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.

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.

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.

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.

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.

Detection and molecular characterization of enteric viruses in breeder turkeys.

The present study was undertaken to detect and characterize enteric viruses (rotavirus, astrovirus, reovirus, and coronavirus) in breeder poults. Five turkey breeder flocks were selected. Faecal samples were collected from all flocks at 1 week of age and then every other week until the poults reached 9 weeks of age. The faecal samples were pooled in groups of five. Of the 193 pools ("samples") tested by reverse transcription-polymerase chain reaction, 47.2%, 30.6%, and 10.4% samples were positive for astrovirus, rotavirus, and reovirus, respectively. No coronavirus was detected in any of the samples. Overall, 118 (61.1%) samples were positive for one or more enteric viruses. Of the 118 samples, 70 (59.3%) were positive for a single virus and 48 (40.7%) for a combination of viruses. Phylogenetic analysis based on the polymerase gene showed that astroviruses clustered into two groups with sequence homology ranging from 85.6 to 100% at the nucleotide level. Based on NSP4 gene sequences, rotaviruses clustered in a group and had 96.3 to 99.9% sequence homology at the nucleotide level. The reoviruses, based on their S4 gene sequences, clustered in a single group with sequence homology of 96.9 to 100%. Differing amino acid sequences of all three viruses may affect the antigenicity and/or pathogenicity of these viruses and may merit further study. The presence of two or three different viruses in combination may affect the dynamics of turkey health and disease.

Identification of H2N3 influenza A viruses from swine in the United States.

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian alpha2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.

Duration of growth depression and pathogen shedding in experimentally reproduced poult enteritis syndrome.

An experimental study was conducted to determine the duration of growth depression and virus shedding in turkey poults after oral inoculation with intestinal contents from birds affected with poult enteritis syndrome (PES). Poults at day 14 of age were divided into four groups (groups A, B, C, and D) of 40 poults each and inoculated orally with unfiltered supernatant, filtered supernatant, sediment suspended in phosphate-buffered saline (PBS), or PBS alone (control), respectively. The poults were observed daily for clinical signs, and their growth response, pathology, and pathogen shedding were examined at 10, 20, 30, 40, and 50 days postinoculation (DPI). Body weights of eight poults in each group were recorded at each of these intervals followed by euthanasia. Dullness, depression, and diarrhea were observed in birds inoculated with supernatant or sediment suspension. All three treatments significantly reduced body weight gain of poults compared with the control group; average weight loss was 14%. Gross pathologic changes consisted of pale distended intestines with watery contents and distended ceca with frothy and watery contents. Astrovirus and rotavirus were detected in the inoculum by reverse transcription (RT)-PCR, whereas Salmonella was identified on bacterial isolation. Both viruses were detected in treated poults by RT-PCR for up to 10 and 40 DPI, respectively. Of the three treatments, sediment suspension caused maximal decrease in weight gain as well as greatest pathologic lesions followed by unfiltered supernatant and filtered supernatant. These findings suggest a role for bacteria in increasing the severity of PES. Lower weight gain in treated poults (compared with controls) at 9 wk of age also indicates that PES-affected poults may not reach normal weight at marketing, leading to economic losses for the producer.

A retrospective study on poult enteritis syndrome in Minnesota.

A retrospective study was conducted to determine the occurrence of poult enteritis syndrome (PES) in Minnesota from January 2002 to December 2007. PES is an infectious intestinal disease of young turkeys between 1 day and 7 wk of age and is characterized by diarrhea, depression, and lethargy with pale intestines and/or excessively fluid cecal contents. During the study period, samples from 1736 turkey flocks were submitted to the Minnesota Veterinary Diagnostic Laboratory for disease investigation. Of these, 151 flocks (8.7%) were PES positive. Cases of PES were seen throughout the year with higher prevalence in fall. The PES was statistically associated with age with higher occurrence in poults less than 3 wk of age. Rotavirus, small round virus (SRV), Salmonella, nonhemolytic Escherichia coli, Enterococcus, and Eimeria oocysts were detected alone or in different combinations. Reovirus and adenovirus were found in one flock each. The most commonly identified pathogens were Salmonella (85 flocks) and rotavirus (73 flocks). Of PES-affected flocks, 39 (25.8%), 66 (43.7%), and 37 (24.5%) had one, two, and three or more pathogens, respectively. Rotavirus, SRV, and reovirus occurred mostly in poults of less than 6 wk of age while Salmonella, E. coli, and Eimeria were seen in poults of all age groups. Minimum age for rotavirus detection was in 2-day-old poults. Histopathologically, moderate to severe mixed intestinal villus or lamina propria inflammatory infiltrates, necrosis of distal villus tips in intestinal specimens, and mild to severe lymphocellular depletion in thymus, bursa, and spleen were seen. Antimicrobial sensitivity patterns of bacterial isolates from PES-affected flocks revealed maximum sensitivity to trimethoprim/sulfamethoxazole and ceftiofur and a varying degree of resistance to other antimicrobials.

Efficacy of disinfectants and hand sanitizers against avian respiratory viruses.

Disinfectants play a major role in the control of animal diseases by decontaminating the farm environment. We evaluated the virucidal efficacy of nine commonly used disinfectants on a nonporous surface contaminated experimentally with avian metapneumovirus (aMPV), avian influenza virus, or Newcastle disease virus (NDV). Phenolic compounds and glutaraldehyde were found to be the most effective against all three viruses. Quaternary ammonium compounds were effective against aMPV but not against the other two viruses. In addition, efficacy of commercially available hand sanitizers was evaluated on human fingers contaminated with aMPV and NDV. All three hand sanitizers tested were found to be effective against both viruses within 1 min of application on fingers.

