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.

Experimental ocular herpesvirus infection in the cat. Sites of virus replication, clinical features and effects of corticosteroid administration.

Experimentally induced ocular feline herpesvirus 1 (FHV-1) infection was studied in 30 specific pathogen-free cats. In ten cats, the ability of five field isolates of FHV-1 to replicate in the epithelium and substantia propria of cornea and conjunctiva was demonstrated by histochemical techniques. Feline herpesvirus 1 was found to preferentially infect and induce necrosis of conjunctival epithelium. Although significant histologic lesions were not induced, all FHV-1 strains were observed to replicate in corneal epithelium; minimal viral antigen was detected in the corneal stroma. The course and clinical features of ocular FHV-1 infection were then studied over a period of 60 days in two groups of ten cats: in one group, infection was preceded by administration of subconjunctival betamethasone. In each of these groups, a distinct clinical syndrome developed. In cats not receiving corticosteroids, a course of epithelial keratitis, characterized by the formation of punctate and dendritic epithelial lesions, persisted for up to 24 days postinfection. In the corticosteroid treated group, a chronic (greater than 60 days) stromal keratitis developed, characterized by geographic epithelial ulceration, interstitial edema and deep vascularization. Other complications observed in corticosteroid-treated animals included decreased tear production, calcific-band keratopathy and a unique stromal disorder of cats termed corneal sequestration. The results of this study indicate that while epithelial keratitis may occur during primary infection, stromal keratitis does not, unless immune responsiveness to FHV-1 is concomitantly suppressed. This feature is similar to naturally occurring HSV-1 keratitis of humans, but contrasts to other animal model systems in which stromal keratitis predictably occurs during primary infection. Study of this animal model, therefore, may allow unique insights into the events preceding the establishment of stromal keratitis.

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.

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.

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.

Partial characterization of a turkey enterovirus-like virus.

Small round viruses, 18 to 24 nm in diameter, were detected by electron microscopy in droppings of young turkeys with enteritis. The virus was propagated in embryonated turkey eggs and tentatively identified as an enterovirus based on size, intracytoplasmic morphogenesis, buoyant density of 1.33 g/ml in CsCl, and a single-stranded RNA genome of approximately 7.5 kb. It was distinguished from avian encephalomyelitis virus by cross-immunofluorescence. These results identify an enterovirus-like virus as a possible etiologic agent of enteric disease of young turkeys. However, its role in this disease remains to be established.

Characterization of an avian adenovirus associated with inclusion body hepatitis in day-old turkeys.

A group I avian adenovirus isolated from day-old turkeys with inclusion body hepatitis (IBH) was identified as turkey adenovirus serotype 2 (TAV2) based on cross-neutralization assays and DNA restriction endonuclease analyses. Yolk sac inoculation of embryonated turkey eggs resulted in embryo mortality and significantly (P < 0.01) decreased hatchability compared with sham-inoculated controls. Embryo mortality occurred primarily between day 24 of incubation and the time embryos hatched. Focal necrosis was detected in livers of 11/52 virus-inoculated embryos that died postinoculation and 1/27 hatchlings; in three embryos, areas of necrosis contained intranuclear inclusion bodies. These findings identify the IBH isolate as TAV2, incriminate the virus as a potential cause of suboptimal hatchability in turkeys, and provide additional evidence for causal involvement in IBH.

Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage.

Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.

An outbreak of influenza (H1N1) in turkey breeder hens.

Outbreaks of influenza were diagnosed in two turkey breeder flocks on the same premises in eastern North Carolina during the "dark-out" period of recycling for a second lay. Clinical history included increased mortality from acute death with no apparent predisposing illness. Mortality attributed to the disease was 4.5% in one flock and 3.3% in the other. Necropsy findings included severe diffuse congestion and edema of both lungs, with little or no pleural exudate. Spleens were moderately to markedly enlarged and mottled, and kidneys were swollen and congested. Microscopic lesions included moderate to severe serofibrinous pneumonia with severe pulmonary congestion. Splenic changes included fibrin deposition and severe congestion, and severe congestion was noted in kidneys. Influenza virus (H1N1) was isolated from pools of tissues including lung, spleen, liver, and kidney, and both flocks seroconverted to influenza (H1N1) virus.

