Runting Stunting Syndrome Associated with Transmissible Viral Proventriculitis in Broiler Chickens.

This report describes an outbreak of transmissible viral proventriculitis (TVP) associated with runting stunting syndrome (RSS) in 25- and 28-day-old broiler chickens, in which chicken proventricular necrosis virus (CNPV) was detected. Clinical signs included poor uniformity, very small birds for their age, increased mortality, and culling of smaller birds. Almost all birds necropsied exhibited moderate to severely enlarged proventriculi with diffusely pale serosa and thickened walls. Microscopically the proventriculi had lesions of degeneration and necrosis of the epithelium of the proventricular glands, accompanied by lymphocytic inflammation and glandular hyperplasia, with occasional formation of lymphoid nodules within the glandular parenchyma. Immunohistochemistry staining for CPNV was positive. Positive staining was generally found in the cytoplasm of glandular epithelial cells in the form of finely granular brown pigment. CPNV RNA was detected in the proventriculi by reverse transcriptase-PCR (RT-PCR). Other findings included mild enteritis in a few birds and small bursa of Fabricius. Direct electron microscopy performed on the intestinal samples was negative for viral particles. RT-PCR analysis of bursae was positive for infectious bursal disease virus (IBDV). In conclusion, this report associates TVP with RSS by describing an outbreak in which TVP attributable to CPNV was the most commonly found lesionin chickens with a clinical history compatible with RSS. Therefore, TVP should be considered as a possible differential diagnosis in cases compatible with RSS.

Parvovirus-associated cerebellar hypoplasia and hydrocephalus in day old broiler chickens.

Cerebellar hypoplasia and hydrocephalus were identified in day old broiler chickens showing nervous signs, impaired mobility, and diarrhea. At postmortem examination, brains of chickens were misshapen and cerebellums were smaller than normal. Microscopically, cerebellar folia were reduced in size and irregularly shaped, and the ventricles were widely distended. Affected cerebellums had focal areas along the base of folia where the internal granular cell layer had been lost, and Purkinje cells were disorganized and located within the molecular layer. Parvovirus DNA was detected by polymerase chain reaction in three of nine brains with oligonucleotide primers designed for amplification of chicken and turkey parvoviruses. On the basis of phylogenetic analyses, the detected virus was most closely related to chicken parvoviruses. These findings suggest that a chicken parvovirus might cause a neurologic disease of young chickens characterized by cerebellar hypoplasia and hydrocephalus; however, its role as the cause of the disease remains to be confirmed.

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.

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome.

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.

Baculovirus expression of turkey coronavirus nucleocapsid protein.

The nucleocapsid (N) gene of turkey coronavirus (TCV) was amplified by reverse transcriptase-polymerase chain reaction, cloned, and expressed in the baculovirus expression system. A recombinant baculovirus containing the TCV N gene (rBTCV/N) was identified by polymerase chain reaction and expression of TCV N protein as determined by western immunoblot analysis. Two TCV-specific proteins, 52 and 43 kDa, were expressed by rBTCV/N; one of these proteins, p52, was comparable in size to native TCV N protein. Baculovirus-expressed N proteins were used as antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for detection of TCV-specific antibodies. The ELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related avian coronavirus, but did not detect antibodies specific for other avian viruses (avian influenza, avian reovirus, avian paramyxovirus 3, avian adenovirus 1, or Newcastle disease virus). These findings indicate that baculovirus-expressed TCV N protein is a suitable source of antigen for ELISA-based detection of TCV-specific antibodies in turkeys.

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.

Characterization of a coronavirus isolated from a diarrheic foal.

A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.

Parvovirus infection of keratinocytes as a cause of canine erythema multiforme.

Erythema multiforme major was diagnosed in a dog with necrotizing parvoviral enteritis. Skin lesions consisted of ulceration of the footpads, pressure points, mouth, and vaginal mucosa; vesicles in the oral cavity; and erythematous patches on the abdomen and perivulvar skin. Microscopic examination of mucosal and haired skin specimens revealed lymphocyte-associated keratinocyte apoptosis at various levels of the epidermis. Basophilic cytoplasmic inclusions were seen in basal and suprabasal keratinocytes. Immunohistochemical staining, performed with canine parvovirus-2-specific monoclonal antibodies, confirmed the parvovirus nature of the inclusions in the nucleus and cytoplasm of oral and skin epithelial cells. This is the first case of canine erythema multiforme reported to be caused by a viral infection of keratinocytes. This case study indicates that the search for epitheliotropic viruses should be attempted in cases of erythema multiforme in which a drug cause cannot be identified.

