Molecular survey of enteric viruses in commercial chicken farms in Korea with a history of enteritis.

Several enteric viruses have increasingly received attention as potential causative agents of runting-stunting syndrome (RSS) in chickens. A molecular survey was performed to determine the presence of a broad range of enteric viruses, namely chicken astrovirus (CAstV), avian nephritis virus (ANV), chicken parvovirus (ChPV), infectious bronchitis virus (IBV), avian rotavirus (AvRV), avian reovirus (ARV), and fowl adenovirus (FAdV), in intestinal samples derived from 34 commercial chicken flocks that experienced enteritis outbreaks between 2010 and 2012. Using techniques such as PCR and reverse-transcription PCR, enteric viruses were identified in a total of 85.3% of investigated commercial chicken flocks in Korea. Furthermore, diverse combinations of 2 or more enteric viruses were simultaneously identified in 51.7% of chicken farms positive for enteric viruses. The rank order of positivity for enteric viruses was as follows: ANV (44.1%), CAstV (38.2%), ChPV (26.5%), IBV (20.6%), ARV (8.8%), AvRV (5.9%), and FAdV (2.9%). Additionally, other pathogens such as Escherichia coli, Salmonella spp., Eimeria spp., and FAdV were detected in 79% of chicken flocks positive for enteric viruses using PCR, bacterial isolation, and microscopic examination. The results of our study indicate the presence of several enteric viruses with various combinations in commercial chicken farms that experienced enteritis outbreaks. Experimental studies are required to further understand the roles of enteric viruses in RSS in commercial chickens.

An outbreak of lymphomas in a layer chicken flock previously infected with fowlpox virus containing integrated reticuloendotheliosis virus.

Visceral lymphomas occurred in a 236-day-old layer flock previously diagnosed with reticuloendotheliosis virus (REV)-integrated fowlpox virus (FPV) infection at the age of 77 days. Common pathologic lesions were multiple neoplastic nodules of homogeneous lymphocytes in the livers and spleens of all submitted chickens. All neoplastic tissues were positive for the REV envelope (env) gene by PCR. In a retrospective molecular study of FPV-infected 77-day-old chickens from the same flock, we identified nearly full-length REV provirus integrated into the genome of FPV as well as the REV env gene in trachea samples, whereas only the REV LTR region was present in the FPV strain used to vaccinate this flock. The 622-bp REV env gene nucleotide sequence derived from the trachea and neoplastic tissues was identical. Commercial ELISA of serum samples revealed that all chickens aged between 17 and 263 days in this flock were positive for REV but not for avian leukosis virus. Taken together, the evidence suggests that the visceral lymphomas were caused by a REV-integrated FPV field strain. FPV infections of commercial chickens should be followed up by careful monitoring for manifestations of REV infection, including lymphomas and immune depression, considering the ease with which the REV provirus appears to be able to integrate into the FPV genome.

Secondary malignant lymphoedema after mastectomy in two dogs.

This case report describes the diagnosis of secondary malignant lymphoedema in two dogs that had undergone a mastectomy. A remarkable severe oedematous lesion associated with lameness in the right hindlimb was observed in both cases. Diagnostic imaging examinations, including direct pedal lymphangiography (case 1) and lymphoscintigraphy (case 2), showed obstruction of lymph flow in the lymphatics of the right hindlimbs. Although the recommended medical management and physiotherapy had been applied to resolve the problems, oedema did not improve in the damaged region in both cases. Results of histopathological examinations suggested that the cause of the obstructed lymph flow was neoplastic cells in the lymphatics of the right hindlimb in both dogs.

Pathogenesis of and immunity to a new influenza A (H5N1) virus isolated from duck meat.

The outbreak of avian influenza H5N1 in Hong Kong in 1997 raised concerns about the potential for the H5 subtype to cause a human pandemic. In 2001 a new H5N1 virus, A/Duck Meat/Anyang/AVL-1/2001 (A/Dkmt), was isolated from imported duck meat in Korea. The pathogenesis of this virus was investigated in mice. A/Dkmt virus had low infectivity but was lethal for mice at high doses, and at lethal doses, the virus replicated in the brains of infected mice. A/Dkmt virus cross-reacted poorly with ferret antisera raised against human H5N1 viruses, but prior infection with A/Dkmt virus protected mice from death after secondary infection with human H5N1 virus.

Evaluation of a high-pathogenicity H5N1 avian influenza A virus isolated from duck meat.

