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.     

Enteric viruses in diarrheic turkey poults

Thirty-three intestinal samples from 10-to-21-day-old diarrheic turkey poults were examined for the presence of enteric viruses by electron microscopy. Samples originated from 32 flocks in six commercial operations located in six states. Mortality in these flocks ranged from 3 to 15%, and birds from recovered flocks varied greatly in size. Rotavirus-like agents (RVLA) were the most common viruses associated with diarrhea outbreaks in the flocks examined, occurring in five out of six operations. Other viruses detected either singly or in combination, in order of prevalence, were astroviruses, reoviruses, rotaviruses, enteroviruses, and adenoviruses. With the exception of RVLA and rotaviruses, the other viruses were identified solely on the basis of morphology. Salmonellae were isolated from only one of the intestinal samples. By electron microscopy, RVLA were morphologically indistinguishable from rotaviruses, occurring as both 55-nm single-shelled and 70-nm double-shelled particles. However, immune electron microscopy was useful for antigenic differentiation of these two viruses. Turkey rotaviruses reacted with antisera to porcine and bovine rotaviruses, whereas turkey RVLA did not. Neither turkey rotaviruses nor RVLA reacted with antisera to porcine para-rotavirus or an antigenically distinct bovine rotavirus (bovine rotavirus-like agent). Similarly, convalescent anti-turkey RVLA serum (from recovered specific-pathogen-free poults) reacted with homologous virus but did not react with mammalian or avian rotaviruses or reoviruses. Further, RVLA were found to possess RNA electrophoretic migration patterns unlike those of conventional rotaviruses or reoviruses. This trait was used as an additional means of differentiating these viruses.     

Studies on orthoreoviruses isolated from young turkeys. I. Isolation and characterization

Four virus isolates were recovered from the intestinal tracts of turkey poults with diarrhea, abnormal feathering, stunted growth, and increased mortality. The isolates were clone-purified and characterized according to cytopathic effect (CPE) in chicken embryo kidney cells, size, morphology, physicochemical properties, and replicative cycle. The isolates were identified as members of the family Reoviridae, genus Orthoreovirus.     

Transmissible enteritis of turkeys: experimental inoculation studies with tissue-culture-adapted turkey and bovine coronaviruses.

Four Quebec isolates of turkey enteric coronaviruses (TCVs) and three isolates of bovine enteric coronaviruses (BCVs) were serially propagated in HRT-18 and compared for their pathogenicity in turkey embryos and turkey poults. By immunoelectron microscopy, hemagglutination-inhibition, and Western immunoblotting assays, tissue-culture-adapted Quebec TCV isolates were found to be closely related to the reference Minnesota strain of TCV and the Mebus strain of BCV. Genomic relationships between TCV isolates and the reference BCV strain were confirmed by hybridization assays with BCV-specific radiolabeled recombinant plasmids containing sequences of the N and M genes. Only TCV isolates could be propagated by inoculation in the amniotic cavity of chicken and turkey embryonating eggs, and induced clinical disease in turkey poults. Nevertheless, coronavirus particles or antigens were detected by electron microscopy or indirect enzyme-linked immunosorbent assay in the clarified intestinal contents of BCV-infected poults up to day 14 PI, and genomic viral RNA was detected by slot-blot hybridization using BCV cDNA probes.     

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.     

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.     

Attachment and infection to MA104 cells of avian rotaviruses require the presence of sialic acid on the cell surface

To determine the characters of receptors on target cells for avian rotaviruses, the receptors on MA104 cells for the pigeon rotavirus PO-13, the turkey rotaviruses Ty-1 and Ty-3, and the chicken rotavirus Ch-1 were analyzed. Pretreatment of MA104 cells with neuraminidase greatly reduced the infection by all of the four avian rotavirus strains. Binding of the cell-attachment protein, purified VP8 expressed in bacteria, of strain PO-13 to MA104 cells was also inhibited by pretreatment of cells with neuraminidase. These findings suggest that avian rotaviruses primarily utilize sialic acid-containing molecules as receptors on MA 104 cells.     

Reciprocal competitive exclusion of salmonella and Escherichia coli by native intestinal microflora of the chicken and turkey

Both the native intestinal microflora of chickens that protected chicks against salmonellae and Escherichia coli and native turkey intestinal microflora were evaluated for their reciprocal protective capacity in both species against Salmonella typhimurium and a pathogenic strain of E. coli. Nalidixic-acid-resistant forms of the S. typhimurium and E. coli strains were used in seeder-bird and individual-bird challenge tests. Reciprocal protection was provided by native chicken and turkey intestinal microflora in chicks and poults against S. typhimurium and the pathogenic strain of E. coli. The chicken and turkey microflora appeared to be equally effective in protecting the two species from S. typhimurium, but protection against E. coli was somewhat greater in the chicken than in the turkey.     

Pathogenicity of Campylobacter jejuni for turkeys and chickens.

