J. Michael Day

Enteric Viruses Detected by Molecular Methods in Commercial Chicken and Turkey Flocks in the United States Between 2005 and 2006

Abstract Intestinal samples collected from 43 commercial broiler and 33 commercial turkey flocks from all regions of the United States during 2005 and 2006 were examined for the presence of astrovirus, rotavirus, reovirus, and coronavirus by reverse transcription-polymerase chain reaction (PCR), and for the presence of groups 1 and 2 adenovirus by PCR. Phylogenetic analysis was performed to further characterize the viruses and to evaluate species association and geographic patterns. Astroviruses were identified in samples from 86% of the chicken flocks and from 100% of the turkey flocks. Both chicken astrovirus and avian nephritis virus (ANV) were identified in chicken samples, and often both viruses were detected in the same flock. Turkey astrovirus type-2 and turkey astrovirus type-1 were found in 100% and 15.4% of the turkey flocks, respectively. In addition, 12.5% of turkey flocks were positive for ANV. Rotaviruses were present in 46.5% of the chicken flocks tested and in 69.7% of the turkey flocks tested. Based upon the rotavirus NSP4 gene sequence, the chicken and turkey origin rotaviruses assorted in a species-specific manner. The turkey origin rotaviruses also assorted based upon geographical location. Reoviruses were identified in 62.8% and 45.5% of chicken and turkey flocks, respectively. Based on the reovirus S4 gene segment, the chicken and turkey origin viruses assorted separately, and they were distinct from all previously reported avian reoviruses. Coronaviruses were detected in the intestinal contents of chickens, but not turkeys. Adenoviruses were not detected in any chicken or turkeys flocks. Of the 76 total chicken and turkey flocks tested, only three chicken flocks were negative for all viruses targeted by this study. Most flocks were positive for two or more of the viruses, and overall no clear pattern of virus geographic distribution was evident. This study provides updated enteric virus prevalence data for the United States using molecular methods, and it reinforces that enteric viruses are widespread in poultry throughout the United States, although the clinical importance of most of these viruses remains unclear.

PERIODIC MONITORING OF COMMERCIAL TURKEYS FOR ENTERIC VIRUSES INDICATES CONTINUOUS PRESENCE OF ASTROVIRUS AND ROTAVIRUS ON THE FARMS

Abstract A longitudinal survey to detect enteric viruses in intestinal contents collected from turkeys in eight commercial operations and one research facility was performed using molecular detection methods. Intestinal contents were collected from turkeys prior to placement, with each flock resampled at 2, 4, 6, 8, 10, and 12 wk of age. The samples were screened for astrovirus, rotavirus, reovirus, and turkey coronavirus (TCoV) by a reverse transcriptase and polymerase chain reaction (RT-PCR), and for groups 1 and 2 adenovirus by PCR. Rotavirus was the only virus detected prior to placement (7 of 16 samples examined). All of the commercial flocks were positive for rotavirus and astrovirus from 2 until 6 wk of age, and most were intermittently positive until 12 wk of age, when the birds were processed. Of the 96 samples collected from birds on the farms, 89.5% were positive for astrovirus, and 67.7% were positive for rotavirus. All flocks were negative for TCoV, reovirus, and group 1 adenovirus at all time points, and positive for group 2 adenovirus (hemorrhagic enteritis virus) at 6 wk of age. All the flocks monitored were considered healthy or normal by field personnel. Turkeys placed on research facilities that had been empty for months and thoroughly cleaned had higher body weights and lower feed conversion rates at 5 wk of age when compared to turkeys placed on commercial farms. Intestinal samples collected at 1, 2, and 3 wk of age from these turkeys were free of enteric viruses. This report demonstrates that astroviruses and rotaviruses may be present within a turkey flock through the life of the flock. Comparison of infected birds with one group of turkeys that were negative for enteric viruses by the methods used here suggests that astrovirus and/or rotavirus may affect production. The full impact on flock performance needs to be further determined.

A Multiplex RT-PCR Test for the Differential Identification of Turkey Astrovirus Type 1, Turkey Astrovirus Type 2, Chicken Astrovirus, Avian Nephritis Virus, and Avian Rotavirus

Abstract Recent studies have revealed the presence of astroviruses and rotavirus in numerous poorly performing and healthy chicken and turkey flocks in the United States. The phylogenetic analysis of the sequence data produced during these studies has identified four groups of avian astroviruses circulating in the United States: turkey astrovirus types 1 and 2 (TAstV-1 and TAstV-2), avian nephritis virus (ANV), and a chicken-origin astrovirus (CAstV). As the molecular epidemiology of poultry enteric disease is poorly understood, the development of updated diagnostic assays is crucial to the continued surveillance and management of enteric disease in affected as well as healthy flocks. This report details the development of a multiplex reverse transcriptase–polymerase chain reaction (RT-PCR) assay specific for astroviruses and avian rotavirus in turkey-origin and chicken-origin samples. The assay consists of two multiplex tests, one for turkey-origin samples and one for chicken-origin samples. The turkey sample test differentially identifies TAstV-1, TAstV-2, ANV, and avian rotavirus. The test for chicken-origin samples differentially identifies CAstV, ANV, and avian rotavirus. Assay sensitivity varied by target sequence between approximately 10 copies for avian rotavirus alone and approximately 2 × 106 copies for TAstV-2 in the presence of a heterologous competitor RNA sequence. Each test was shown to be specific for the intended target by testing for cross-reaction with other common avian enteric viruses. The specificity was further shown by testing 109 chicken specimens and 32 turkey specimens from commercial flocks with the appropriate test and sequencing the RT-PCR amplicons to confirm amplification of the correct target.

