Viral agents associated with outbreaks of diarrhea in turkey flocks in Quebec.

The relative importance of various enteric viruses associated with diarrhea of turkey poults was investigated by an evaluation of specimens received since 1982. Specimens originated from one to eight week old turkey poults, with mild to severe diarrhea, from 114 flocks in 42 commercial operations located in southern Quebec. The acute phase of enteritis occurred usually in poults between two and four weeks of age. Clarified intestinal contents were examined by direct electron microscopy and enzyme immunoassays. Enzyme-linked immunosorbent assays were performed with antisera to bovine rotavirus group antigen, avian reovirus types 1 to 5, and the prototype strain of the turkey enteric coronavirus. The presence of viruses could be demonstrated by electron microscopy in 55.3% of the specimens, and at least five different viruses were incriminated either alone or in combination. The coronavirus was by far the most common enteric virus with a prevalence of 47.5%. By enzyme-linked immunosorbent assay, rotavirus, reovirus and turkey coronavirus were detected in 14.5%, 18.1% and 61.4% of the specimens, respectively. By electron microscopy, 56.6% of these cases were positive for at least one virus.     

A survey of enteric viruses of turkey poults

Intestinal samples from 91 turkey flocks between 1 day and 5 weeks of age were examined for enteric viruses using electron microscopy and electropherotyping. These flocks originated from eight operations in six states. Individual flocks were sampled only once. At the time of sampling, 31 flocks were considered normal/healthy and 60 were considered to have enteric disease. The most frequently identified viruses from diseased flocks were astroviruses (78%) and rotavirus-like viruses (RVLVs) (67%). Far less frequent were rotaviruses (22%), atypical rotaviruses (12%), enteroviruses (5%), and reoviruses (2%). Only 10% of the samples from diseased flocks were negative, but 48% of the samples from normal/healthy flocks were negative. Astroviruses and RVLVs were far less frequent in normal/healthy flocks than in diseased flocks, but rotaviruses were identified slightly more often. No viruses were detected from flocks sampled within the first few days of life. Astrovirus infections seemed to occur at an earlier age than other virus infections. Seldom was only one type of virus identified. Astrovirus + RVLV was the most frequently identified combination in diseased flocks.     

Isolation and serial propagation of turkey rotaviruses in a fetal rhesus monkey kidney (MA104) cell line

Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.     

Isolation and characterization of avian rotaviruses

Rotaviruses were isolated from intestinal contents obtained from flocks of turkey poults and pheasant poults with diarrhea and from different age groups of chickens showing various signs of intestinal disorders. The incorporation of 5 micrograms trypsin/ml in the inoculum and medium was essential for virus isolation in chicken kidney cells. All isolates were identified as rotaviruses by fluorescent-antibody technique using a National Institutes of Health reference rotavirus antiserum against human rotavirus strain "D," Type 2. Negative-staining and transmission electron microscopy showed the presence of rotavirus particles. Furthermore, polyacrylamide gel electrophoresis pattern of viral RNA segments of our isolates confirmed that they are rotaviruses. Seven-day-old turkey poults could be infected with turkey and chicken rotavirus isolates; in contrast, chicks of the same age were refractory.     

Astrovirus: a cause of an enteric disease in turkey poults

Virus particles of 30 nm diameter and star-shaped morphology were detected in intestinal contents of turkey poults and were identified as astroviruses. Seventy-six intestinal samples from 65 commercial turkey flocks between 6 and 35 days of age were evaluated for the presence of astroviruses by immune electron microscopy. Astroviruses were frequently detected in intestinal samples from poults that had enteritis and diarrhea of undetermined etiology. Astroviruses were geographically widespread and were present in poults from all six operations evaluated. Astroviruses were inoculated into specific-pathogen-free poults. Changes observed in the gastrointestinal tract were: dilatated ceca containing yellowish frothy contents, gaseous fluid in the intestinal tract, and loss of tone of the intestinal tract (gut thinness). Poults experimentally inoculated with astrovirus gained significantly less body weight and absorbed significantly less D-xylose than uninoculated controls.     

Detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses

A double-antibody ELISA for the detection of coronaviruses in intestinal contents from turkey poults with diarrhea was developed. Antibodies were raised in rabbits and guinea pigs against a Minnesota isolate of turkey enteric coronavirus (TCV) propagated in embryonating turkey eggs and were purified by density-gradient centrifugation. The specificity of antisera was confirmed by hemagglutination-inhibition and immunoelectron microscopy. Absorption of anti-TCV hyperimmune sera with egg extracts or egg ovalbumin and the use of different dilution and blocking buffers influenced the sensitivity and specificity of the ELISA. Reciprocal cross-reactivity was detected among turkey, chicken, bovine, and murine coronaviruses. Antisera to the transmissible gastroenteritis virus of swine, the rabbit enteric coronavirus, or the human coronavirus strain 299E failed to react with TCV. The TCV cross-reacted only moderately with the avian infectious bronchitis virus and the hemagglutinating encephalomyelitis virus of swine. Investigations with samples from 47 commercial turkey flocks in Quebec with episodes of transmissible enteritis revealed that the ELISA was more sensitive than was electron microscopy for detection of TCV.     

Demonstration of rotavirus and rotavirus-like virus in the intestinal contents of diarrheic pheasant chicks

Intestinal content samples from four flocks of pheasant chicks experiencing diarrhea and increased mortality were evaluated using immune electron microscopy and genome electropherotyping techniques. Enteric viruses serologically and electropherotypically indistinguishable from turkey rotaviruses and turkey rotavirus-like viruses (RVLVs) were detected. Rotavirus-only infections, RVLV-only infections, and simultaneous rotavirus and RVLV infections were demonstrated in these flocks.     

Association of Reoviridae particles in an enteric syndrome of poults observed in turkey flocks during 1988

An enteric syndrome of turkey poults, characterized by enteritis, crop mycosis, intestinal changes (pale, thin-walled ballooning with watery contents), and rickets, occurred during 1988 in 74 turkey flocks from different farms belonging to 9 California turkey growers. The flocks ranged in size from 9,000 to 120,000 birds. Pools of intestine sections from 618 birds, representing 78 field cases, were examined. Histopathological examination of the intestines showed a mild to severe atrophy with a reduced depth of crypts, which was more prominent in the distal part of the small intestine. Viral isolation attempts with primary cell cultures of chicken embryo kidney cells were negative. Examination by electron microscopy of negatively stained intestinal specimens revealed the presence of Reoviridae particles of 58.8 to 80 nm in diameter. Enzyme-linked immunosorbent assay results on the intestinal pools for mammalian and group A avian rotaviruses were negative. A statistically significant relationship was found for the presence of Reoviridae particles in the intestines of 10-21-day-old birds. Of the 7 most common pathological conditions analyzed, 2, rickets and intestinal changes (thin-walled ballooning intestine with watery contents), showed a statistically significant association with the presence of Reoviridae particles.     

Characterization of turkey coronavirus from turkey poults with acute enteritis.

The present study was to characterize turkey coronavirus associated with turkey poult enteritis and mortality. Intestinal contents or intestines from affected turkey poults and inoculated turkey embryos contained coronaviruses as revealed by electron microscopy or were positive for turkey coronavirus by immunofluorescent antibody assay. Sucrose density gradient ultracentrifugation of the virus-containing intestinal homogenate yielded two opalescent bands corresponding to the buoyant densities of 1.14-1.15 and 1.18-1.20 g/ml, respectively. Coronaviral particles from intestinal contents or the sucrose density gradient preparation were mainly spherical in shape and had envelope and central depression. They were surrounded by a fringe of regularly spaced petal-shaped projections attached to the particles by a short stalk. Purified viruses hemagglutinated rabbit erythrocytes with a titer of 16. Major protein bands of purified viruses analyzed by SDS-PAGE were located at 200, 100-110, 50-60, and 30-35 kDa. The patterns of protein bands were consistent with those of Minnesota or Quebec turkey coronavirus isolates. A 568 bp nucleotide fragment of turkey coronavirus spike protein gene was amplified from RNA of inoculated turkey embryo intestine or purified virus. Sequence analysis of the 568 bp PCR product revealed high degree of identity with the corresponding spike protein gene sequence of human and bovine coronaviruses. The results indicated that turkey coronavirus was associated with turkey poults with acute enteritis.     

Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.     

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.     