The introduction of an influenza A virus possessing a novel hemagglutinin (HA) into an immunologically naive human population has the potential to cause severe disease and death. Such was the case in 1997 in Hong Kong, where H5N1 influenza was transmitted to humans from infected poultry. Because H5N1 viruses are still isolated from domestic poultry in southern China, there needs to be continued surveillance of poultry and characterization of virus subtypes and variants. This study provides molecular characterization and evaluation of pathogenesis of a recent H5N1 virus isolated from duck meat that had been imported to South Korea from China. The HA gene of A/Duck/Anyang/AVL-1/01 (H5N1) isolate was found to be closely related to the Hong Kong/97 H5N1 viruses. This virus also contained multiple basic amino acids adjacent to the cleavage site between HA1 and HA2, characteristic of high-pathogenicity avian influenza viruses (HPAI). The pathogenesis of this virus was characterized in chickens, ducks, and mice. The DK/Anyang/AVL-1/01 isolate replicated well in all species and resulted in 100% and 22% lethality for chickens and mice, respectively. No clinical signs of disease were observed in DK/Anyang/AVL-1/01-inoculated ducks, but high titers of infectious virus could be detected in multiple tissues and oropharyngeal swabs. The presence of an H5N1 influenza virus in ducks bearing a HA gene that is highly similar to those of the pathogenic 1997 human/poultry H5N1 viruses raises the possibility of reintroduction of HPAI to chickens and humans.

Pathogenicity and vaccine efficacy of a thymidine kinase gene deleted infectious laryngotracheitis virus expressing the green fluorescent protein gene.

The deletion of the thymidine kinase (TK) gene of herpesviruses causes a reduction in their virulence. However, the effects of the TK gene in infectious laryngotracheitis virus (ILTV) have not been clearly elucidated. In the present study, we constructed a TK gene-deleted recombinant ILTV expressing the green fluorescent protein (GFP) gene as a marker. The GFP gene was inserted in place of the TK gene in both virulent and low virulence strains of ILTV. The GFP gene in the recombinants was expressed in chicken kidney cells, LMH cells and in the chorioallantoic membrane of embryonated chicken eggs. The recombinants produced cytopathic effects in chicken kidney cells and LMH cells and formed pocks in the chorioallantoic membrane of embryonated chicken eggs. The growth rate of the recombinant in chicken kidney cells was similar to that of wild type viruses. The recombinants showed a reduction of virulence compared to that of parental viruses and induced protection against virulent ILTV in specific pathogen free chickens. The recombinant expressing GFP gene may be a candidate for a genetically engineered vaccine and provide a means to study growth kinetics and mechanism of latent infection and reactivation of ILTV. In this study, we confirmed that the TK gene is directly related to virulence of ILTV. This is the first report to show the evidence that the TK gene is a major gene related to virulence of ILTV.

Competitive exclusion against Salmonella gallinarum of Salmonella enteritidis infected chickens.

To evaluate the degree of competitive exclusion against Salmonella gallinarum(S. gallinarum) of Salmonella enteritidis(S. enteritidis) infected chickens, fifty-six, 4-week old Hyline layer suspected of S. enteritidis infection were challenged with S. gallinarum. All chickens were tested for S. enteritidis isolation using cloacal swabs and serum plate agglutination test using S. enteritidis Ag. before challenge and classified into four groups(SE isolated, SE nonisolated, SE seropositive and SE seronegative). None of the SE isolated and the SE seropositive groups died after challenge and the average weight gains were 245.5g and 254.6g, respectively. But in the SE nonisolated and the SE seronegative groups, mortality was 18.2% and 20.6% and the average weight gains were 150.1g and 111.2g. The incidence of reisolation of S. gallinarum of the SE isolated and the SE seropositive groups were 41.7% and 47.6% from liver, 33.3% and 47.6% from spleen and 8.3% and 14.3% from cecum, respectively, and the SE nonisolated and the SE seronegative group were 63.6% and 64.7% from liver, 84.1% and 88.2% from spleen and 47.7% and 52.9% from cecum. The serological response of the SE isolated and the SE seropositive groups hardly changed from 75.0 and 81.8% before challenge to 75.0 and 85.7% after. But, the other two groups were found to be significantly higher after challenge and increased from 0 and 18.2% to 100%. Consequently, S. enteritidis preinfected chickens were found to be significant different in terms of mortality, weight gain, reisolation of S. gallinarum and serological response compared to noninfected chickens. Moreover, our study shows that S. enteritidis infected chickens appear strong competitive exclusion against the colonization of S. gallinarum.

Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96.

