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.

Genotyping of turkey coronavirus field isolates from various geographic locations in the Unites States based on the spike gene.

Turkey flocks have experienced turkey coronaviral enteritis sporadically in the United States since the 1990s. Twenty-four field isolates of turkey coronavirus (TCoV) from multiple states in the United States were recovered from 1994 to 2010 to determine the genetic relationships among them. The entire spike (S) gene of each TCoV isolate was amplified and sequenced. Pairwise comparisons were performed using the Clustal W program, revealing 90.0% to 98.4% sequence identity in the full-length S protein, 77.6% to 96.6% in the amino terminus of the S1 subunit (containing one hypervariable region in S1a), and 92.1% to 99.3% in the S2 subunit at the deduced amino acid sequence level. The conserved motifs, including two cleavage recognition sequences of the S protein, two heptad repeats, the transmembrane domain, and the Golgi retention signal were identified in all TCoV isolates. Phylogenetic analysis based on the full-length S gene was used to distinguish North American TCoV isolates from French TCoV isolates. Among the North American TCoV isolates, three distinct genetic groups with 100% bootstrap support were observed. North Carolina isolates formed group I, Texas isolates formed group II, and Minnesota isolates formed Group III. The S genes of 24 TCoV isolates from the United States remained conserved because they contained predominantly synonymous substitutions. The findings of the present study suggest endemic circulation of distinct TCoV genotypes in different geographic locations.

Investigating turkey enteric coronavirus circulating in the Southeastern United States and Arkansas during 2012 and 2013.

Periodic monitoring of poultry flocks in the United States via molecular diagnostic methods has revealed a number of potential enteric viral pathogens in continuous circulation in turkeys and chickens. Recently turkey integrators in the Southeastern United States and Arkansas experienced an outbreak of moderate to severe enteritis associated with turkey enteric coronavirus (TCoV), and numerous enteric samples collected from turkey flocks in these areas tested positive for TCoV via real-time reverse-transcriptase PCR (RRT-PCR). This report details the subsequent sequence and phylogenetic analysis of the TCoV spike glycoprotein and the comparison of outbreak-associated isolates to sequences in the public database. TCoVs investigated during the present outbreak grouped geographically based upon state of origin, and the RRT-PCR assay was a good indicator of subsequent seroconversion by TCoV-positive turkey flocks.

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.

Detection and molecular characterization of gene 3 and 5 of turkey coronavirus from turkeys with severe enteritis in Brazil.

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3' UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3' UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Use of recombinant S1 spike polypeptide to develop a TCoV-specific antibody ELISA.

Turkey coronavirus (TCoV) causes diarrhoea in young turkey poults but little is known about its prevalence in the field. To address this, a portion of the S1 region of the spike glycoprotein of TCoV carrying relevant B cell epitopes (amino acid positions 54-395) was cloned and expressed in Escherichia coli. This protein was purified and used to develop an indirect ELISA for detection of antibodies against TCoV. Using experimentally derived positive and negative turkey serum samples this ELISA showed high sensitivity (95%) and specificity (92%) for TCoV. To further evaluate the potential of the ELISA, 360 serum samples from commercial turkey farms in Ontario were tested for TCoV-specific antibodies using the recombinant TCoV ELISA. High seroprevalence of TCoV was found with 71.11% of breeders and 56.67% of meat turkeys testing seropositive. Although there was significant positive correlation with a TCoV-N protein-based ELISA, there was little to no correlation with the whole IBV antigen-based ELISA when field sera were tested for antibodies against TCoV.

Seroprevalence of turkey coronavirus in North American turkeys determined by a newly developed enzyme-linked immunosorbent assay based on recombinant antigen.

Turkey coronavirus (TCoV) causes diarrhea in young turkey poults, but little is known about its prevalence in the field. To address this, the complete nucleocapsid gene was cloned and expressed in Escherichia coli. Expressed nucleocapsid gene produced two distinct proteins (52 and 43 kDa); their specificity was confirmed by Western blotting using two different monoclonal antibodies. Recombinant N protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay (ELISA) for the serological detection of TCoV that was then validated using experimentally derived turkey serum. The N-based ELISA showed (97%) sensitivity and (93%) specificity for TCoV, which was significantly higher than an infectious bronchitis coronavirus-based commercial test for TCoV. To assess the utility of this recombinant ELISA, 360 serum samples from turkey farms in Ontario, Canada, and 81 serum samples from farms in Arkansas were tested for TCoV-specific antibodies. A high seroprevalence of TCoV was found in turkeys from the Ontario farms with 73.9% of breeders and 60.0% of meat turkeys testing seropositive using the N-based ELISA. Similarly, a high field prevalence was found in the turkeys from Arkansas, for which 64.2% of the serum samples tested seropositive.

Detection of turkey coronavirus in commercial turkey poults in Brazil.

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality. In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabrícius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV. Histopathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabrícius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Risk factors associated with poult enteritis mortality syndrome-positive turkey flocks.

