Antigenicity of two turkey astrovirus isolates

Astroviruses are positive-sense single-stranded RNA viruses. These viruses cause gastroenteritis in humans and in a variety of animal species, including turkey poults. Only human astroviruses are well characterized antigenically. In the current study, two turkey astrovirus isolates, TAstV1987 and TAstV2001, were antigenically compared using cross-neutralization tests in turkey embryos, as well as cross-reactivity of the two isolates by an enzyme-linked immunosorbent assay (ELISA). The antigenic relatedness values (R) were calculated using the Archetti and Horsfall formula. The R value based on the cross-neutralization tests was 0.56%, which indicates that TAstV1987 and TAstV2001 belong to different serotypes; the R value of the two viruses based on ELISA was 70.7%, which suggests these two viruses share common antigen(s).     

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.     

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.     

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.     

Molecular characterization of the capsid gene of two serotypes of turkey astroviruses

Astrovirus infections mainly cause acute gastroenteritis in children and young animals. Human astroviruses are well characterized antigenically and genetically. However, information on turkey astroviruses is limited. We isolated two astroviruses (TAstV1987 and TAstV2001) from turkeys and classified them as two different serotypes using a virus neutralization test. To elucidate the differences between these two isolates at the molecular level, further genetic characterization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were carried out. The sequences of the complete capsid protein gene of these two isolates were obtained by cloning and sequencing. The percentage nucleotide and predicted amino acid identities for these two sequences along with those of 16 other capsid protein gene sequences from human and animal astroviruses retrieved from GenBank were calculated using MegAlign. The results showed that TAstV1987 and TAstV2001 had 73.3% nucleotide and 82.8% amino acid identities, respectively. An unrooted Neighbor-joining phylogenetic tree of these sequences was generated using MEGA 3 software with 1000 bootstrap replicates. The results of evolutionary analysis showed that TAstV1987 was closely related genetically to another virus, designated TAstV-2, whereas TAstV2001 was not as close to TAstV-2 as TAstV1987. The analysis of the capsid proteins of the two viruses by SDS-PAGE revealed that they had different band patterns, indicating that their capsid proteins consisted of different viral proteins. The findings in this study revealed the molecular differences in the capsid protein gene of TAstV1987 and TAstV2001, which may provide the molecular basis of the antigenic differences between these two serotypes of turkey astroviruses.     

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.     

Field studies with convalescent serum and infectious bursal disease vaccine to control turkey coryza

Convalescent serum given to 1-day-old poults delayed clinical signs of turkey coryza by several days and reduced mortality on infected farms. Turkey breeders immunized with cell-culture-adapted infectious bursal disease virus (IBDV) or turkey infectious bursal disease virus (TIBDV) had a marked increase in virus-neutralization (VN) antibody titers. The VN antibody titer was significantly higher in progeny poults than in poults from unimmunized breeders. Clinical turkey coryza and mortality was considerably less in poults from IBD- or TIBD-vaccinated breeders than in control poults. They also responded more favorably to hemorrhagic enteritis and fowl cholera vaccination.     

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。     

Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.     

Evidence for bursal involvement in the pathogenesis of hemorrhagic enteritis of turkeys

The pathogenesis of hemorrhagic enteritis in turkey poults infected with hemorrhagic enteritis virus (HEV) at 3 days or at 2 or 5 weeks of age was compared with pathogenesis in poults that had been chemically bursectomized neonatally and exposed to cell-culture-propagated virus at 2 or 5 weeks of age. Conventional poults exposed to HEV at 2 or 5 weeks developed clinical disease, and mortality ranged from 38% to 100%. In addition to the splenic and intestinal lesions usually seen with HEV infection, the pancreas, bursa of Fabricius, and thymus were also affected. In contrast, although they were free from detectable maternal antibody, poults infected with HEV at 3 days of age failed to develop clinical disease or mortality; however, virus was demonstrated by histological and electron microscopic examinations in spleens of these poults. Neonatal chemical bursectomy completely prevented the clinical signs, gross lesions, and mortality induced by HEV in poults at 2 or 5 weeks of age. These findings strongly suggest that an intact bursa is necessary for HEV to induce disease in turkeys.     