Comparison of virus isolation, immunohistochemistry, and reverse transcriptase-polymerase chain reaction procedures for detection of turkey coronavirus.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure and two monoclonal antibody (MAb)-based immunohistochemical procedures were developed for detection of turkey coronavirus (TCV) in tissues and intestinal contents/dropping samples. The RT-PCR, MAb-based fluorescent antibody (FA), and MAb-based immunoperoxidase (IP) procedures were compared with virus isolation (VI) for detection of TCV in experimentally infected turkeys. TCV was detected in experimentally infected turkeys as early as day 1 postexposure (PE) by each of the four detection procedures. TCV was detected as late as day 35 PE by FA or IP and days 42 and 49 PE by VI and RT-PCR, respectively. With VI as a reference, sensitivity and specificity of RT-PCR were 93% and 92%, respectively; specificity of both FA and IP was 96%, and sensitivities were 69% and 61%, respectively. Each of the examined procedures was highly specific, but the RT-PCR procedure was also highly sensitive. These findings demonstrate the utility of both immunohistochemistry and RT-PCR for detection of TCV. In addition, the findings indicate that RT-PCR is a highly sensitive and specific alternative to conventional diagnostic procedures.

Avian infectious laryngotracheitis.

Avian infectious laryngotracheitis (ILT) herpesvirus continues to cause sporadic cases of respiratory disease in chickens world-wide. Sources of transmission of ILT infection are three-fold, namely: chickens with acute upper respiratory tract disease, latently infected 'carrier' fowls which excrete infectious laryngotracheitis virus (ILTV) when stressed, and all fomites (inanimate articles as well as the personnel in contact with infected chickens). Infectious laryngotracheitis virus infectivity can persist for weeks to months in tracheal mucus or carcasses. Rigorous site biosecurity is therefore critical in ILT disease control. Furthermore, while current (modified live) ILT vaccines can offer good protection, the strains of ILTV used in vaccines can also produce latent infections, as well as ILT disease following bird-to-bird spread. The regional nature of reservoirs of ILTV-infected flocks will tend to interact unfavourably with widely varying ILT control practices in the poultry industry, so as to periodically result in sporadic and unexpected outbreaks of ILT in intensive poultry industry populations. Precautions for trade-related movements of chickens of all ages must therefore include an accurate knowledge of the ILT infection status, both of the donor and recipient flocks.

Poult enteritis complex.

Poult enteritis complex (PEC) is a general term that encompasses the infectious intestinal diseases of young turkeys. Some diseases, such as coronaviral enteritis and stunting syndrome, are relatively well characterised, while others, such as transmissible viral enteritis, poult growth depression and poult enteritis mortality syndrome, remain ill-defined. All forms of PEC are multifactorial, transmissible and infectious. Salient clinical features include stunting and poor feed utilisation that result from enteritis. In the more severe forms, runting, immune dysfunction and mortality are reported. Gross and microscopic lesions of enteritis are present in all forms but tend to be non-specific. Other lesions may be present, depending on the agents involved. The basic pathogenesis involves the following: a) alteration of the intestinal mucosa, generally by one or more viruses infecting enterocytes; b) inflammation; c) proliferation of secondary agents, usually bacteria. Non-infectious factors interplay with infectious agents to modulate the course and severity of disease. Diarrhoea is believed to be primarily osmotic because of maldigestion and malabsorption, but may also have a secretory component. Transmission is primarily faecal-oral. No public health significance is recognised or suspected. Prevention is based on eliminating the infectious agents from contaminated premises and preventing introduction into flocks. This is accomplished by an effective cleaning, disinfection and biosecurity programme. All-in/all-out production or separate brooding and finishing units are helpful. Control may require regional co-ordination among all companies producing turkeys, especially if the production is highly concentrated, and a quarantine programme for more severe forms of PEC. No vaccines or specific measures for controlling the organisms involved in PEC are available. Treatment is supportive for the viral component, while antibiotics, especially those with efficacy against Gram positive bacteria, may help to reduce the impact to bacterial infections. Evidence suggests that PEC occurs wherever turkeys are raised commercially, but this is not well documented and distribution of the various organisms that have been associated with PEC is largely unknown. The disease causes enormous economic loss, mostly from failure of the turkey to reach its genetic potential.

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.

Limited transmission of turkey coronavirus in young turkeys by adult Alphitobius diaperinus (Coleoptera: Tenebrionidae).

We examined the role of lesser mealworm, Alphitobius diaperinus (Panzer), in the transmission of an enteric disease of turkeys caused by a coronavirus. Turkey coronavirus (TCV) from two sources was studied, one isolate (NC95) was embryo propagated, the second was TCV infected material from turkeys diagnosed with poult enteritis mortality syndrome (PEMS). Beetles were fed virus-infected feces mixed with chicken feed. Transmission of virus was effectively halted by surface sterilization of the beetles. Turkey poults administered beetle homogenates infected with TCV+ PEMS that had not been surface sterilized had reduced weight gains and 50% mortality. Mortality and weight gains were not effected in the NC95 group. Virus isolation procedures were performed to determine NC95 viability at varying time intervals. Beetles were dissected and the guts removed 1, 12, and 24 h after the initial viral feeding. Whole beetles were also examined for comparison. Whole beetles and beetle guts were homogenized and injected into turkey eggs for embryo propagation. Direct immunofluorescence was used to determine the presence of TCV. A. diaperinus were capable of mechanical transmission of TCV. However, only turkey embryos receiving whole beetle and beetle gut homogenates within 1 h of feeding on the virus were positive for TCV. Laboratory studies demonstrating PEMS transmission by A. diaperinus are continuing.

Turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review.

Turkey coronavirus (TCoV) is the cause of an acute highly contagious enteric disease of turkeys. In recent years, TCoV has been increasingly recognized in North America as an important pathogen of young turkeys, resulting in economic loss due to impaired growth and poor feed conversion. While the epidemiology and pathogenesis of TCoV have been extensively studied, TCoV remains one of the least characterized of the known coronaviruses. Avian and mammalian coronaviruses have been subdivided into distinct antigenic/genotypic groups; however, classification of TCoV has been controversial. Previous studies indicated that TCoV was closely related to bovine coronavirus and other group 2 mammalian coronaviruses, but more recent antigenic and genome sequence analyses contradict these findings and, instead, provide evidence that TCoV is closely related to avian infectious bronchitis virus (IBV). Additionally, experimental studies have indicated that the host range of TCoV, once thought to be restricted to turkeys, includes chickens. These studies have raised additional questions regarding the classification of TCoV; particularly, whether IBV and TCoV are taxonomically distinct viruses, or whether TCoV is merely a variant of IBV. Sequence analyses of TCoV have given credence to the idea that TCoV is a variant of IBV, as these studies have shown that TCoV and IBV are very closely related. However, these studies have been limited to only three TCoV strains and relatively small portions of the TCoV genome. TCoV is readily distinguished from IBV based on antigenic and biological differences, and these differences suggest that TCoV should be considered a distinct virus species. Additional studies will be needed to better define the relationship between TCoV and IBV, and to resolve this taxonomic question. Based on our current understanding, it seems prudent to consider TCoV and IBV as distinct virus species that share a close phylogenetic relationship and together comprise group 3 of the coronavirus major antigenic groups.

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

Sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus.

A reverse transcriptase, polymerase chain reaction (RT-PCR) procedure was used to amplify a segment of the genome of turkey coronavirus (TCV) spanning portions of the matrix and nucleocapsid (MN) protein genes (approximately 1.1 kb). The MN gene region of three epidemiologically distinct TCV strains (Minnesota, NC95, Indiana) was amplified, cloned into pUC19, and sequenced. TCV MN gene sequences were compared with published sequences of other avian and mammalian coronaviruses. A high degree of similarity (>90%) was observed between the nucleotide, matrix protein, and nucleocapsid protein sequences of TCV strains and published sequences of infectious bronchitis virus (IBV). The matrix and nucleocapsid protein sequences of TCV had limited homology (<30%) with MN sequences of mammalian coronaviruses. These results demonstrate a close genetic relationship between the avian coronaviruses, IBV and TCV.

Hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection.

Six weeks after vaccination with modified live feline parvovirus vaccine, a cat gave birth to five kittens, three of which died soon afterwards. The remaining two kittens (A and B) survived, but at 8 weeks of age were unable to walk and showed abnormal behaviour, with lack of menace and oculovestibular responses, and severe dysmetria. These signs suggested multifocal disease associated with the cerebrum and cerebellum. Magnetic resonance imaging demonstrated severe bilateral (kitten A) or unilateral (kitten B) hydrocephalus or hydranencephaly, combined with cerebellar agenesis (kitten A) or severe hypoplasia (kitten B). Hydranencephaly was confirmed histopathologically in both kittens. Parvovirus was isolated from the kidney of one kitten. Parvoviral DNA was amplified by the polymerase chain reaction (PCR) from paraffin wax-embedded brain of both kittens. The severe malformations observed in these kittens presumably resulted from an in-utero parvovirus infection, possibly due to vaccination, that occurred late in the first, or early in the second, trimester of pregnancy.

Virus infections of the gastrointestinal tract of poultry.

Several different viruses have been identified as causes of gastrointestinal tract infections in poultry. These include rotaviruses, coronaviruses, enteroviruses, adenoviruses, astroviruses, and reoviruses. In addition, a number of other viruses of unknown importance have been associated with gastrointestinal diseases in poultry based on electron microscopic examination of feces and intestinal contents. Viral infections of the gastrointestinal tract of poultry are known to negatively impact poultry production, and they likely contribute to the development of other, extragastrointestinal diseases. Our current understanding of the viruses that cause gastrointestinal tract infections in poultry is reviewed, with emphasis given to those of greatest importance.

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.