Sequence analysis of the hemagglutinin (HA) gene of five Korean H9N2 avian influenza virus (AIV) isolates showed that these viruses were closely related and possibly came from the same source. Phylogenetic analysis of the HA1 subunit of H9 subtype isolates revealed that Korean AIV isolates were different from isolates from the poultry markets in Hong Kong in 1997. None of the Korean AIVs had multiple basic amino acids at the HA cleavage site that confer high pathogenicity to some H5 and H7 AIVs. Phylogenetic analysis of the nucleoprotein and matrix gene demonstrated that Korean isolates cluster with Eurasian origin AIVs. The pathogenic potential of one of the isolates (MS96) was assessed after several passages in 14-day-old embryonated chicken eggs (ECE). Fourteen-day-old ECE derivatives of MS96 showed increased HA titer and embryo mortality in eggs; this was apparent after the third passage in 14-day-old ECE. Sequence analysis of the cleavage site of MS96 after the third and tenth passages in 14-day-old ECE revealed no changes in the amino acid sequence. The pathogenicity of MS96 after the tenth passage in 14-day-old eggs (MS96p10(ECE14)) was tested with 4-wk-old specific-pathogen-free chickens. The 14-day-old derivative, MS96p10(ECE14), showed wider tissue tropism and induced more severe clinical signs than the parent virus. Furthermore, after intranasal inoculation of 86-wk-old broiler breeders and 30-wk-old layers, the MS96p10(ECE14) derivative induced more severe signs of depression than the parent virus as well as a transient drop in egg production.

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia.

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.

Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus.

A recombinant baculovirus containing the S1 glycoprotein gene of the virulent nephropathogenic KM91 strain of infectious bronchitis virus (IBV) was constructed in order to investigate protective immunity in vaccinated chickens. Results from the protection test were evaluated by re-isolation of virus from the kidneys and tracheas of vaccinated chickens after challenge with strain KM91. After three immunizations, the recombinant S1 (rS1) glycoprotein induced 50% protection of the kidney, whilst inactivated KM91 induced 88% and 50% protection of the kidney and trachea, respectively. In chickens primed with the attenuated H120 vaccine strain, which is heterologous to KM91, the rS1 glycoprotein induced 83% protection of the kidney after two immunizations. Haemagglutination-inhibition titres were also increased in chickens immunized with the rS1 glycoprotein after three immunizations, and significantly higher titres were detected after challenge. These data indicate that the expressed rS1 glycoprotein alone can induce a protective immune response as well as an antibody response.

Comparative pathology of chickens experimentally inoculated with avian influenza viruses of low and high pathogenicity.

Pathologic changes and distribution of viral antigen as determined by immunohistochemistry were compared among 4-wk-old specific-pathogen-free chickens inoculated intratracheally with avian influenza virus (AIV) isolates of either low or high pathogenicity. Viruses of low pathogenicity, previously characterized as mildly pathogenic (MP), included A/chicken/Pennsylvania/21525/83 (H5N2) (MP-Penn) and A/chicken/Alabama/7395/75 (H4N8) (MP-Alab). Viruses of high pathogenicity included A/chicken/Pennsylvania/1370/83 (H5N2), A/chicken/Victoria/A185/85 (H7N7), and A/turkey/Ontario/7732/66 (H5N9). Extremely variable clinical signs ranging from mild respiratory distress to high mortality were present among chickens inoculated with these viruses. Chickens inoculated with highly pathogenic (HP) virus had histologic lesions of necrosis and inflammation in cloacal bursa, thymus, spleen, heart, pancreas, kidney, brain, trachea, lung, and skeletal muscle, whereas chickens inoculated with MP virus had histologic lesions most frequently in lung and trachea or lacked histologic lesions. Immunospecific staining for avian influenza viral proteins was most common in cells within heart, lung, kidney, brain, and pancreas of chicken inoculated with HP viruses, but immunospecific staining was present only and infrequently in trachea and lung of chickens inoculated with MP-Penn AIV. MP-Alab did not produce lesions nor have viral antigen in inoculated chickens but did produce serologic evidence of infection. The pattern of organ involvement and viral antigen distribution in chickens intratracheally inoculated with HP AIV isolates indicates a common capability to spread beyond the respiratory tract and confirms the pantrophic replicative, pathobiologic, and lethal nature of the viruses. However, variability in severity and lesion distribution exists between different HP AIVs. By contrast, MP viruses had the ability to replicate in respiratory or enteric tracts or both and produce lesions within the respiratory tract. These MP viruses exhibited a restricted ability to replicate or produce lesions or both in nonrespiratory or nonenteric tissues; such effects were associated with only sporadic deaths.

Myocardial sarcocystosis in a grand eclectus parrot (Eclectus roratus) and a Moluccan cockatoo (Cacatua moluccensis).

Cardiac sarcocystosis is described in a grand eclectus parrot and a Moluccan cockatoo. Many cysts containing metrocytes were observed within cardiac muscle fibers on tissue sections stained with hematoxylin and eosin. Characteristic ultrastructural features of the cyst walls included the presence of villous projections containing microtubules. Compartmentalization of the cysts resulted from inward extensions of the cyst wall. The differential diagnosis of sarcocystosis, the life cycle of the parasite, and control measures are discussed.