Virulence of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates.

Virulence of six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses was compared with that of 11 field isolates (indistinguishable from vaccine viruses by DNA restriction endonuclease analyses) by intratracheal exposure of 4-week-old, specific-pathogen-free chickens. Virulence of ILT viruses was based on an intratracheal pathogenicity index, mortality, and tracheal lesions. Intratracheal pathogenicity indices for ML vaccine viruses ranged from 0.0 to 0.14, while those for field isolates were 0.20 to 0.82. Mortality was a consistent clinical feature of field isolates; all produced mortality, with seven of the 11 isolates causing two or more deaths per inoculation group. In contrast, only one of six ML vaccine viruses produced mortality (one death per inoculation group). In general, tracheal lesions were more severe in chickens inoculated with field isolates and were produced more consistently than in chickens inoculated with vaccine viruses. These studies indicate that virulence of ILT field isolates was greater than that of ML vaccine viruses. Together with previous restriction endonuclease analyses, these findings suggest the possibility that field isolates originated from ML vaccine viruses through reversion to parental-type virulence.

Inclusion-body hepatitis in day-old turkeys.

Inclusion-body hepatitis was diagnosed in 1-day-old turkeys experiencing above-average mortality. At necropsy, turkeys appeared anemic and had pale yellow livers. Histopathologic examination of affected livers revealed diffuse hepatic degeneration and multifocal necrosis, with approximately 70% of the hepatocytes containing large, basophilic, intranuclear inclusion bodies. An adenovirus was isolated from affected livers and identified as a group I avian adenovirus by indirect immunofluorescence.

Experimental infection of young broiler chickens with eastern equine encephalitis virus and Highlands J virus.

Two-week-old broiler chickens were experimentally infected with either eastern equine encephalitis (EEE) virus or Highland J (HJ) virus. Mortality rates were 24/30 (80%) in EEE-virus-inoculated chickens and 2/30 (7%) in HJ-virus-inoculated chickens. Chickens inoculated with EEE virus exhibited severe depression and somnolence on days 1-6 postexposure (PE), with 17/30 birds dying during this period. After day 6 PE, EEE-virus-inoculated chickens exhibited abdominal distention, depression, and growth retardation, and an additional seven chickens died. Pathologic changes in EE-virus-inoculated chickens dying on days 1-6 PE consisted of multifocal necrosis in the heart and liver, as well as lymphoid depletion and necrosis in the thymus, spleen, and bursa of Fabricius. Ascites, pericardial effusion, and right ventricular dilatation of the heart were the predominant lesions in chickens dying after day 6 PE. No clinical signs were observed in sham-inoculated controls or in most HJ-virus-inoculated chickens. Ascites, pericardial effusion, and multifocal myocardial necrosis were observed in 2/30 HJ-virus-inoculated chickens that died or were euthanatized after development of clinical signs. These findings indicate that both EEE virus and HJ virus are pathogenic for young chickens.

Restriction endonuclease analysis of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates.

Six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, three reference strains, and 18 field isolates were compared by restriction endonuclease analysis of their DNA. Viral DNA digestion patterns were established for vaccine viruses using restriction endonucleases PstI, BamHI, KpnI, and HindIII. Using these enzymes, five of six ML vaccine viruses had identical restriction endonuclease cleavage patterns. Vaccine viruses had distinct patterns compared with ILT virus reference strains Illinois-N71851, Cover, and NVSL. Restriction endonuclease cleavage patterns of 18 field isolates of ILT virus, obtained from ILT outbreaks in North Carolina, were indistinguishable from vaccine viruses. These results suggest a possible role of vaccine or vaccine-like viruses in recent ILT outbreaks.

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.

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.

Interaction of reovirus and Cryptosporidium baileyi in experimentally infected chickens.

