Necrotic enteritis in a commercial turkey flock coinfected with hemorrhagic enteritis virus.

Four turkeys from a commercial flock with acutely elevated mortality levels were submitted for postmortem examination and diagnostic workup. No clinical signs had been observed before death. On gross examination, hemorrhage and necrosis were present throughout the intestinal tracts, and the spleens were markedly enlarged and speckled. Microscopically, numerous, large basophilic-to-amphophilic intranuclear inclusion bodies were observed in mononuclear cells of the spleen and the lamina propria of the small intestine. In addition, there were lesions of diffuse villus blunting and necrosis of the small intestine, with large numbers of rod-shaped bacteria adhered to the epithelium and in the intestinal lumen. Hemorrhagic enteritis virus (HEV) infection was confirmed via PCR on the spleen. Clostridium perfringens was demonstrated in the small intestine by anaerobic culture and immunohistochemistry. The C. perfringens isolate was type F by PCR and, to our knowledge, necrotic enteritis in turkeys has not been described in association with C. perfringens type F infection.

Targeted detection and molecular epidemiology of turkey coronavirus spike gene variants in turkeys and chickens.

Turkey coronavirus (TCoV) is a member of the Avian coronavirus species with infectious bronchitis virus (IBV), which is considered to be the source of TCoV. These 2 viruses are highly similar in all regions of their genomes, except for the spike gene, which is necessary for virus attachment. Although TCoV causes severe enteric disease in turkey poults, it does not cause clinical disease in chickens. However, considering that TCoV can infect chickens, it is important to distinguish TCoV from IBV in chickens. This is particularly true for chickens that are housed near turkeys and thus might be infected with TCoV and serve as a silent source of TCoV for turkeys. We developed and validated a real-time PCR assay to detect the spike gene of TCoV and sequenced a portion of this gene to evaluate the molecular epidemiology of TCoV infections associated with a commercial turkey premises in the United States in 2020-2021. We identified natural infections of TCoV in chickens, and based on the molecular epidemiology of the viruses detected, these chickens may have served as a source of infection for the commercial turkey premises located nearby.

Characterization of the Cross-Species Transmission Potential for Porcine Deltacoronaviruses Expressing Sparrow Coronavirus Spike Protein in Commercial Poultry.

Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and their gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-SHKU17, and icPDCoV-RBDISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-SHKU17- and icPDCoV-RBDISU virus infections. In 5-day-old ECEs, significant mortality was observed in PDCoV inoculated eggs with less in the spike chimeras, while in 11-day-old ECEs there was no evidence of viral replication, suggesting that PDCoV is better adapted to cross species infection and differentiated ECE cells are not susceptible to PDCoV infection. Collectively, we demonstrate that the SpCoV chimeric viruses are not more infectious in turkeys, nor ECEs than wild type PDCoV. Therefore, understanding the cell and host factors that contribute to resistance to PDCoV and avian-swine chimeric virus infections may aid in the design of novel antiviral therapies against DCoVs.

Molecular Epidemiology of Turkey Coronaviruses in Poland.

The only knowledge of the molecular structure of European turkey coronaviruses (TCoVs) comes from France. These viruses have a quite distinct S gene from North American isolates. The aim of the study was to estimate the prevalence of TCoV strains in a Polish turkey farm during a twelve-year period, between 2008 and 2019, and to characterize their full-length S gene. Out of the 648 flocks tested, 65 (10.0%, 95% CI: 7.9-12.6) were positive for TCoV and 16 of them were molecularly characterized. Phylogenetic analysis showed that these strains belonged to two clusters, one formed by the early isolates identified at the beginning of the TCoV monitoring (from 2009 to 2010), and the other, which was formed by more recent strains from 2014 to 2019. Our analysis of the changes observed in the deduced amino acids of the S1 protein suggests the existence of three variable regions. Moreover, although the selection pressure analysis showed that the TCoV strains were evolving under negative selection, some sites of the S1 subunit were positively selected, and most of them were located within the proposed variable regions. Our sequence analysis also showed one TCoV strain had recombined with another one in the S1 gene. The presented investigation on the molecular feature of the S gene of TCoVs circulating in the turkey population in Poland contributes interesting data to the current state of knowledge.

