Experimental bovine coronavirus in turkey poults and young chickens

The DB2 calf strain of bovine coronavirus (BCV) was used to inoculate 1-day-old specific-pathogen-free (SPF) turkey poults in three trials. In all trials, the birds developed clinical signs of enteritis at 48-72 hr postinoculation. Birds euthanatized at 3, 5, and 7 days postinoculation (DPI) had flaccid, pale intestines with watery contents, and the ceca were markedly enlarged with frothy contents. Coronavirus particles were detected by immune electron microscopy with BCV antibodies from the intestinal contents of birds killed at 3, 5, 7, and 12 DPI. Body weights of inoculated poults killed at 3, 5, and 7 DPI were significantly reduced as compared with controls. Hemagglutinating antibodies were detected in sera of convalescent birds at 12 DPI. However, experimental inoculation of 1-day-old SPF chicks in two trials with the same virus resulted in no clinical signs or macroscopic or microscopic lesions. No coronaviruses were detected from intestinal contents, and there were no significant differences in body weights of inoculated and noninoculated control chicks.     

Pathogenicity of turkey coronavirus in turkeys and chickens

We designed this study to compare the replication potential of turkey coronavirus (TCV) and its effect in chickens and turkeys and to study the effect of singleand combined infection of turkey poults with TCV and astrovirus. We studied the pathogenicity of TCV in experimentally inoculated turkey poults and chickens by observing the dinical signs and gross lesions. Two trials were conducted with 1-day-old and 4-wk-old specific-pathogen-free turkey poults and chickens. One-day-old turkey poults developed diarrhea at 48 hr postinoculation. Poults euthanatized at 3, 5, and 7 days postinoculation had flaccid, pale, and thin-walled intestines with watery contents. The 4-wk-old turkeys had no clinical signs or gross lesions. One-day-old and 4-wk-old chicks developed no clinical signs or gross lesions although the TCV was detected in gut contents of the birds throughout the experimental period (14 days). In another experiment, mean plasma D-xylose concentrations in 3-day-old turkey poults inoculated with TCV, turkey astrovirus, or a combination of both viruses were significantly lower than in the uninoculated controls.     

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。     

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.     

Intestinal cryptosporidiosis and reovirus isolation from bobwhite quail (Colinus virginianus) with enteritis

An acute enteric disease of young pen-raised bobwhite quails was studied. Affected quails had white, watery diarrhea accompanied by dehydration and subsequent death. Mortality from hatch to 17 days of age ranged from 30 to 45% in the three flocks examined. Small intestines were thin-walled and distended with fluid and gas. Microscopic lesions in the intestinal tract consisted of villus atrophy, villus fusion, and sloughing of cells at the tip of the villi in duodenum, jejunum, and ileum. Cryptosporidium sp. and reovirus were identified in affected quails.     

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.     

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.     

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.     

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.     

Infectious stunting and pancreatic fibrosis in broiler chickens in Saskatchewan

Two broiler flocks contained 0.5 and 3% small chickens. The small chickens were approximately one-third the size of their penmates, were very active, and retained much of their chick down. They had distended abdomens with full intestinal tracts, often containing undigested feed. In many, the pancreases were thin, white, and firm because of loss of exocrine tissue and replacement by fibrous tissue. Many had skeletal changes suggestive of rickets. In a survey of 48 broiler flocks for small birds and pancreatic lesions, five flocks had a noticeable incidence of small chickens, varying from less than 1% to a maximum of 2%. In 33 of the flocks surveyed, a low incidence of pancreatic lesions was found in birds at the processing plant. No correlation was found between a noticeable number of small birds at the farm and pancreatic lesions found at processing.     