Metagenomic analysis of the turkey gut RNA virus community

Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses.

The diversity of the orthoreoviruses: Molecular taxonomy and phylogentic divides

The family Reoviridae is a diverse group of viruses with double-stranded RNA genomes contained within icosahedral, non-enveloped, double-layered protein capsids. Within the Reoviridae, the Orthoreovirus genus includes viruses that infect reptiles, birds and mammals (including humans). Recent sequencing efforts have produced a great deal of new molecular data for the fusogenic orthoreoviruses, a group of reoviruses that induce cell-cell fusion during an infection. This new data has allowed a fresh look at the phylogenetic relationships among the members of the Orthoreovirus genus, and has provided insight into the evolution of orthoreovirus species and species groups. This review mainly focuses on the molecular taxonomy of the fusogenic orthoreoviruses, and aims to provide insight into their relationships with the non-fusogenic orthoreoviruses and other selected Reoviridae genera.

Development of a Polymerase Chain Reaction Procedure for Detection of Chicken and Turkey Parvoviruses

Abstract Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A polymerase chain reaction (PCR) assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detecting parvoviruses in experimentally infected chickens. In a nationwide survey, a total of 138 field enteric samples from poultry flocks were tested by PCR for parvovirus presence. Of the tested chicken samples that were collected in 54 farms, 77% showed the presence of parvovirus, while 78% of the turkey samples that were received from 29 farms were parvovirus positive. For the first time, our data clearly demonstrate that parvoviruses are widely distributed in commercial poultry flocks in the United States. The high prevalence of parvovirus infection in birds from enteric disease-affected flocks suggests a potential role of these viruses in the etiology of enteric disease of poultry. Phylogenetic analyses comparing NS gene segments showed that most of the chicken and turkey parvovirus isolates formed separate phylogenetic groups. These findings suggest that the chicken and turkey parvoviruses might have diverged from a common ancestor and have subsequently undergone host-specific adaptation.

Determination and analysis of the full-length chicken parvovirus genome

Abstract Viral enteric disease in poultry is an ongoing problem in many parts of the world. Many enteric viruses have been identified in turkeys and chickens, including avian astroviruses, rotaviruses, reoviruses, and coronaviruses. Through the application of a molecular screening method targeting particle-associated nucleic acid (PAN), we recently described the detection and partial characterization of a novel enteric parvovirus in chickens. Subsequent surveys of intestinal homogenates from turkeys and chickens in the United States revealed widespread occurrence of parvovirus in poultry. Here we report the first full genome sequence of a novel chicken parvovirus, ChPV ABU-P1. ChPV ABU-P1 genome organization, predicted amino acid sequence, and phylogenetic relationships with other described parvoviruses are discussed.

Pathogenesis of type 2 turkey astroviruses with variant capsid genes in 2-day-old specific pathogen free poults.

The pathogenicity of three different type 2 turkey astroviruses (TAstV-2) was studied in specific pathogen free turkeys. These viruses differ based on sequence analysis of the capsid gene. Poults were inoculated at 2 days of age and examined during 14 days for clinical signs and virus shedding. All inoculated poults presented signs of enteric disease including diarrhoea and growth depression. Virus presence and shedding was detected by real-time reverse transcriptase-polymerase chain reaction from intestinal contents and cloacal swabs collected at 3, 7 and 14 days post-inoculation. Viraemia was also confirmed by this method. Common lesions observed at necropsy were dehydration; distended intestines filled with watery contents and undigested feed, and dilated caeca with foamy contents. Microscopic lesions present in the intestines consisted of mild crypt hyperplasia, villous atrophy and lymphocytic infiltration, and were most common in the jejunum. Presence of the viruses was demonstrated by immunohistochemistry and by in situ hybridization in both villi and crypt enterocytes in the jejunum and, less frequently, the duodenum, ileum and caeca. Mild lesions consisting mainly of lymphocytic infiltration were also observed in other organs including the pancreas, liver, spleen and kidneys. Mild to moderate bursal atrophy occurred in all TAstV-2-infected poults examined; however, no specific viral staining was observed in this organ or any other tissues examined apart from the intestines. In conclusion, TAstV-2 viruses with variant capsids produce a similar enteric disease in young turkeys and may also affect the immune system of the birds by causing bursal lymphoid depletion.

