Pathogenic evaluation of a turkey coronavirus isolate (TCoV NC1743) in turkey poults for establishing a TCoV disease model.

Turkey coronavirus (TCoV) can cause a highly contagious enteric disease in turkeys with severe economic losses in the global turkey industry. To date, no commercial vaccines are available for control of the disease. In the present study, we isolated a field strain (NC1743) of TCoV and evaluated its pathogenicity in specific-pathogen-free (SPF) turkey poults to establish a TCoV disease model. The results showed that the TCoV NC1743 isolate was pathogenic to turkey poults with a minimal infectious dose at 106 EID50/bird. About 50 % of one-day-old SPF turkeys infected with the virus's minimal infectious dose exhibited typical enteric disease signs and lesions from 6 days post-infection (dpi) to the end of the experiment (21 dpi). In contrast, fewer than 20 % of older turkeys (1- or 2-week-old) infected with the same amount of TCoV displayed enteric disease signs, which disappeared after 15-18 dpi. Although all infected turkeys, regardless of age, shed TCoV, the older turkeys shed less virus than the younger birds, and 50 % of the 2-week-old birds even cleared the virus at 21 dpi. Furthermore, the viral infection caused day-old turkeys more body-weight-gain reduction than older birds. The overall data demonstrated that the TCoV NC1743 isolate is a highly pathogenic strain and younger turkeys are more susceptible to TCoV infection than older birds. Thus, one-day-old turkeys infected with the minimal infectious dose of TCoV NC1743 could be used as a TCoV disease model to study the disease pathogenesis, and the TCoV NC1743 strain could be used as a challenge virus to evaluate a vaccine protective efficacy.

Age-Related Susceptibility of Turkeys to Enteric Viruses.

Several different enteric viruses have been identified as the causes of gastrointestinal infections in poultry. Enteric virus infections are well characterized in poults, but limited studies have been conducted in older birds. The susceptibility of 2-, 7-, 12-, 30-, and 52-wk-old turkeys to turkey coronavirus (TCoV) and turkey astrovirus (TAstV) was evaluated, as well as the effect of combined infection of TAstV and TCoV in 2-wk-old poults and turkey hens. From cloacal swabs and intestines, TCoV was consistently detected by reverse transcriptase-PCR throughout the experimental period (1-21 days postinoculation [DPI]) from all age groups. In contrast, the last detection point of TAstV gradually decreased to 21, 16, and 12 DPI in birds inoculated at 2, 7, and 12 wk of age, respectively, and viral RNA was rarely detected from cloacal swabs or intestinal contents in turkey hens within 3 DPI. Infection with TAstV alone did not affect body weight in poults or egg production in hens. The combined infection of TAstV and TCoV did not induce more severe clinical signs and pathology than the TCoV infection alone. However, a severe prolonged decrease in egg production (about 50%) was observed in turkey hens in the combined infection group compared with a transient egg production drop in the TCoV-infected hens alone. The underlying mechanism regarding the age-related TAstV susceptibility and the pathogenesis of the TAstV and TCoV coinfection in layer hens needs to be further elucidated.

First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

An increasing incidence of enteric disorders clinically suggestive of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time reverse transcriptase-polymerase chain reaction assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37% of the intestinal samples collected from diseased turkey flocks. The full-length spike (S) gene of these viruses was amplified, cloned and sequenced from three samples. The French S sequences shared 98% identity at both the nucleotide and amino acid levels, whereas they were at most 65% and 60% identical with North American (NA) TCoV and at most 50% and 37% identical with IBV at the nucleotide and amino acid levels, respectively. Higher divergence with NA TCoV was observed in the S1-encoding domain. Phylogenetic analysis based on the S gene revealed that the newly detected viruses form a sublineage genetically related with, but significantly different from, NA TCoV. Additionally, the RNA-dependent RNA polymerase gene and the N gene, located on the 5' and 3' sides of the S gene in the coronavirus genome, were partially sequenced. Phylogenetic analysis revealed that both the NA TCoV and French TCoV (Fr TCoV) lineages included some IBV relatives, which were however different in the two lineages. This suggested that different recombination events could have played a role in the evolution of the NA and Fr TCoV. The present results provide the first S sequence for a European TCoV. They reveal extensive genetic variation in TCoV and suggest different evolutionary pathways in North America and Europe.

Emergence of a group 3 coronavirus through recombination.

Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.

Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada.

