Comparison of capsid gene sequences of turkey astrovirus-2 from poult-enteritis-syndrome-affected and apparently healthy turkeys.

This study was conducted to determine genetic variations in the capsid gene of turkey astrovirus-2 (TAstV-2) detected in apparently healthy and poult enteritis syndrome (PES)-affected turkeys. Capsid genes of astroviruses obtained from 30 PES-affected and 45 apparently healthy turkey flocks had sequence homologies of 73.4-100% and 72.4-100% at the nucleotide levels, respectively. The analysis of deduced amino acid sequences revealed one amino acid deletion at position 552 in 28 (93.3%) of 30 PES-affected cases. However, there were two deletions (at positions 551 and 552) in 31 (68.9%) of 45 TAstV-2 from apparently healthy flocks. The TAstV-2 (6.7%) from two PES-affected cases had two amino acid insertions each between positions 552 and 553, while TAstV-2 from 14 (31.1%) of 45 healthy flocks had two insertions at the same position. Phylogenetic analysis based on nucleotide sequences revealed that the astroviruses in this study were closely related to most of the previously published TAstV-2 isolates. The sequence homology of TAstV-2 in this study ranged from 70.4% to 99.4% at the nucleotide level with those of previously published TAstV-2 isolates. The variations at the amino acid level in the capsid gene suggest the possibility of the existence of different serotypes of turkey astrovirus. The close relationship of turkey astroviruses from apparently healthy flocks to those from PES-affected cases in capsid gene phylogeny necessitates further studies to compare complete capsid gene sequences from both types of flocks from different geographic areas for better understanding of TAstV circulating in turkeys.

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

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.

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.

Electron microscopic identification of viruses associated with poult enteritis in turkeys grown in California 1993-2003.

Poult enteritis (PE) is one of the most common diseases seen in young turkey flocks. Since 1993, more than 1800 cases of suspected PE have been submitted for examination by negative stain electron microscopy; this has involved more than 2400 individual results, because in many cases more than one virus was identified; at least 1500 individual results were positive for viruses. Viruses have been identified in poults as young as 3 days and up to 9 wk of age. The most commonly found viruses are rotavirus-like viruses and small round viruses ranging from 15 nm to 30 nm, either alone or in combination. Reovirus, birnavirus, and adenovirus have also been detected. There has been no evidence to suggest the presence of coronaviruses. This report summarizes our findings.

Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006.

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.

Detection of Turkey astrovirus in young poults affected with poult enteritis complex in Brazil.

Reverse transcription polymerase chain reaction (RT-PCR) of turkey astrovirus (TAstV) capsid and polymerase genes was applied to the bursa of Fabricius (BF), thymus (TH), spleen (SP) and cloacal swabs (CS) of young poults with "Poult enteritis complex" (PEC). The histological lesions included atrophy, lymphoid depletion, cellular infiltration and necrosis of the BF, TH and SP, respectively. The RT-PCR reactions were positive for the polymerase gene of TAstV-2 in all 100 CSs, 7 out of 10 of BFs and 10 out of 20 THs and SPs, respectively. Five out of 10 THs and SPs samples, considered to be negative by RT-PCR, were positive when specific primers designed for the TAstV-2 capsid gene were applied. This is the first description of turkey astrovirus infection presenting PEC in Latin America.

Isolation of a reovirus from poult enteritis and mortality syndrome and its pathogenicity in turkey poults.

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.

Interactions of poult enteritis and mortality syndrome-associated reovirus with various cell types in vitro.

An avian reovirus, ARV-CU98, has recently been isolated from poults experiencing poult enteritis and mortality syndrome (PEMS). To further understand ARV-CU98 and its role in PEMS, the current study investigates interactions of ARV-CU98 with various cell types in vitro. When macrophages, B cells, T cells, and liver cells of chicken or turkey origin were co-incubated with ARV-CU98, only cells of liver origin demonstrated cytopathic effects, the presence of viral antigen, and reduced metabolic activity over time. Furthermore, distinctive pockets of viral particles were evident in electron microscopic examination of a chicken hepatocellular carcinoma (LMH) cell line, but not in a chicken macrophage cell line (MQ-NCSU) co-incubated with virus. Additional evidence of viral replication in LMH, cells but not MQ-NCSU cells was demonstrated by the presence of two viral bands (43 and 145 kD size) in cell lysates from LMH cells exposed to ARV-CU98. Although not capable of being infected by ARV-CU98, MQ-NCSU cells do appear to be activated by the virus since IL-1 mRNA expression is increased in MQ-NCSU cells 2 h after addition of the virus. LMH cells exposed to the virus demonstrate a decrease in IL-1 mRNA expression by 8 to 10 h after addition of the virus, perhaps corresponding to the initiation of infection by the virus. In conclusion, this study demonstrates that ARV-CU98 actively infects and replicates in LMH cells, but not in lymphocytes or macrophages, suggesting that the liver may be a target and site of replication of ARV-CU98 in poults experiencing PEMS.

Astrovirus induces diarrhea in the absence of inflammation and cell death.

Astroviruses are a leading cause of infantile viral gastroenteritis worldwide. Very little is known about the mechanisms of astrovirus-induced diarrhea. One reason for this is the lack of a small-animal model. Recently, we isolated a novel strain of astrovirus (TAstV-2) from turkeys with the emerging infectious disease poult enteritis mortality syndrome. In the present studies, we demonstrate that TAstV-2 causes growth depression, decreased thymus size, and enteric infection in infected turkeys. Infectious TAstV-2 can be recovered from multiple tissues, including the blood, suggesting that there is a viremic stage during infection. In spite of the severe diarrhea, histopathologic changes in the intestine were mild and there was a surprising lack of inflammation. This may be due to the increased activation of the potent immunosuppressive cytokine transforming growth factor beta during astrovirus infection. These studies suggest that the turkey will be a useful small-animal model with which to study astrovirus pathogenesis and immunity.