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.

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 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.

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.

High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus.

Six-day-old turkeys were inoculated with turkey coronavirus (TCV) and an enteropathogenic Escherichia coli (EPEC) (isolate R98/5) that were isolated from poult enteritis and mortality syndrome (PEMS)-affected turkeys. Turkeys inoculated with only R98/5 did not develop clinically apparent disease, and only mild disease and moderate growth depression were observed in turkeys inoculated with only TCV. Turkeys dually inoculated with TCV and R98/5 developed severe enteritis with high mortality (38/48, 79%) and marked growth depression. R98/5 infection resulted in attaching/effacing (AE) intestinal lesions characteristic of EPEC: adherence of bacterial microcolonies to intestinal epithelium with degeneration and necrosis of epithelium at sites of bacterial attachment. AE lesions were more extensive and were detected for a prolonged duration in dually inoculated turkeys compared with turkeys inoculated with only R98/5. An apparent synergistic effect in dually inoculated turkeys was indicated by increased mortality, enhanced growth depression, and enhanced AE lesion development. The results suggest that TCV promoted intestinal colonization by R98/5; however, R98/5 did not appear to alter TCV infection. The present study provides a possible etiologic explanation for PEMS.

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.

Antigenic characterization of a turkey coronavirus identified in poult enteritis- and mortality syndrome-affected turkeys.

A turkey coronavirus (TCV [NC95]) was characterized by antigenic comparison with other avian and mammalian coronaviruses using immunofluorescence (FA) and immunoperoxidase (IP) procedures. Based on FA and IP procedures, TCV (NC95) was determined to be antigenically indistinguishable from turkey enteric (bluecomb) coronavirus (TECV). In addition, TCV (NC95) and TECV were found to be closely related to infectious bronchitis virus (IBV); a one-way antigenic relationship was demonstrated. Polyclonal antibodies specific for TECV and IBV reacted strongly against TCV (NC95), as determined by FA procedures. Monoclonal antibodies (MAbs) specific for IBV matrix protein (MAb 919) reacted strongly against TCV (NC95) and TECV as determined by FA and IP procedures; an IBV peplomer protein-specific MAb (MAb 94) did not recognize the two viruses. These studies suggest an identification of TCV (NC95) as a strain of TECV, and provide evidence of a close antigenic relationship between these viruses and IBV.

Spiking mortality of turkey poults: 1. Experimental reproduction in isolation facilities.

Spiking mortality of turkeys (SMT) is an infectious disease of 5-to-25-day-old turkey poults characterized by acute enteritis and bursal and thymic atrophy. Brooding 1-day-old poults on litter taken from naturally occurring cases successfully reproduced SMT 5 days postexposure. Oral exposure to an organ homogenate made of tissue samples from naturally occurring cases successfully reproduced SMT 5 days postinoculation. Coronaviruses were present in intestinal and bursal contents taken from poults with naturally occurring SMT. They were also present 5 days after exposure in the experimentally reproduced disease. Severe intestinal villus atrophy, bursal follicular lymphoid depletion, and thymic cortical atrophy were present histologically in naturally occurring SMT and in SMT reproduced by either experimental method.

Spiking mortality of turkey poults: 2. Effect of six different in vitro disinfection techniques on organ homogenates capable of reproducing SMT.

Spiking mortality of turkeys (SMT) is an infectious disease of 5-to-25-day-old turkey poults characterized by acute enteritis and bursal and thymic atrophy. It is reproducible by exposure to organ homogenates or contaminated litter. We studied methods potentially useful for decontamination of turkey houses contaminated with SMT. Organ homogenates capable of producing SMT and containing turkey intestinal coronavirus were exposed in vitro for 5 hr to either 5.0% NaCl, pH 2.0, pH 12, 1.0% formaldehyde, 57 C, or lyophilization. Results were assessed by oral gavage of treated inocula into 1-day-old turkeys and measurement of subsequent coronavirus shedding, growth rate, feed conversion, and mortality from 1 to 21 days of age. Formaldehyde treatment rendered the inoculum nonpathogenic, whereas other treatments failed to ameliorate its negative effects.

A monoclonal antibody-based immunoperoxidase method for rapid detection of haemorrhagic enteritis virus of turkeys.

An indirect immunoperoxidase (IP) technique involving avidin-biotin peroxidase complex, using a monoclonal antibody was developed for the detection of haemorrhagic enteritis (HE) virus antigen in frozen and formalin-fixed paraffin-embedded tissue sections. This IP procedure was compared with an indirect immunofluorescence antibody technique (IFAT) and an agar gel precipitation test (AGPT). Spleens from turkeys experimentally infected with HE virus were collected and examined for the presence of viral antigen. The IP staining procedure detected HE viral antigen as early as 48 hours after infection and continued to demonstrate the presence of viral antigen for up to 11 days after infection at which time the experiment was terminated. The antigen was detected from three to seven days and from two to nine days after infection by the AGPT and IFAT, respectively. The IFAT and AGPT had sensitivities of 74.19 and 48 per cent, respectively, compared with IP. Because of its high sensitivity and specificity, the IP technique could be useful for studying the pathogenesis and rapid laboratory detection of HE virus.