Chicken Parvovirus–Induced Runting-Stunting Syndrome in Young Broilers

SUMMARY. Previously we identified a novel parvovirus from enteric contents of chickens that were affected by enteric diseases. Comparative sequence analysis showed that the chicken parvovirus (ChPV) represented a new member in the Parvoviridae family. Here, we describe some of the pathogenic characteristics of ChPV in young broilers. Following experimental infection, 2-day-old broiler chickens showed characteristic signs of enteric disease. Runting-stunting syndrome (RSS) was observed in four of five experimental groups with significant growth retardation between 7 and 28 days postinoculation (DPI). Viral growth in small intestine and shedding was detected at early times postinoculation, which was followed by viremia and generalization of infection. ChPV could be detected in most of the major tissues for 3 to 4 wk postinoculation. Immunohistochemistry staining revealed parvovirus-positive cells in the duodenum of inoculated birds at 7 and 14 DPI. Our data indicate that ChPV alone induces RSS in broilers and is important determinant in the complex etiology of enteric diseases of poultry. RESUMEN. Nota de Investigación—Síndrome de mala absorción en pollos de engorde jóvenes inducido por un parvovirus de los pollos. Anteriormente se identificó un parvovirus nuevo de muestras de contenidos intestinales de pollos que estaban mostrando trastornos entéricos. El análisis comparativo de las secuencias mostró que el parvovirus de pollo (ChPV) representa a un nuevo miembro de la familia de parvovirus. En este trabajo, se describen algunas de las características patogénicas de este parvovirus en pollos jóvenes. Después de la infección experimental, pollos de engorde de dos días de edad mostraron signos característicos de enfermedad entérica. El síndrome de mala absorción se observó en cuatro de los cinco grupos experimentales con retraso en el crecimiento significativo después de la inoculación entre los siete y 28 días después de la inoculación. Se detectaron replicación y eliminación viral en el intestino delgado en etapas tempranas después de la inoculación, que fueron seguidos por viremia y generalización de la infección. El parvovirus de los pollos pudo ser detectado en la mayoría de los tejidos principales de tres a cuatro semanas después de la inoculación. La tinción por inmunohistoquímica reveló células positivas a la presencia de parvovirus en el duodeno de aves inoculadas de siete a catorce días después de la inoculación. Nuestros datos indican que el parvovirus de los pollos solo induce el síndrome de mala absorción en pollos de engorda y es un determinante importante en la etiología compleja de las enfermedades entéricas de las aves de corral.

Recent Progress in the Characterization of Avian Enteric Viruses

SUMMARY Despite the importance of the poultry gut, remarkably little is known about the complex gut microbial community. Enteric disease syndromes such as runting-stunting syndrome in broiler chickens and poult enteritis complex in young turkeys are difficult to characterize and reproduce in the laboratory. A great deal of work has been done to characterize the bacterial population in the poultry gut, leading to useful performance-based interventions such as direct-fed microbial preparations. Advances in the application of rapid molecular diagnostics and the advent of the next generation of nucleic acid sequencing have allowed researchers to begin to decipher the microbial community in complex environmental samples. Researchers have made great strides recently in placing names to some of the unknown and undescribed small viruses in the poultry gut such as parvoviruses, picornaviruses, picobirnavirus, and calicivirus. Investigation into the novel avian astroviruses continues, and recent progress has been made in the molecular characterization of the avian rotaviruses. This review will focus on the recent advances that have been made in the discovery, description, and characterization of the multitude of viruses that reside in the poultry gut. RESUMEN Estudio Recapitulativo—Avances recientes en la caracterización de los virus entéricos aviares. A pesar de la importancia del tracto intestinal en las aves, se conoce muy poco acerca de la compleja comunidad microbiana intestinal. Los síndromes de enfermedades entéricas, tales como el síndrome de mala absorción y el complejo de enteritis de los pavipollos, son difíciles de caracterizar y reproducir en el laboratorio. Una gran parte de los esfuerzos se han enfocado para caracterizar la población bacteriana en el intestino de las aves comerciales, lo que ha conducido a la aplicación de prácticas útiles basadas en el desempeño, tales como la alimentación directa de preparaciones microbianas. Los avances en la aplicación de métodos diagnósticos moleculares rápidos y el advenimiento de la secuenciación de ácidos nucleicos de nueva generación han permitido a los investigadores el comenzar a descifrar la comunidad microbiana en muestras ambientales complejas. Los investigadores han hecho grandes avances recientemente en asignar los nombres de algunos de los virus pequeños desconocidos y que no habían sido descritos en el intestino de las aves comerciales, tales como parvovirus, picornavirus, picobirnavirus, y calicivirus. Las investigaciones sobre los nuevos astrovirus aviares continúa, y se han logrado avances recientes en la caracterización molecular de los rotavirus aviares. Esta revisión se centrará en los últimos avances sobre el descubrimiento, la descripción y caracterización de la multitud de virus que se encuentran en el intestino de las aves comerciales.