No clinical signs, gross lesions, or increased mortality were observed in specific-pathogen-free chickens orally inoculated at 5 days of age with Cryptosporidium baileyi, reovirus 2035, reovirus 2408, or combinations of these agents. Weight gain of chickens inoculated with only reovirus 2408 was depressed 0-8 days postinoculation (PI) (P less than 0.01) but not for the 21-day period PI. Weight gain of chickens inoculated with only reovirus 2035 was not affected. Cryptosporidium baileyi infection significantly depressed weight gain 8-14 days PI but not for the entire 21-day period PI. Weight gain of chickens infected with both C. baileyi and reovirus 2035 was significantly depressed 0-14 days PI and for the entire 21-day period PI. Dual infection with C. baileyi and either reovirus appeared to promote shedding of both agents. Cryptosporidia were found principally in the rectum 2-10 days PI and in the bursa of Fabricius 6-10 days PI. Reovirus infection did not cause any microscopic lesions and did not modify lesions caused by C. baileyi infection.

Experimental infection of young turkeys with eastern equine encephalitis virus and highlands J virus.

Depression, somnolence, and increased mortality were observed in 2-week-old turkeys inoculated intramuscularly with either eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus. Mortality rates in EEE virus- and HJ virus-inoculated turkeys were 7/30 (23%) and 9/30 (27%), respectively; no sham-inoculated controls died. Both EEE virus- and HJ virus-inoculated turkeys developed viremia that lasted 2 days; peak mean titers were 5.5 and 3.2 log10 plaque-forming units per ml of blood, respectively. Pathologic changes in both EEE virus- and HJ virus-inoculated turkeys consisted primarily of multifocal necrosis in the heart, kidney, and pancreas, and lymphoid necrosis and depletion in the thymus, spleen, and bursa of Fabricius. The findings indicate that EEE virus and HJ virus are pathogenic for young turkeys.

High mortality of domestic turkeys associated with Highlands J virus and eastern equine encephalitis virus infections.

High mortality occurred in two flocks of commercial turkey hens placed in southern North Carolina in fall 1991. Daily mortality peaked at 3.19% in Flock 1 and 3.79% in Flock 2. Clinical signs included restlessness, somnolence, vocalization, and acute death. Gross lesions included atrophy of the bursa of Fabricius, thymus, and spleen, and watery intestinal contents. Microscopic changes included moderate to marked lymphocyte necrosis and depletion in the bursa, thymus, and spleen, widely scattered necrosis of pancreatic acinar cells, and mild villous atrophy and fusion in the jejunum and ileum with cuboidal to low columnar epithelial cells covering the villous tips. In Flock 1, at 27 days of age, reovirus and picornavirus particles were detected in the feces. One week later, togavirus-like particles were observed in fecal contents, and two of seven serum samples showed seroconversion to Highlands J virus. Eleven days later, five of six serum samples were positive for antibodies against Highlands J virus, with a fourfold increase in the geometric mean titer. In Flock 2, seroconversion to eastern equine encephalitis virus was observed in four of 10 serum samples 11 days after the onset of clinical signs. Based on the above observations, it is suspected that these alphaviruses were the cause of the clinical syndrome.

Egg-production drop in turkeys associated with alphaviruses: eastern equine encephalitis virus and Highlands J virus.

Alphaviruses were isolated from tracheas of turkey breeders in two North Carolina flocks experiencing a severe drop in egg production. Highlands J virus was isolated from one of the breeder flocks, in which production decreased by as much as 72.6% in selected houses over a 48-to-96-hour period. Eastern equine encephalitis virus was isolated from the second breeder flock, which experienced an egg-production drop of 44.5%. Clinical signs in both flocks were similar, with inactivity and the egg-production drop being the only clinical signs observed. Eggs from affected breeders were small and white, and a few were soft-shelled. Sera collected from the flocks 2 to 3 weeks after production began dropping confirmed the presence of antibodies to the viruses recovered. In the first flock, egg production failed to return to above 50%, although heat stress may have played a role in production recovery. The second flock was taken out of production and recycled.