Antigenic and genomic relationships among turkey and bovine enteric coronaviruses.

Antigenic and genomic relationships among tissue culture-adapted turkey enteric coronavirus (TCV) isolates, three strains of avian infectious bronchitis virus (IBV), and mammalian coronaviruses were investigated. Immunoblotting and immunoprecipitation experiments using polyclonal antisera showed that the four major structural proteins of TCV cross-reacted with the four homologous proteins of bovine enteric coronavirus (BCV), the N and M proteins of mouse hepatitis virus serotype 3, and the N protein of IBV. Close antigenic relationships between TCV and BCV were also established by seroneutralization and hemagglutination-inhibition. Of 49 monoclonal antibodies produced against either TCV or BCV, 11 differentiated the two viruses. Five of these monoclonal antibodies had neutralizing activities and were directed to either the peplomeric S (gp200-gp100) or hemagglutinin HE (gp140-gp65) glycoproteins. BCV cDNA probes tested on purified viral preparations and coronavirus-positive (by electron microscopy) fecal samples from diarrheic turkey poults confirmed the relatedness of TCV and BCV. The two viruses produced distinct cytopathic changes in HRT-18 cells in the presence of trypsin, whereas only TCV isolates were able to reproduce the clinical symptoms in turkey poults. Their matrix (M) proteins undergo different glycosylation processes.

Field outbreaks of colibacillosis of turkeys associated with hemorrhagic enteritis virus.

In a study of field material and a survey conducted by the authors, typical signs of colibacillosis of 6-to-12-week-old poults included sudden onset, listlessness, rales, and high mortality. Signs persisted for about 2 weeks and were often followed by a low incidence of lameness caused by Escherichia coli. Gross lesions included enlarged and congested spleens and livers, and dilated discolored black or purple duodenal loops. Microscopic lesions included splenic and hepatic congestion. In some birds (freshly killed and fixed immediately), the epithelium at the tips of the duodenal villi was sloughing, but in other birds the villi were intact and normal in appearance. Splenic enlargement, the presence of intranuclear splenic inclusions similar to those found in hemorrhagic enteritis (HE), and the isolation of HE virus from some of the field spleens all indicated that inapparent HE infection often occurs at approximately the same time as this type of colibacillosis. It is therefore believed that HE infection often exacerbates colibacillosis of older poults.

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

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.

Scanning electron, light, and immunofluorescent microscopy of coronaviral enteritis of turkeys (Bluecomb).

Intestinal sections from both experimental and field cases of turkey coronaviral enteritis (TCE) were examined by scanning electron microscopy and light microscopy through 10 days after inoculation and by a direct fluorescent antibody test for TCE through 12 days. Serums were collected for an indirect fluorescent antibody test for TCE through 160 days after inoculation. Lesions observed with the scanning electron microscopy were catarrhal enteritis with hemorrhage per diapedesis, epithelial desquamation, and villous atrophy which developed and regressed within 6 days after inoculation. Light microscopy demonstrated similar lesions, except that villus-to-crypt ratios remained depressed 10 days. The direct fluorescent antibody test demonstrated the presence of coronaviral antigen throughout the sampling period, and serum antibodies to TCE were present until at least 160 days, when the experiment was terminated.

Histopathology of fasting and bluecomb disease in turkey poults and embryos experimentally infected with bluecomb disease coronavirus.

The histopathology of fasting and bluecomb disease in one-day-old turkey poults inoculated with bluecomb disease coronavirus (BCDCV) was studied. Uninoculated fasting poults produced clinical signs similar to those observed in BCDCV-inoculated poults. No histological changes in the intestines were observed in the fasted poults whereas definite lesions were observed in the BCDCV-inoculated poults. The lesions did not differ significantly with whether they were fed or fasted. The severity of the lesions in the intestinal epithelium was in decreasing order in the jejunum, ileum, and cecum. The lesions first appeared 24 hours postinoculation (PI) and progressed through 96 hours PI, as marked shortening of the villi, loss of microvilli, granular appearance of the cytoplasm of epithelial cells with nuclear margination of chromatin, and accentuation of the nucleolus. Similar lesions were observed in the jejunum, ileum, and cecum of turkey embryos inoculated at 24 days old as well as poults from these embryos. Signs of healing were first seen at 120 hours PI. No histopathological changes were observed in the pancreas, brain, kidneys, liver, adrenal, and bursa of Fabricius. The intestinal lesions observed should be a useful histological technique for differentiating fasting from bluecomb disease in turkey poults.