducted to investigate the effect of temperature on the survival of PCV2b in fresh pork. Several pieces of longissimus dorsi muscle were injected with 100 µL of a suspension containing 10(5.2) TCID50 (50% tissue culture infective doses) of the virus. Virus-inoculated pieces of pork were stored at 25 °C, 4 °C and -20 °C and tested for the presence of infectious virus after different times of storage. PCV2b was found to survive in fresh pork for up to 2 days post inoculation (dpi) at room temperature, for 6 dpi at 4 °C and for up to 30 dpi at -20 °C indicating that the survival of PCV2b in fresh pork depends on temperature of storage.

A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys.

This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting "poult enteritis syndrome" were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

Detection and molecular characterization of Porcine astrovirus strains associated with swine diarrhea.

Astrovirus has been reported to be associated with diarrhea in pigs. The current study was conducted for the detection and molecular characterization of astroviruses in diarrheic pigs submitted to the Veterinary Diagnostic Laboratory, University of Minnesota. Intestinal contents from 269 pigs were examined by reverse transcription polymerase chain reaction (RT-PCR), and 62% were found positive for astroviruses. Of the positive samples, 20% were positive for astrovirus alone while astrovirus with rotavirus was detected in 58% of the samples. The remaining 22% revealed the presence of astrovirus along with Porcine hemagglutinating encephalomyelitis virus, Transmissible gastroenteritis virus, or Porcine circovirus-2. Sequencing the capsid gene of 56 randomly selected samples confirmed them to be Porcine astrovirus type 4 (PAstV-4) with 58-100% nucleotide identity within these viruses. Phylogenetic analysis revealed 2 possible subgroups. The results indicate that PAstV is present on swine farms in the United States and that it may be associated with diarrhea either alone or in combination with other enteric viruses. Further studies are needed to determine strain diversity among porcine astroviruses so that appropriate control strategies can be devised and implemented.

Ring test evaluation of the repeatability and reproducibility of a Porcine reproductive and respiratory syndrome virus oral fluid antibody enzyme-linked immunosorbent assay.

The precision of a Porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody enzyme-linked immunosorbent assay (ELISA) was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples of known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (phase 1) and then using an ELISA kit and reagents configured in their respective laboratory (phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples of known PRRSV antibody status ranged from 0.724 to 0.997 in phase 1 and from 0.953 to 0.998 in phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for 3 conditions: case 1--samples of unknown status (n = 224); case 2--samples of known status (n = 39), and case 3--all samples (n = 263). Among the 3 cases, reliability coefficients ranged from 0.937 to 0.964 in phase 1 and from 0.922 to 0.935 in phase 2. For case 3, it was estimated that 96.67% of the total variation in phase 1 and 93.21% in phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. The current study supports the routine use of this test in laboratories providing diagnostic service to pig producers.

Avian metapneumovirus subtypes circulating in Brazilian vaccinated and nonvaccinated chicken and turkey farms.

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.