Upregulation of INF-γ, IL-6, and IL-8 expression during replication of turkey coronavirus in nonepithelial cells obtained from Meleagris gallopavo.

Mesenchymal stromal cells (MSCs) are considered multipotent progenitors with the capacity to differentiate into mesoderm-like cells in many species. The immunosuppressive properties of MSCs are important for downregulating inflammatory responses. Turkey coronavirus (TCoV) is the etiological agent of a poult mortality syndrome that affects intestinal epithelial cells. In this study, poult MSCs were isolated, characterized, and infected with TCoV after in vitro culture. The poult-derived MSCs showed fibroblast-like morphology and the ability to undergo differentiation into mesodermal-derived cells and to support virus replication. Infection with TCoV resulted in cytopathic effects and the loss of cell viability. TCoV antigens and new viral progeny were detected at high levels, as were transcripts of the pro-inflammatory factors INFγ, IL-6, and IL-8. These findings suggest that the cytokine storm phenomenon is not restricted to one genus of the family Coronaviridae and that MSCs cannot always balance the process.

Susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2.

The susceptibility of turkeys, chickens and chicken embryos to SARS-CoV-2 was evaluated by experimental infection. Turkeys and chickens were inoculated using a combination of intranasal, oral and ocular routes. Both turkeys and chickens did not develop clinical disease or seroconvert following inoculation. Viral RNA was not detected in oral swabs, cloacal swabs or in tissues using quantitative real-time RT-PCR. In addition, chicken embryos were inoculated by various routes including the yolk sac, intravenous, chorioallantoic membrane and allantoic cavity. In all instances, chicken embryos failed to support replication of the virus. SARS-CoV-2 does not affect turkeys or chickens in the current genetic state and does not pose any potential risk to establish an infection in both species of domestic poultry.

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.

Porcine Deltacoronavirus Infection and Transmission in Poultry, United States1.

Coronaviruses cause respiratory and gastrointestinal diseases in diverse host species. Deltacoronaviruses (DCoVs) have been identified in various songbird species and in leopard cats in China. In 2009, porcine deltacoronavirus (PDCoV) was detected in fecal samples from pigs in Asia, but its etiologic role was not identified until 2014, when it caused major diarrhea outbreaks in swine in the United States. Studies have shown that PDCoV uses a conserved region of the aminopeptidase N protein to infect cell lines derived from multiple species, including humans, pigs, and chickens. Because PDCoV is a potential zoonotic pathogen, investigations of its prevalence in humans and its contribution to human disease continue. We report experimental PDCoV infection and subsequent transmission among poultry. In PDCoV-inoculated chicks and turkey poults, we observed diarrhea, persistent viral RNA titers from cloacal and tracheal samples, PDCoV-specific serum IgY antibody responses, and antigen-positive cells from intestines.

Spotlight on avian coronaviruses.

Coronaviruses (CoVs) mainly cause enteric and/or respiratory signs. Mammalian CoVs including COVID-19 (now officially named SARS-CoV-2) belong to either the Alphacoronavirus or Betacoronavirus genera. In birds, the majority of the known CoVs belong to the Gammacoronavirus genus, whilst a small number are classified as Deltacoronaviruses. Gammacoronaviruses continue to be reported in an increasing number of avian species, generally by detection of viral RNA. Apart from infectious bronchitis virus in chickens, the only avian species in which CoV has been definitively associated with disease are the turkey, pheasant and guinea fowl. Whilst there is strong evidence for recombination between gammacoronaviruses of different avian species, and between betacoronaviruses in different mammals, evidence of recombination between coronaviruses of different genera is lacking. Furthermore, the recombination of an alpha or betacoronavirus with a gammacoronavirus is extremely unlikely. For recombination to happen, the two viruses would need to be present in the same cell of the same animal at the same time, a highly unlikely scenario as they cannot replicate in the same host!