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.

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.

The immune response of young turkeys to haemorrhagic enteritis virus infection at different levels and sources of methionine in the diet.

BACKGROUND: Haemorrhagic enteritis (HE) of turkeys was first described in 1937 in the USA, while in Poland it was first diagnosed in 1987. Polish haemorrhagic enteritis virus (HEV) isolates are usually low pathogenic and trigger a subclinical disease. Unfortunately, even the low- pathogenic HEV strains cause severe immunosuppression leading to secondary bacterial infections and huge economic losses. The objective of this study was to evaluate if the influence of Met on HEV infected turkeys immune response can be differentiated by both its level and source. Met is one of the amino acids that not only play a nutritional role but also participate in and regulate key metabolic pathways and immune response. In our study, the birds were assigned to 4 dietary treatments which differed in Met levels (0.55 and 0.78% in weeks 1-4 of age and 0.45 and 0.65% in weeks 5-8 of age, respectively) and sources (DL-methionine (DLM) or DL-methionine hydroxy analogue (MHA)). RESULTS: The HEV added the percentage of CD4+ cells and decreased the percentage of IgM+ cells in the blood, spleen and caecal tonsils (CTs) of turkeys. In addition, it increased the percentage of CD4+CD25+ cells in blood, and interleukin-6 (IL-6) level in plasma. The higher dose of Met led to a significant decrease in the percentages of CD4+, CD8+ and CD4+IL-6+ cell subpopulations in the blood of HEV-infected and uninfected turkeys and to an increase in the percentage of IgM+ B cells in CTs. Turkeys administered feeds with an increased Met content displayed a decrease in plasma IL-6 levels and an increase in plasma IgA levels. CONCLUSIONS: The results of this study indicate that HEV infection impairs the immune function in turkeys. Met content in the feed has a moderate effect on the immune response in HEV-infected turkeys. The source of this amino acid appears not be as important as its dose, because value of the analysed parameters did not differ significantly between turkeys receiving feeds with DLM or MHA. In the uninfected turkeys, the higher by 40% (than recommended by NRC) level of Met in the feeds had a positive effect on humoral immunity parameters.

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.

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.

Longitudinal field studies of avian metapneumovirus and turkey hemorrhagic enteritis virus in turkeys suffering from colibacillosis associated mortality.

The aim of this study was to evaluate if the exposure to Avian metapneumovirus (aMPV) and/or to Turkey hemorrhagic enteritis virus (THEV) was significant for the induction of episodes of colibacillosis in aMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. aMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques for viruses detection and antibody titres were evaluated. Field subtype B aMPVs were detected in all flocks at different ages of life always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems does not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field aMPV infection is directly correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of aMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of aMPV. It would be interesting to further investigate this issue through experimental trials in secure isolation conditions.

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.

A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge.

The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750µg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200µg 4F/4R S fragment. One dose of 100µg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750µg DNA vaccine and one dose of 200µg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100µg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.

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 enteric viruses in breeder turkeys.

The present study was undertaken to detect and characterize enteric viruses (rotavirus, astrovirus, reovirus, and coronavirus) in breeder poults. Five turkey breeder flocks were selected. Faecal samples were collected from all flocks at 1 week of age and then every other week until the poults reached 9 weeks of age. The faecal samples were pooled in groups of five. Of the 193 pools ("samples") tested by reverse transcription-polymerase chain reaction, 47.2%, 30.6%, and 10.4% samples were positive for astrovirus, rotavirus, and reovirus, respectively. No coronavirus was detected in any of the samples. Overall, 118 (61.1%) samples were positive for one or more enteric viruses. Of the 118 samples, 70 (59.3%) were positive for a single virus and 48 (40.7%) for a combination of viruses. Phylogenetic analysis based on the polymerase gene showed that astroviruses clustered into two groups with sequence homology ranging from 85.6 to 100% at the nucleotide level. Based on NSP4 gene sequences, rotaviruses clustered in a group and had 96.3 to 99.9% sequence homology at the nucleotide level. The reoviruses, based on their S4 gene sequences, clustered in a single group with sequence homology of 96.9 to 100%. Differing amino acid sequences of all three viruses may affect the antigenicity and/or pathogenicity of these viruses and may merit further study. The presence of two or three different viruses in combination may affect the dynamics of turkey health and disease.

Pathology and tissue distribution of turkey coronavirus in experimentally infected chicks and turkey poults.

Twenty 1-day-old specific pathogen free chicks and 20 1-day-old commercially derived turkey poults were inoculated with a Brazilian strain of turkey coronavirus (TCoV) to study the pathogenicity and virus distribution up to 14 days post-inoculation by histopathology, immunohistochemistry, reverse transcriptase polymerase chain reaction and sequencing. At 2-14 dpi, TCoV antigens were detected in the paranasal sinus and lachrymal accessory gland (Harderian gland) of infected chicks and in the ileum, ileocaecal junction and caecum of infected poults. Lymphocytic inflammation was present in these tissues. TCoV was re-isolated from pooled tissue suspensions of nasal concha, Harderian gland and paranasal sinus from chicks, as well as from the ileum, ileocaecal junction and caecum of poults, after three consecutive passages in 28-day-old embryonated turkey eggs. Viral RNA corresponding to the spike gene region (1178-2073 genome position) was amplified from the upper respiratory tract of chickens and from the intestinal tract of poults and phylogenetic analysis confirmed the identity as TCoV. This is the first description of TCoV antigens and mRNA in upper respiratory tissues in experimentally infected chickens.

Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys.

This study was conducted to detect and characterize enteric viruses [rotavirus, turkey astrovirus-2 (TAstV-2), reovirus, and turkey coronavirus] from cases of poult enteritis syndrome (PES) in Minnesota turkeys. Of the intestinal contents collected from 43 PES cases, 25 were positive for rotavirus and 13 for small round viruses by electron microscopy (EM). Of the enteric virus-positive cases by EM (n=27), 16 cases had rotavirus or small round viruses alone and the remaining 11 cases had both viruses. None of the cases were positive for reovirus or coronavirus by EM. However, with reverse transcription-PCR (RT-PCR), 40 cases (93%) were positive for rotavirus, 36 (84%) for TAstV-2, and 17 (40%) for reovirus. None of the cases were positive for turkey coronavirus by RT-PCR. The viruses from all cases were detected either alone or in combination of 2 or 3 by RT-PCR. Thus, 8 (19%) cases were positive for a single virus, whereas a combination of viruses was detected in the remaining 35 (81%) cases. The rota-TAstV-2 combination was the most predominant (n=18 cases). Fifteen cases were positive for all 3 viruses. The rotaviruses had sequence homology of 89.8 to 100% with previously published sequences of turkey rotaviruses at the nucleotide level. The TAstV-2 had sequence homology of 84.6 to 98.7% with previously published TAstV-2, whereas reoviruses had sequence homology of 91.6 to 99.3% with previously published sequences of turkey reoviruses. Phylogenetic analysis revealed that rota- and reoviruses clustered in a single group, whereas TAstV-2 clustered in 2 different groups. In conclusion, a larger number of PES cases was positive for rotavirus, TAstV-2, and reovirus by RT-PCR than with EM. The presence of more than one virus and changes at the genetic level in a virus may affect the severity of PES in turkey flocks.

Specific real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus.

Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in the turkey poults, leading to significant economic loss in the U.S. turkey industry. Rapid detection, differentiation, and quantitation of TCoV are critical to the diagnosis and control of the disease. A specific one-step real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay for detection and quantitation of TCoV in the turkey tissues was developed using a dual-labeled fluorescent probe. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamin) and a quencher dye (AbsoluteQuencher) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3' end of spike gene of TCoV. The assay was performed on different avian viruses and bacteria to determine the specificity as well as serial dilutions of TCoV for the sensitivity. Three animal trials were conducted to further validate the assay. Ten-day-old turkey poults were inoculated orally with 100 EID(50) of TCoV. Intestinal tissues (duodenum, jejunum, ileum, cecum), feces from the cloacal swabs, or feces from the floor were collected at 12 h, 1, 2, 3, 5, 7, and/or 14 days post-inoculation (DPI). RNA was extracted from each sample and subjected to the RRT-PCR. The designed primers and probe were specific for TCoV. Other non-TCoV avian viruses and bacteria were not amplified by RRT-PCR. The assay was highly sensitive and could quantitate between 10(2) and 10(10) copies/microl of viral genome. The viral RNA in the intestine segments reached the highest level, 6x10(15) copies/microl, in the jejunum at 5 DPI. Eighty-four intestine segments assayed by the developed RRT-PCR and immunofluorescence antibody assay (IFA) revealed that there were 6 segments negative for TCoV by both assays, 45 positive for TCoV by IFA, and 77 positive for TCoV by RRT-PCR. Turkey coronavirus was detected in the feces from the cloacal swabs or floor 1-14 DPI; however, the viral RNA load varied among different turkey poults at different intervals from different trials. The highest amount of viral RNA, 2.8x10(10) copies/microl, in the feces was the one from the cloacal swab collected at 1 DPI. The average amount of TCoV RNA in the cloacal fecal samples was 10 times higher than that in the fecal droppings on the floor. Taken together, the results indicated that the developed RRT-PCR assay is rapid, sensitive, and specific for detection, differentiation, and quantitation of TCoV in the turkey tissues and should be helpful in monitoring the progression of TCoV induced acute enteritis in the turkey flocks.

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.

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.

Lack of evidence of avian adenovirus infection among turkey workers.

Zoonotic infections constitute a major public health concern. Outbreaks of the SARS (severe acute respiratory syndrome) and avian influenza viruses are but recent examples. Although there are many animal-specific adenoviruses and occasionally they have been noted to infect man, rarely have they been studied as potential zoonotic pathogens. In this study, the authors hypothesized that the hemorrhagic enteritis virus (HEV), an avian adenovirus that causes illness among turkeys, might infect humans. Using an enzyme immunosorbent assay, the authors compared sera from 95 turkey-exposed individuals with sera from 82 nonexposed controls for serologic evidence of infection with HEV. Multivariate modeling revealed no statistical difference in elevated antibody titers against HEV between the two groups. These data do not support the hypothesis that avian adenoviruses cross the species barrier to infect humans.

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.