Turkey origin reovirus-induced immune dysfunction in specific pathogen free and commercial turkey poults

Recently, pathogenesis studies, using genetically distinct turkey-origin reoviruses (TRVs), revealed that poults infected with certain TRV isolates had moderate to severe bursal atrophy, suggesting virus-induced immune dysfunction. In order to characterize the effect of TRV infection on the turkey immune system, classical assays were undertaken to quantify the humoral and cell-mediated immune responses in small Beltsville and broad-breasted white poults infected with the TRV isolate NC/SEP-R44/03. A marked effect on the cutaneous basophil hypersensitivity response, and on the antibody response to Newcastle disease virus (NDV) exposure, was noted in commercial and specific pathogen free (SPF) poults inoculated with NC/SEP-R44/03 at three days of age. Moderate to severe bursal atrophy, similar to that noted previously in SPF poults, occurred in commercial poults inoculated at three days of age. This immune dysfunction and bursal atrophy was not present in commercial poults inoculated at three weeks of age.     

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.     

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.     

Experimental transmission of turkey viral hepatitis to day-old poults and identification of associated viral particles resembling picornaviruses.

Turkey viral hepatitis (TVH) was experimentally reproduced in two experiments in 1-day-old poults. In the first experiment, an infectious inoculum was prepared from filtered yolk materials harvested from dead embryonating chicken eggs (ECE) previously inoculated with suspensions of liver and pancreas tissues collected from TVH-affected birds in commercial turkey flocks. One-day-old poults given a yolk-sac inoculation or oral gavage with this preparation developed lesions in the liver and pancreas characteristic of TVH at 20 days postinoculation (PI) in 60% and 14% of the experimentally infected birds, respectively. With the identical inoculum, embryo mortality occurred at 8 and 10 days PI in embryonating turkey eggs (ETE) inoculated into the yolk sac. In the second experiment, an infectious inoculum was prepared from filtered yolk materials from dead ETE harvested in the first experiment. One-day-old poults given a yolk-sac inoculation with this filtered yolk material developed lesions in the liver and pancreas within 5 days PI. At 20 days PI, 67% of the experimentally infected birds had similar lesions. With the inoculum given to these poults, embryo mortality occurred at 6, 8, and 10 days PI in ETE inoculated into the yolk sac. Virus particles 26-28 nm in diameter with icosahedral morphology typical of picornaviruses were identified by EM in the yolk sacs of ETE that died in both experiments, and inoculated ETE that died following passage of filtered suspensions of pancreatic tissues collected from affected birds in the first experiment.     

Effect of Bordetella avium infection on electrocardiograms in turkey poults.

Electrocardiograms (ECGs) were recorded from turkey poults infected with the W isolate of Bordetella avium. Strip chart data (amplitudes and intervals) were measured at 7, 14, 21, and 28 days postinoculation and compared with data from uninoculated controls. B. avium infection altered P-, RS-, and T-wave amplitudes, as well as P-R and S-T intervals throughout the 4-week experimental period. Heart rates were similar over the 4-week period in control poults, while rates in the infected birds were variable. Alterations in ECGs associated with B. avium infection appeared to be the result of disturbances in the cardiovascular system and may be associated with the pathogenicity of the organism in turkey coryza.     

An enzyme-linked immunosorbent assay for detection of Bordetella avium infection in turkey flocks: sensitivity, specificity, and reproducibility.

An enzyme-linked immunosorbent assay (ELISA) for detection of Bordetella avium infection in turkey poults was developed. One-week-old poults challenged intratracheally with 10(12) colony-forming units of B. avium had detectable titers (greater than or equal to 11), with an average of 13.6% positive samples when the birds were 6 to 11 weeks old. The method was sensitive enough to detect maternal antibodies to B. avium in poults up to 3 weeks of age. The same poults challenged at 1 week of age had 100% tracheal infection up to 3 weeks of age, which dropped to 0% by 6 weeks. The method resulted in no false-positive samples (titer = 0) from birds not infected with B. avium and tested weekly between 4 and 11 weeks of age. Antibodies in turkey flocks infected with Newcastle disease virus, hemorrhagic enteritis virus, and Mycoplasma meleagridis, and birds infected with Escherichia coli had no apparent cross-reactivity to the B. avium antigens used in the ELISA. The percentages of B. avium-positive serum samples collected from different turkey flocks did not significantly differ (P greater than 0.05) when samples were tested by the developed ELISA at different times, an indication of the reproducibility of the method.