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.

Isolation and Propagation of Coronaviruses in Embryonated Eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

Transmission Kinetics and histopathology induced by European Turkey Coronavirus during experimental infection of specific pathogen free turkeys.

Numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (PEC). In 2008 a coronavirus (Fr-TCoV 080385d) was isolated in France from turkey poults exhibiting clinical signs compatible with this syndrome. In the present study, the median infectious dose (ID50 ), transmission kinetics and pathogenicity of Fr-TCoV were investigated in 10-day-old SPF turkeys. Results revealed a titre of 104.88 ID50 /ml with 1 ID50 /ml being beyond the limit of genome detection using a well-characterized qRT-PCR for avian coronaviruses. Horizontal transmission of the virus via the airborne route was not observed however, via the oro-faecal route this proved to be extremely rapid (one infectious individual infecting another every 2.5 hr) and infectious virus was excreted for at least 6 weeks in several birds. Histological examination of different zones of the intestinal tract of the Fr-TCoV-infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of Fabricius. Viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for Fr-TCoV to replicate in antigen-presenting cells. Together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds.

Isolation and propagation of coronaviruses in embryonated eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, yolk). The developing embryo and its membranes provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). The embryonated egg also has been shown to be suitable for isolation and propagation of pheasant coronavirus, a proposed member of the Gammacoronavirus genus. IBV and pheasant coronavirus replicate well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as these viruses replicate well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius, thus amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection and characterization of other, novel coronaviruses.

Effect of coronavirus infection on reproductive performance of turkey hens.

Turkey coronavirus (TCoV) infection causes enteritis in turkeys of varying ages with high mortality in young birds. In older birds, field evidence indicates the possible involvement of TCoV in egg-production drops in turkey hens. However, no experimental studies have been conducted to demonstrate TCoV pathogenesis in turkey hens and its effect on reproductive performance. In the present study, we assessed the possible effect of TCoV on the reproductive performance of experimentally infected turkey hens. In two separate trials, 29- to 30-wk-old turkey hens in peak egg production were either mock-infected or inoculated orally with TCoV (Indiana strain). Cloacal swabs and intestinal and reproductive tissues were collected and standard reverse-transcription PCR was conducted to detect TCoV RNA. In the cloacal swabs, TCoV was detected consistently at 3, 5, 7, and 12 days postinoculation (DPI) with higher rates of detection after 5 DPI (> 90%). All intestinal samples were also positive for TCoV at 7 DPI, and microscopic lesions consisting of severe enteritis with villous atrophy were observed in the duodenum and jejunum of TCoV-infected hens. In one of the trials TCoV was detected from the oviduct of two birds at 7 DPI; however, no or mild microscopic lesions were present. In both experimental trials an average of 28%-29% drop in egg production was observed in TCoV-infected turkey hens between 4 and 7 DPI. In a separate trial we also confirmed that TCoV can efficiently transmit from infected to contact control hens. Our results show that TCoV infection can affect the reproductive performance in turkey hens, causing a transient drop in egg production. This drop in egg production most likely occurred as consequence of the severe enteritis produced by the TCoV. However, the potential replication of TCoV in the oviduct and its effect on pathogenesis should be considered and further investigated.

An experimental study of the survival of turkey coronavirus at room temperature and +4°C.