Turkey coronavirus (TCoV) is an important viral pathogen causing diarrhoea of young turkey poults that is associated with sizeable economic losses for the turkey industry. Using a field isolate that was found to be free from turkey astrovirus and avian reovirus we were able to reproduce the clinical disease associated with TCoV. Clinical signs and weight gain of poults during experimental infections were compared with age-matched, uninfected controls. Poults infected at 2 days of age had 100% morbidity and 10% mortality, and birds infected at 28 days of age showed 75% morbidity and no mortality. Diarrhoea was consistently seen in infected poults at 2 to 3 days post infection (d.p.i.) with a duration of about 3 to 5 days. Mean body weights of birds infected at 2 or 28 days of age were significantly reduced compared with uninfected birds by 7 d.p.i. and remained significantly lower for the duration of the study. At 44 days of age, poults infected at 2 or 28 days of age weighed only 68.1% or 77.7%, respectively, compared with uninfected turkeys of the same age on the same diet, a mean difference in body weights of 683 or 477g, respectively. Infected birds had profound villus atrophy with some compensatory crypt hyperplasia at 5 to 7 d.p.i. Villus heights in the duodenum were significantly reduced at 7 d.p.i. We were able to reproduce enteric disease using only a pathogenic field isolate (MG10) of TCoV that negatively affected growth performance and intestinal morphology of young turkey poults.

A reverse transcriptase-polymerase chain reaction assay for the diagnosis of turkey coronavirus infection.

This study reports on the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific detection of turkey coronavirus (TCoV). Of the several sets of primers tested, 1 set of primers derived from the P gene and 2 sets derived from the N gene of TCoV could amplify the TCoV genome in the infected samples. The RT-PCR was sensitive and specific for TCoV and did not amplify other avian RNA and DNA viruses tested except the infectious bronchitis virus (IBV). To overcome the problem of IBV amplification, a set of separate primers was designed from the spike protein gene of IBV. The RT-PCR under the same conditions as above could effectively differentiate between TCoV and IBV. The closely related bovine coronavirus and transmissible gastroenteritis virus of pigs were differentiated from TCoV using the same RT-PCR with slight modifications. The results of RT-PCR correlated well with the results of the immunofluorescent test for the same samples tested at the Purdue University Animal Disease Laboratory, West Lafayette, Indiana. The nucleotide sequence and projected amino acid sequence comparison of the P gene of different isolates of TCoV from 5 different states in the United States revealed a close association among the different isolates of TCoV.

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.

Immunopathogenesis of haemorrhagic enteritis virus (HEV) in turkeys.

Infection of turkeys with the haemorrhagic enteritis virus (HEV), a type II avian adenovirus, results in varying rates of morbidity and mortality. The disease is characterised by splenomegaly, intestinal haemorrhage, sudden death and immunosuppression. The mechanisms of HEV immunopathogenesis and immunosuppression are not fully understood. Recent studies indicate that immune responses play a central role in disease pathogenesis. HEV infects B cells and macrophages and induces necrosis as well as apoptosis in infected and possibly in by-stander cells. The ability of the infected birds to mount an optimum humoral immune response as well as normal macrophage functions such as phagocytosis may be impaired. Elevated numbers of splenic CD4(+) cells during the acute phase of infection may be associated with viral clearance. Types I and II interferons (IFN) and pro-inflammatory cytokines such as interleukin-6 and tumour necrosis-like factors (TNF) are released at the peak of the infection. Cytokines may play a protective as well as a destructive role. While a massive release of proinflammatory cytokines may lead to systemic shock associated with haemorrhagic enteritis and death, release of IFNs may protect turkeys from the disease. Treatment with thalidomide, which is a potent TNF down-regulatory drug, prevented HEV-induced intestinal haemorrhage and treatment with an IFN-inducing chemical prevented HEV-replication and inhibited HEV-induced pathological and histopathological lesions.

Limited transmission of turkey coronavirus in young turkeys by adult Alphitobius diaperinus (Coleoptera: Tenebrionidae).

We examined the role of lesser mealworm, Alphitobius diaperinus (Panzer), in the transmission of an enteric disease of turkeys caused by a coronavirus. Turkey coronavirus (TCV) from two sources was studied, one isolate (NC95) was embryo propagated, the second was TCV infected material from turkeys diagnosed with poult enteritis mortality syndrome (PEMS). Beetles were fed virus-infected feces mixed with chicken feed. Transmission of virus was effectively halted by surface sterilization of the beetles. Turkey poults administered beetle homogenates infected with TCV+ PEMS that had not been surface sterilized had reduced weight gains and 50% mortality. Mortality and weight gains were not effected in the NC95 group. Virus isolation procedures were performed to determine NC95 viability at varying time intervals. Beetles were dissected and the guts removed 1, 12, and 24 h after the initial viral feeding. Whole beetles were also examined for comparison. Whole beetles and beetle guts were homogenized and injected into turkey eggs for embryo propagation. Direct immunofluorescence was used to determine the presence of TCV. A. diaperinus were capable of mechanical transmission of TCV. However, only turkey embryos receiving whole beetle and beetle gut homogenates within 1 h of feeding on the virus were positive for TCV. Laboratory studies demonstrating PEMS transmission by A. diaperinus are continuing.