Localization of astrovirus in experimentally infected turkeys as determined by in situ hybridization

Twenty-one 3-day-old turkey poults from British United Turkeys of America were orally inoculated with a recently characterized astrovirus, TAstV-2, isolated from turkeys with poult enteritis and mortality syndrome. At 1, 2, 3, 4, 5, 7, and 9 days postinfection (dpi), three inoculated birds were euthanatized, and tissues (intestines, spleen, bursa, and thymus) were collected immediately into 10% neutral buffered formalin. Inoculated birds were diarrheic by 3 dpi, and frothy feces persisted throughout the experimental period. Histologically, there was only slight evidence of enteric damage, which was characterized by mild epithelial necrosis, lamina propria infiltrates, minimal villus atrophy, and mild crypt hyperplasia. In situ hybridization, using a negative sense digoxigenin-labeled riboprobe to the capsid gene of TAstV-2, revealed viral RNA in intestinal epithelial cells at the basal margins of the villi, in distal small intestine, and in cecum at 2 dpi, with subsequent extension to epithelium of the large intestine and proximal small intestine (3-5 dpi). Minimal virus remained by 9 dpi.     

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.     

Identifying agent(s) associated with poult enteritis mortality syndrome: importance of the thymus

Poult enteritis mortality syndrome (PEMS), a highly infectious disease of young turkeys, causes serious financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, growth depression, and immunosuppression. Although many viruses, bacteria, and parasites are found in PEMS-infected birds, the inciting agent remains unknown. Experimentally, PEMS can be reproduced by exposing na?ve poults to the intestinal contents from infected birds. Previous reports suggest that extraintestinal tissues fail to reproduce the disease. Histopathologic examination of tissues from PEMS-infected poults suggested that the thymus exhibited the earliest signs of pathology. On the basis of these observations, we hypothesized that the thymus harbors an agent(s) involved in PEMS. In these studies, na?ve turkey poults were orally inoculated with a bacteria-free filtrate composed of either the intestines and feces or the thymus from PEMS-infected birds and were monitored for clinical signs of PEMS. Poults exposed to a filtrate composed solely of the thymus from PEMS-infected birds exhibited diarrhea, growth depression, mortality, pathology, and, most importantly, immunosuppression similar to poults exposed to the intestinal filtrate. The results of this study suggest that the thymus of infected birds harbors the agent(s) that can reproduce a PEMS-like disease in turkey poults.     

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.     

Phenotypic and genotypic characterization of Salmonella arizonae from an integrated turkey operation

Fifty cases submitted between 2000 and 2002 were selected for retrospective analysis to evaluate possible relationships between Salmonella arizonae isolated from breeder flocks, hatching eggs, and meat bird flocks belonging to a single turkey integrator. In all the meat bird cases selected for this study, arizonosis was the primary diagnosis. In birds under 1 month of age, clinical signs and pathologic changes were observed in older birds. The Salmonella arizonae isolates were analyzed by antibiotic resistance pattern and serotype and genotyped by pulsed-field gel electrophoresis (PFGE). Serotyping and PFGE yielded similar results, but the antibiotic resistance patterns did not correspond to either serotyping or PFGE typing. The presence of common pulsed-field patterns in breeder flocks, eggs, and meat bird flocks suggested that S. arizonae was being transmitted vertically from the breeder flock.     

Distribution of immunoglobulin-bearing cells in the gut-associated lymphoid tissues of the turkey: effect of oral treatment with intestinal microflora

One-day-old turkeys (Meleagris gallopavo) were orally inoculated with the intestinal contents of an adult turkey, and the intestinal tissues were studied by immunofluorescence for immunoglobulin (Ig)-bearing cells at 3, 7, 14, and 21 days of age. Microflora inoculation increased numbers of Ig-bearing cells in the gut-associated lymphoid tissues; the most uniform effects were observed at 3 days of age. As the birds grew older, this uniformity in response to the microflora inoculation was not evident in all the tissues. In the bursa of Fabricius and the large intestine of the inoculated birds, IgM-bearing cells were more numerous throughout the study period. Compared with noninoculated control turkeys, IgA-bearing cells in the cecal tonsils, and IgG- and IgA-bearing cells in the small intestine were increased at all age intervals.