Devi P. Patnayak

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 α2,6Gal-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.

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

Abstract 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.

Detection of Influenza a Virus in Porcine Oral Fluid Samples

Porcine oral fluids have been used for the detection of Porcine reproductive and respiratory syndrome virus and Porcine circovirus-2. The objective of the present study was to determine whether Influenza A virus (FLUAV) is present in porcine oral fluids at detectable levels and to validate a standard FLUAV molecular diagnostic test for porcine oral fluids. Pen-based oral fluid samples were collected on 3, 4, 5, and 6 days postinfection (DPI) from 4 groups of 6 pigs each that were inoculated intratracheally with A/Swine/ Iowa/00239/2004 H1N1 and from 2 untreated or mock-inoculated groups of 6 pigs each that served as negative controls. Individual nasal swabs were also collected from these 36 pigs on 3 and 7 DPI. All oral fluid samples were examined for the presence of FLUAV by matrix gene real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and virus isolation. Nasal swabs were tested initially by virus isolation followed by retest of negative samples with real-time RT-PCR. No oral fluid sample from virus-inoculated pigs was positive by virus isolation, but 15 of 16 positive (94%) oral fluids were positive by real-time RT-PCR. In contrast, virus was isolated from 32 of 48 (67%) nasal swabs collected from virus-inoculated pigs. In addition, 382 of 910 porcine oral fluids collected from pigs in the field between August 1, 2009, and January 31, 2010, were positive by real-time RT-PCR. The results of the present study indicate that pen-based oral fluids provide an easy, effective, and safe collection method for the detection of FLUAV with rapid testing methods such as real-time RT-PCR.

Porcine reproductive and respiratory syndrome virus (PRRSV) in serum and oral fluid samples from individual boars: will oral fluid replace serum for PRRSV surveillance?

The purpose of this study was to determine whether oral fluid samples could be used to monitor individually-housed adult boars for porcine reproductive and respiratory syndrome virus (PRRSV) infection. In 3 trials, 24 boars were intramuscularly (IM) inoculated with a modified-live PRRSV (MLV) vaccine (Trial 1), a Type 1 PRRSV isolate (Trial 2), or a Type 2 isolate (Trial 3). Oral fluid samples were collected daily and serum samples were collected twice weekly. Following the completion of the study, samples were randomized and blind-tested for PRRSV by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). PRRSV was detected in oral fluids at DPI 1 and all oral fluid specimens were PRRSV qRT-PCR positive at DPI 4. Although PRRSV was detected in both serum and oral fluid specimens through DPI 21, a comparison of matched samples from individual boars showed that oral fluid was equal to serum for the detection of PRRSV at DPI 7 and more likely to be positive than serum on DPI 14 and 21. Overall, oral fluid was superior to serum for the detection of PRRSV using PCR over the 21-day observation period in this study. The results of this experiment suggest that individually-penned oral fluid sampling could be an efficient, cost-effective approach to PRRSV surveillance in boar studs and other swine populations.

Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys

ABSTRACT This study was conducted to detect and characterize enteric viruses [rotavirus, turkey astrovirus-2 (TAstV-2), reovirus, and turkey coronavirus] from cases of poult enteritis syndrome (PES) in Minnesota turkeys. Of the intestinal contents collected from 43 PES cases, 25 were positive for rotavirus and 13 for small round viruses by electron microscopy (EM). Of the enteric virus-positive cases by EM (n = 27), 16 cases had rotavirus or small round viruses alone and the remaining 11 cases had both viruses. None of the cases were positive for reovirus or coronavirus by EM. However, with reverse transcription-PCR (RT-PCR), 40 cases (93%) were positive for rotavirus, 36 (84%) for TAstV-2, and 17 (40%) for reovirus. None of the cases were positive for turkey coronavirus by RT-PCR. The viruses from all cases were detected either alone or in combination of 2 or 3 by RT-PCR. Thus, 8 (19%) cases were positive for a single virus, whereas a combination of viruses was detected in the remaining 35 (81%) cases. The rota-TAstV-2 combination was the most predominant (n = 18 cases). Fifteen cases were positive for all 3 viruses. The rotaviruses had sequence homology of 89.8 to 100% with previously published sequences of turkey rotaviruses at the nucleotide level. The TAstV-2 had sequence homology of 84.6 to 98.7% with previously published TAstV-2, whereas reoviruses had sequence homology of 91.6 to 99.3% with previously published sequences of turkey reoviruses. Phylogenetic analysis revealed that rota- and reoviruses clustered in a single group, whereas TAstV-2 clustered in 2 different groups. In conclusion, a larger number of PES cases was positive for rotavirus, TAstV-2, and reovirus by RT-PCR than with EM. The presence of more than one virus and changes at the genetic level in a virus may affect the severity of PES in turkey flocks.

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.

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 100.5 tissue culture infective dose (50%) (TCID50)/ml, 101.2 TCID50/ml, and 100.7 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.

A Retrospective Study on Poult Enteritis Syndrome in Minnesota

Abstract 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.

Experimental reproduction of poult enteritis syndrome: Clinical findings, growth response, and microbiology

ABSTRACT Poult enteritis syndrome (PES) is an infectious disease of turkey poults characterized by diarrhea, dullness, and depression. Five experiments were conducted to reproduce the disease in turkey poults using intestinal contents of PES-affected birds. In all experiments, poults at 14 d of age were divided into 4 groups and were orally given 2 mL of unfiltered supernatant, filtered supernatant, sediment dissolved in PBS, or PBS alone. Inocula in experiments 1, 3, and 5 consisted of intestinal contents from PES-affected birds of less than 2 wk of age, whereas those in experiments 2 and 4 consisted of intestinal contents from PES-affected birds of 4 to 6 wk of age. Poults in all groups were observed daily for clinical signs. The BW and microbiological criteria in experiments 1, 3, and 5 were evaluated at 5, 10, and 15 d postinoculation, whereas in experiments 2 and 4, these observations were made at 10 and 20 d postinoculation. Rotavirus, astrovirus, and Salmonella were present in all 5 inocula. Diarrhea and depression were the major signs in poults given PES material. Significant retardation of growth was observed in poults given any of the 3 PES materials, but this effect was more pronounced in poults given the sediment inoculum. Rotavirus, astrovirus, and Salmonella were detected in poults given PES material. In some cases, enterovirus was also detected. No major difference was noticed in experimental reproduction of PES when intestinal contents from different age birds were used as the inoculum.

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.