Comparative pathogenesis of haemorrhagic enteritis virus (HEV) infection in turkeys and chickens.

The pathogenesis of haemorrhagic enteritis virus (HEV) infection in chickens 3-4 days post-infection was compared with that in turkeys. As expected, infected turkeys showed HEV-specific lesions that included enlargement and mottling of the spleen, as well as haemorrhagic enteritis. In infected chickens, only splenomegaly was observed. The number of HEV-infected cells in the spleen was significantly (P < 0.05) higher in the turkey than in the chicken. In both species, the immunohistochemical labelling of B-cell surface determinants was diminished and the splenic B-cell areas were undetectable after HEV infection. Infection with HEV resulted in an increase in nitric oxide production by macrophages in chickens but not in turkeys.

Response of specific-pathogen-free turkeys to vaccines derived from marble spleen disease virus and hemorrhagic enteritis virus.

Tissue-culture-propagated marble spleen disease virus (MSDV-TC) and two preparations of spleen homogenate (MSDV-SH and MSDV-SH-TC) were compared as anti-hemorrhagic enteritis virus (HEV) vaccines in specific-pathogen-free turkeys. Both types of vaccines spread horizontally among turkeys, induced anti-HEV antibodies, and protected turkeys against challenge with virulent HEV. Antibody development and horizontal spread of virus occurred earlier in turkeys given MSDV-SH or MSDV-SH-TC than in those given MSDV-TC. Virulent HEV was serially passed in MDTC-RP19 cells. The 30th passage virus (HEV-P30) was nonpathogenic for turkeys but was immunogenic. Turkeys exposed to HEV-P30 had viral antigen in the spleen, developed neutralizing antibodies, and resisted virulent HEV. The principal difference between MSDV-TC and HEV-P30 vaccines was that MSDV-TC caused well-defined splenomegaly in turkeys, whereas HEV-P30 protected turkeys without causing spleen enlargement.

Hemorrhagic enteritis of turkeys.

Hemorrhagic enteritis (HE), an economically important disease of turkeys is caused by a type II adenovirus. The virus is ubiquitous and is liable to infect most field turkeys. In unprotected turkey flocks, infection with virulent hemorrhagic enteritis virus (HEV) may result in variable mortality and immunodepression. Turkeys younger than 2-4 weeks of age are resistant to clinical HE. This age-related resistance is expressed in the presence or absence of maternal antibodies against HEV. Clinical disease is characterized by HE and splenomegaly. The virus causes intranuclear inclusions in the reticuloendothelial cells. Bursectomy or splenectomy abrogate clinical HE. Field data and laboratory studies indicate that HEV causes immunodepression in the humoral as well as the cellular immune functions of turkeys. The mechanism of immunodepression is not known.

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.

Immunosuppressive effects of virulent strain of hemorrhagic enteritis virus in turkeys vaccinated against Newcastle disease.

One week after infection with a virulent strain of hemorrhagic enteritis virus (HEV), turkeys were vaccinated for Newcastle disease. The effect of a virulent strain of HEV on turkeys' immune response to Newcastle disease vaccine and the mitogenic response of their whole blood peripheral lymphocytes were examined. The results revealed a statistically significant difference (P less than .01) in the Newcastle disease hemagglutination inhibition (NDHI) antibody titers from turkeys infected with virulent HEV. The NDHI antibody titers were lower in turkeys exposed to virulent HEV before vaccination. There was an initial depression in phytohemagglutinin (PHA) response 1 week postinfection in turkeys infected with virulent HEV strain.

Enteritis Hemorragica por Adenovirus en Pavos / Hemorrhagic Enteritis by Adenovirus in Turkeys

Se describe por primera vez en Colombia la enteritis hemorragica (EH), en pavos de 4 a 11 semanas de edad, mediante estudios macroscopicos, histologicos y de microscopia electronica. Se demostro una enteritis hemorragica severa, y esplenomegalia con inclusiones intranucleares en la mayoria de las celulas de la pulpa blanca esplenica y en algunos mononucleares en el intestino delegado. Las inclusiones son caracteristicas; consisten de un centro eosinofilico, rodeado de cromatina densa y condensada sobre la membrana nuclear y de una estructura muy basofila y densa sobre un polo nuclear que parece corresponder a un nucleolo. Al microscopio electronico se aprecian fases del desarrollo viral, desde un material filamentoso y amorfo hasta la formacion de grandes acumulos de viriones y capsides de 70-80 nm de diametro con morfologia tipica de adenovirus. Los viriones se demostraron facilmente en macerados de bazo fijado en formol, tenidos negativamente con fosfotungstato de potasio; no se visualizaron en el contenido intestinal hemorragico estudiado con el mismo metodo. El estudio de microscopia de luz de bazo e intestino es suficiente para establecer el diagnostico de esta entidad

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.