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.

Permissibility of different cell types for the growth of avian metapneumovirus.

Vero cells are commonly used for the growth of avian metapneumovirus subtype C (aMPV-C). This study was conducted to evaluate 17 different cell types for the growth of a Minnesota strain of aMPV-C. The virus was inoculated into these cell types and virus growth was monitored by the development of cytopathic effects (cpe) and immunofluorescence. Virus growth was obtained in 6 of 17 cell types tested with the highest virus titers observed in BGM and DF-1 cells. The flow cytometric analysis of cells at 72 h post inoculation found the highest number of infected cells in BGM cells followed by QT-35 cells. At 48 h post inoculation, DF-1 and BGM cells showed the highest number of infected cells. These results suggest that BGM, QT-35, and DF-1 cells can be used for high titer propagation of aMPV-C.

Survival of two avian respiratory viruses on porous and nonporous surfaces.

The transmission of pathogens from infected to susceptible hosts may occur through contaminated fomites and inanimate objects. This type of transmission depends on the ability of the pathogens to survive in the environment. In this report, we describe the survivability of two avian respiratory viruses, e.g., avian metapneumovirus and avian influenza virus on 12 different porous and nonporous surfaces. The viruses survived on some of the surfaces for up to 6 days postcontamination but not after 9 days. Both viruses survived longer on nonporous surfaces than on porous ones. One of the reasons for poor survival on porous surfaces could be inefficient elution of virus from these surfaces. These results should be helpful in determining how long the premises should be left vacant after an outbreak of these viruses has occurred in poultry houses.

Duration of immunity engendered by a single dose of a cold-adapted strain of Avian pneumovirus.

The duration of immunity after a single dose of a cold-adapted strain of Avian pneumovirus (APV) was studied. Turkeys were vaccinated at 1 wk of age and challenged with virulent virus 3, 7, 10, and 14 wk later. Nonvaccinated groups were also challenged at the same times. No clinical signs were observed in the vaccinated birds after vaccination or after any challenge. No viral RNA was shed by the vaccinated birds after any challenge. The nonvaccinated birds shed viral RNA after all challenges. Avian pneumovirus-specific humoral antibodies were detected in the vaccinated birds until 14 wk after vaccination. The results of this preliminary study indicate that inoculation with a single dose of a cold-adapted strain of APV at 1 wk of age provides protection until 15 wk of age.

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.

Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.

Detection of three avian respiratory viruses by single-tube multiplex reverse transcription-polymerase chain reaction assay.

Acute respiratory tract infections are leading causes of morbidity in poultry farms throughout the world. Avian pneumovirus (APV), avian influenza virus (AIV), and Newcastle disease virus (NDV) have been recognized as the most important pathogens of both chicken and turkeys. Single-virus reverse transcription-polymerase chain reaction (sRT-PCR) assays are used extensively to detect these viruses in clinical samples. This study reports the development and evaluation of a single-tube multiplex RT-PCR (mRT-PCR) assay for simultaneous and specific detection of APV, AIV, and NDV. Specific primers for each virus were selected that amplified products of predicted sizes from each virus in the mRT-PCR as well as in the sRT-PCR assays (438, 218, and 532 bp for APV, AIV, and NDV, respectively). The sensitivity and specificity of mRT-PCR assay were compared with those of the sRT-PCR. The mRT-PCR assay was as sensitive as the sRT-PCR assays because virus detection limits were similar in both assays. The detection limits of mRT-PCR assay were 10(0.5) tissue culture infective dose (50%) (TCID50)/ml, 10(1.2) TCID50/ml, and 10(0.7) TCID50/ml for APV, AIV, and NDV, respectively. Overall, there was an excellent correlation between mRT-PCR and sRT-PCR assays. No product amplification was obtained with nucleic acid from infectious bronchitis virus and reovirus using these primer sets. In summary, mRT-PCR assay holds potential to be an economical and rapid diagnostic method for the simultaneous detection of 3 avian respiratory viruses in chickens and turkeys.

The effect of pooling sera on the detection of avian pneumovirus antibodies using an enzyme-linked immunosorbent assay test.

Pooling of samples is a cost-effective approach to estimate disease prevalence and to identify infected individuals. The objective of this study was to evaluate the use of serum pools for the detection of avian pneumovirus infection in turkey flocks by enzyme-linked immunosorbent assay, so that a minimum number of tests can be performed without compromising the sensitivity and specificity of the test. A total of 900 field samples were tested; 20 samples from each of 45 flocks. All samples were tested individually followed by pool testing in groups of 3, 4, 5, and 7 samples each. The number of positive pools for a given pool size was positively associated with the number of positive samples. In a separate experiment, the effect of dilution was examined by pooling 1 positive sample with different numbers of negative samples to form pools of sizes 2-7. These laboratory results were analyzed and integrated into a simulation model aimed at evaluating cost-efficient testing procedures. The probability of detecting an infected flock depended on prevalence of infection, size of serum pool, and the cutoff value used for optical density difference. At a theoretical prevalence of 20%, the probability of detecting an infected flock was 0.93 and 0.86 for a pool of 2 and 7, respectively. The probability of detecting positive flocks increased with increased prevalence and decreased cutoff. Pooling of samples represented a significant reduction in the cost of testing, suggesting that pooling is more advantageous and cost effective than testing individual samples.