Poult enteritis mortality syndrome (PEMS) has been an economically devastating disease in North Carolina since the early 1990s. Though much is known about the disease, many questions remain unanswered about the syndrome, including its cause, transmission of causative agent(s), and control methods. This study was designed to investigate the association between PEMS and farm management factors. A prospective longitudinal study was conducted by collecting farm data and monitoring weekly mortality in 54 commercial turkey flocks raised in PEMS-affected regions. Univariate and multivariate statistical analyses revealed that enhancing rodent control methods was negatively associated (P = 0.0228) with PEMS.

Mortality patterns associated with poult enteritis mortality syndrome (PEMS) and coronaviral enteritis in turkey flocks raised in PEMS-affected regions.

Poult enteritis mortality syndrome (PEMS) is an economically devastating disease. To date, many questions about the syndrome remain unanswered, including its cause, transmission of causative agent(s), and control methods. Turkey coronavirus (TCV) infection has been associated with some outbreaks of PEMS, with areas having a higher prevalence of TCV infection also experiencing an increased incidence of PEMS. This study was designed to establish mortality patterns for flocks experiencing excess mortality and TCV infection in PEMS-affected regions and to delineate the possible role of TCV in PEMS-affected flocks. Fifty-four commercial turkey flocks on farms in areas with and without a history of TCV infection were monitored for weekly mortality and for antibodies to TCV. Flocks were chosen on the basis of placement dates and were monitored from day of placement until processing. All flocks were tested for TCV by an indirect fluorescent antibody assay. PEMS status was determined with the use of the clinical definition of mortality greater than 2% during any 3-wk period from 2 wk of age through the end of brooding due to unknown cause. Of the 54 flocks, 24 remained healthy, 23 experienced PEMS, and 7 tested positive for TCV but did not experience PEMS. Ten flocks experienced PEMS and tested positive for TCV, whereas 13 flocks experienced PEMS and did not test positive for TCV. Four health status groups were evident: healthy, PEMS positive, TCV positive, and PEMS + TCV positive. Distinct mortality patterns were seen for each of the four health status groups. Whereas TCV was associated with PEMS in 43% of PEMS cases, 13 cases (57%) of PEMS did not involve TCV. Additionally, 7 out of 17 cases of TCV (41%) did not experience excess mortality (PEMS) at any time during brooding of the flock. The results of this study indicate that TCV can be associated with PEMS but is neither necessary nor sufficient to cause PEMS.

Infectious bursal disease and hemorrhagic enteritis.

Infectious bursal disease (IBD) of chickens and hemorrhagic enteritis (HE) of turkeys are caused by infectious bursal disease virus (IBDV) and hemorrhagic enteritis virus (HEV), respectively. Both diseases have common features, including an acute stage followed by immunosuppression, resulting in lowered resistance to a variety of infectious agents and poor response to commonly used vaccines. The IBDV and HEV infections are widespread in commercial chicken and turkey flocks, respectively. The acute stage of the disease, the immunosuppression that follows, and the widespread distribution of both diseases, are major factors contributing to the economic significance of both diseases. The mechanism of immunosuppression for both infections has similarities, both affect lymphocytes and macrophages, and both are lymphocidal. In this report, an overview of both diseases with emphasis on some of the recent findings will be presented. There has been greater research activity on IBD than on HE, reflecting the relative economic importance of the species affected and the recent changes in the antigenic make up and pathogenicity of the IBDV.

Hemorrhagic enteritis of turkeys in California: serologic study of hemorrhagic enteritis virus antibody with an enzyme-linked immunosorbent assay.

The incidence of hemorrhagic enteritis (HE) infection in California turkeys was studied by testing 2220 turkey blood samples from 173 flocks for HE virus (HEV) antibody by the enzyme-linked immunosorbent assay (ELISA). Maternal antibody was detected at 1 day of age in all flocks tested, and it vanished after 3 weeks. Acquired HEV antibody appeared at 8 to 10 weeks, and 100% of the meat and breeder turkey flocks were positive after 11 weeks of age. HEV infection occurred earlier in the meat flocks than in the breeder flocks, and it also occurred earlier during summer than during the fall and winter months.

Detection of turkey coronaviral enteritis (bluecomb) in field epiornithics, using the direct and indirect fluorescent antibody tests.

In Minnesota, efforts have been made over the past 10 years to eliminate turkey coronaviral enteritis (TCE, bluecomb) by controlled depopulation and decontamination with a rest period before restocking. In 1973, clinical observations indicated that bluecomb was restricted to one limited area in Minnesota. Five epiornithics occurred during late 1973 and 1974, involving 5 different farms in this limited geographic area. During 1975, 3 epiornithics of TCE were investigated, involving 185,000 turkeys in 17 flocks, of which approximately 17,000 died. Naturally infected turkeys representing 7 operations between 1973 and 1976 were examined by both the direct fluorescent antibody test and indirect fluorescent antibody test (IFAT). The direct fluorescent antibody test detected coronaviral antigen in intestinal tissues during the acute phase of the disease, and the IFAT was highly useful in detecting TCE serum antibodies of turkey flocks that had recovered and were potential carriers. Therefore, an IFAT surveillance program was instituted for replacement flocks on farms where clinical epiornithics of TCE had occurred in 1974 through 1976. Operation 5 involved TCE epiornithics over a 2-year period and illustrate the importance of complete depopulation with an intensive decontamination program.