Observations on Alcaligenes faecalis infection in turkeys

Experiments were initiated to study the pathogenicity of 5 Alcaligenes faecalis isolates in specific-pathogen-free poults. The isolates were recovered from commercial flocks suffering from a respiratory disease. There were no differences between cultural or biochemical characteristics of the isolates, but differences in antibiotic sensitivity were detected. All 5 isolates were capable of initiating a respiratory disease in poults similar to that seen in the early stages of turkey coryza. The infection, clinical signs, and lesions were limited to the upper part of the respiratory tract, but there were substantial differences in the severity of disease initiated by different isolates. There were also differences in the persistence of infection in the host. Secondary infections in the tracheas and sinuses were higher in poults infected with A. faecalis. The disease observed in the experimentally infected birds was milder than in 4 naturally infected flocks that also had complicating Escherichia coli infections. There was no evidence of infection with infectious bursal disease virus in 4 naturally occurring outbreaks in Ohio. It is proposed that the term turkey coryza be used to describe the disease initiated by A. faecalis.     

Coronaviruses associated with outbreaks of transmissible enteritis of turkeys in Quebec: hemagglutination properties and cell cultivation

Coronaviruses were observed by electron microscopy in the intestinal contents of turkeys in Quebec flocks where repeated outbreaks of enteritis occurred. Three isolates could be serially propagated in turkey embryos inoculated by the amniotic route with clarified intestinal contents. Purification and concentration of viral particles contained in intestinal contents of infected embryos were achieved by precipitation with polyethylene glycol and ultracentrifugation on sucrose density gradients. Three particle types were demonstrated: intact virions with a density of 1.18 to 1.20 g/ml and incomplete particles with densities of 1.14 and 1.24 g/ml. Hemagglutination of rabbit and guinea pig erythrocytes was demonstrated with the intact viral particles; the hemagglutinin was not dependent on incubation temperature. All the isolates were antigenically related, as shown by hemagglutination-inhibition. The turkey coronaviruses did not cross-react with antisera against coronaviruses of avian infectious bronchitis, porcine transmissible enteritis, bovine neonatal calf diarrhea, or mouse hepatitis. One of the Quebec isolates was shown to induce syncytia formation on its third passage in primary chicken-embryo kidney cell cultures. Electron-microscopic examination of infected cell-culture fluids revealed characteristics coronavirus particles identical to those found in intestinal contents of infected turkeys.     

Alterations in levels of various host antioxidant factors in turkey knockdown syndrome

Five components (selenium, glutathione peroxidase, copper, superoxide dismutase, and vitamin E) of the antioxidant system of turkey poults were examined to determine if they play any role in the knockdown (KD) syndrome. All flocks were provided with feed formulated to contain monensin at 54-60 g/ton. Flock data were analyzed as a case-control study with three treatment groups (KD-affected and unaffected turkey poults from a KD flock and poults from an unaffected flock [control]). Affected turkey poults had lower (P < 0.001) serum vitamin E levels compared with unaffected poults from KD flocks or poults from unaffected flocks. No significant differences were observed for the other parameters evaluated but there was a trend towards lower copper and superoxide dismutase values in affected birds. It appears that serum vitamin E concentrations in turkey poults may play a significant role in susceptibility to or protection against KD syndrome. Other components of the antioxidant system may also be involved, and complex interactions among several body systems may be critical.     

Comparison of electron microscopy and polyacrylamide gel electrophoresis in the diagnosis of avian reovirus and rotavirus infections.

Electron microscopy (EM) and genome electropherotyping by polyacrylamide gel electrophoresis (PAGE) for the detection of avian rotaviruses and reoviruses in intestinal specimens and cell cultures were compared. Fifty-eight field samples of intestine with intestinal contents, referred to as direct specimens, from turkey and chicken flocks located in different regions of California and submitted during 1989 for virus isolation were randomly selected as test samples. Also, 38 field intestinal specimens with suspected viral infection that had been passaged three times in primary chicken embryo kidney (CEK) cell cultures were used in their third passage. The percentage of agreement and the Kappa statistic of positive and negative results between these two tests were calculated. In the comparison, EM was considered the standard test. By statistical analysis, an agreement of 87% was observed in cell-culture samples analyzed by the two virus-detection methods, as contrasted with an agreement of 72% for direct specimens. The analysis of the number of segments and band migration profiles of reference and field virus strains indicated that only reoviruses replicated in CEK cell cultures and mainly rotaviruses were detected by both tests in direct specimens. The Kappa statistic analysis indicated substantial agreement (0.69) between the two tests for CEK samples, with moderate agreement (0.45) for the direct specimens examined.     

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.