Turkey coronavirus (TCoV) is a gammacoronavirus (Coronaviridae, Nidovirales) responsible for digestive disorders in young turkeys. TCoV has been associated with poult enteritis complex, a syndrome that severely affects turkey production. No medical prophylaxis exists to control TCoV, therefore sanitary measures such as cleaning and disinfection are essential. It is thus important to evaluate temperatures that allow persistence of TCoV in the environment. Two series of aliquots of a suspension of a French isolate of TCoV (Fr TCoV) were stored at room temperature or +4°C for 0 to 40 days. As TCoV does not grow in cell culture, the presence of residual infectious TCoV in the stored samples was tested by inoculating embryonated specific pathogen free turkey eggs. As TCoV does not induce lesions in the embryo, virus replication in the jejunum and ileum of the embryos was detected 4 days post inoculation, using RNA extraction and a real-time reverse transcriptase-polymerase chain reaction based on the nucleocapsid gene. No surviving virus was detected after 10 days storage at +21.6±1.4°C or after 40 days storage at +4.1±1.6°C, these temperatures being representative of the mean summer and winter temperatures, respectively, in the major French poultry-producing region. The relatively short survival of the virus at room temperature should contribute to limited virus survival during summer months. However, infectious virus was still detected after 20 days storage at the cooler temperatures, a finding that suggests prolonged survival of Fr TCoV and easier transmission between poultry farms in a cool environment are possible.

Enhancement of enteropathogenic Escherichia coli pathogenicity in young turkeys by concurrent turkey coronavirus infection.

In a previous study, turkey coronavirus (TCV) and enteropathogenic Escherichia coli (EPEC) were shown to synergistically interact in young turkeys coinfected with these agents. In that study, inapparent or mild disease was observed in turkeys inoculated with only TCV or EPEC, whereas severe growth depression and high mortality were observed in dually inoculated turkeys. The purpose of the present study was to further evaluate the pathogenesis of combined TCV/EPEC infection in young turkeys and determine the role of these agents in the observed synergistic interaction. Experiments were conducted to determine 1) effect of EPEC dose, with and without concurrent TCV infection, and 2) effect of TCV exposure, before and after EPEC exposure, on development of clinical disease. Additionally, the effect of combined infection on TCV and EPEC shedding was determined. No clinical sign of disease and no attaching and effacing (AE) lesions characteristic of EPEC were observed in turkeys inoculated with only EPEC isolate R98/5, even when turkeys were inoculated with 10(10) colony forming units (CFU) EPEC (high dose exposure). Only mild growth depression was observed in turkeys inoculated with only TCV; however, turkeys inoculated with both TCV and 10(4) CFU EPEC (low dose exposure) developed severe disease characterized by high mortality, marked growth depression, and AE lesions. Inoculation of turkeys with TCV 7 days prior to EPEC inoculation produced more severe disease (numerically greater mortality, significantly lower survival probability [P < 0.05], increased frequency of AE lesions) than that observed in turkeys inoculated with EPEC prior to TCV or simultaneously inoculated with these agents. Coinfection of turkeys with TCV and EPEC resulted in significantly increased (P < 0.05) shedding of EPEC, but not TCV, in intestinal contents of turkeys. These findings indicate that TCV infection predisposes young turkeys to secondary EPEC infection and potentiates the expression of EPEC pathogenicity in young turkeys.

Hemorrhagic enteritis virus induced changes in the lymphocyte subpopulations in turkeys and the effect of experimental immunodeficiency on viral pathogenesis.

We examined the changes in the lymphocyte subpopulations in the spleen and peripheral blood of turkeys and the effects of experimental immunodeficiency in the B and T cell compartments on the pathogenesis of hemorrhagic enteritis (HE) in turkeys. Inoculation of turkeys with hemorrhagic enteritis virus (HEV) induced a drop in the relative proportions of IgM bearing cells on Day 2, 3, and 9 post-infection and an elevation in the relative proportions of CD4+ cells on Day 4 and 6 post-infection. Elevated levels of CD8+ cells were observed in the infected turkeys only on Day 16 after infection. Marked depletion of IgM+ cells may play a role in immunodepression caused by HEV. Cyclophosphamide (CY) treatment induced B cell deficiency in turkeys severely impaired HEV replication in the spleen suggesting that B lymphocytes are important for viral replication. Cyclosporin A (CsA) selectively impaired T cell mitogenesis and protected the turkeys against HEV-induced intestinal hemorrhages. CsA did not prevent viral replication in the spleen or the associated splenomegaly. This result suggested that T cell immunity may be important for intestinal hemorrhaging induced by HEV.

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.