A Campylobacter jejuni isolate obtained from a turkey liver, designated C101, and a C. jejuni isolate obtained from the feces of a chicken, designated C111, were used to inoculate their respective hosts. Isolate C101 depressed weight gain by 20% when inoculated into newly hatched poults or 4-day-old poults. It also caused death, hepatic necrosis, and generalized hemorrhages in turkey embryos. The chicken-derived isolate, C111, did not reduce weight gain in newly hatched chicks, but it did induce mortality in chicken embryos. The supernatant of the cultures of both C. jejuni isolates also caused mortality in embryos.     

The effect of infectious bursal disease virus on the immune system of turkeys

In 1979 it was reported that an infectious bursal disease virus (IBDV) isolated from a case of respiratory disease of turkeys differed antigenically from the chicken isolates of this virus. We injected turkey poults with the turkey-originating TY89 and chicken-originating BD/6 isolates of IBDV and studied their effects on antibody production to the virus, serum immunoglobulin G (IgG), antibody response to sheep erythrocytes, in vitro response of peripheral blood lymphocytes to mitogens, and microscopic structure of the bursa of Fabricius. The chicken isolate BD/6 caused a significant decrease in the response to sheep erythrocytes, lower serum IgG, transient decrease in the response of lymphocytes to PHA, and mild microscopic lesions in the bursa of Fabricius. The turkey isolate TY89, however, caused no obvious damage to the immune system of the infected poults. We suggest that a partial and transient functional disorder of the immune system of poults can occur after infection with IBDV originating from chickens, even if the poults exhibited no clinical signs.     

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.     

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.     

火鸡冠状病毒的分离和初步鉴定

2003年国内某火鸡场发生了一种以侵害15～25日龄雏火鸡为主的急性传染病，主要表现为腹泻，十二指肠、直肠充血和出血，盲肠肿大，肠道内充满黄绿色内容物，死亡率约为10％～20％。取病死火鸡肝、脾、肠匀浆，取上清液通过尿囊腔接种15日龄SPF鸡胚。连续传代至第5代，收集接种后72h内死亡或存活鸡胚的卵黄和肠道，用于病毒分离和提纯。试验中发现该病毒能凝集兔红细胞，不能凝集鸡红细胞。经电镜观察，在病毒提纯液中发现有圆形或椭圆形、带花冠状纤突的病毒粒子，初步诊断为火鸡冠状病毒感染。进而设计针对火鸡冠状病毒S2基因引物，进行RT-PCR扩增，结果扩增出预期大小的片段。运用所分离病毒进行动物回归试验，感染火鸡出现与自然病例一致的临床症状和病理变化，并能从发病火鸡分离出该病毒。以上结果表明所分离的病毒为火鸡冠状病毒。此病毒的分离在国内尚属首例。     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

Pathogenicity for chickens of a reovirus isolated from turkeys

A viral agent that was isolated from livers of commercial turkey poults that died at approximately two weeks of age was characterized as a reovirus. Experimental infection of day-old chickens with this reovirus isolate resulted in the development of tenosynovitis, hepatitis, and myocarditis. In vitro neutralization of the turkey reovirus isolate by antiserum against chicken reovirus correlated with in vivo protection of maternally immune chickens from day-old oral challenge with the turkey reovirus isolate.     

PREVENTION OF SALMONELLA TYPHIMURIUM INFECTION IN POULTRY BY PRETREATMENT OF CHICKENS AND POULTS WITH INTESTINAL EXTRACTS

Day-old chickens or turkey poults when pretreated with an oral dose of intestinal fluid froma healthy adult bird, were considerably more resistant to the subsequent establishment of Salmonella typhimurium in their intestinal tract than were non-treated chickens or turkey poults. Caecal fluid was more effective as a pretreatment than were washings taken from other parts of the gastro-intestinal tract of a healthy adult bird. This increased resistance of pretreated chickens or poults is thought to result from the rapid establishment of a conventional indigenous microflora which inhabits establishment and growth by the invading enteric pathogen.     

Isolation, identification and characterization of group A rotavirus from a chicken: the inner capsid protein sequence shows only a distant phylogenetic relationship to most other avian group A rotaviruses

Rotavirus particles were identified in the intestinal content of a 35-day-old stunted chicken. The virus was isolated, RNA pattern was analysed and the viral genome segment 6 was sequenced. In particular, the sequence data showed a very close similarity to the chicken rotavirus isolate Ch-1 (99.2% amino acid homology), this is distantly related to all known avian rotaviruses and supports the existence of different VP6 types amongst avian group A rotaviruses.     

A small round virus associated with enteritis in turkey poults

In a natural outbreak of enteric disease in turkey poults, Salmonella, group D rotavirus, astrovirus, and a small (18-24 nm) round virus were detected in the gut contents. Except for the small virus, the pathogenic potential of the other agents is recognized. In experiments, the small round virus was shown to be transmissible and pathogenic in specific-pathogen-free turkey poults.     

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.