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Avipoxvirus is the etiological agent of the avian pox, a well-known disease of captive and wild birds, and it has been associated with tumor-like lesions in some avian species. A white-faced whistling duck (Dendrocygna viduata) raised in captivity was referred to a Veterinary Teaching Hospital in Northeast due to cutaneous nodules present in both wings. A few days after the clinical examination, the animal died naturally. Once submitted to necropsy, histopathological evaluation of the lesions revealed clusters of proliferating epithelial cells expanding toward the dermis. Some of these cells had round, well-defined, intracytoplasmic eosinophilic material suggestive of poxvirus inclusion (Bollinger bodies). PCR performed on the DNA extracted from tissue samples amplified a fragment of the 4b core protein gene (fpv167), which was purified and sequenced. This fragment of Avipoxvirus DNA present in these tumor-like lesions showed high genetic homology (100.0%) with other poxviruses detected in different avian species in several countries, but none of them were related to tumor-like lesions or squamous cell carcinoma. This is the first report of Avipoxvirus detected in tumor-like lesions of a white-faced whistling duck with phylogenetic analysis of the virus. RESUMO: Avipoxvirus é o agente etiológico da varíola (bouba) aviária, uma doença bem descrita em aves de cativeiro e selvagens, tendo sido associada a lesões semelhantes a tumores em algumas dessas espécies. Uma marreca piadeira (Dendrocygna viduata), criada em cativeiro, foi atendida em um Hospital Veterinário na região nordeste devido à presença de nódulos cutâneos em ambas as asas. Alguns dias após o exame clínico, o animal veio a óbito naturalmente. A ave foi submetida à necropsia e coletados fragmentos das lesões para análise histopatológica, que revelou proliferação de células epiteliais expandindo para a derme. Algumas dessas células possuíam material eosinofílico intracitoplasmático e bem definido, sugestivo de inclusão de poxvírus (corpúsculos de Bollinger). A PCR realizada a partir do DNA extraído de amostras das lesões amplificou um fragmento do gene da proteína do núcleo 4b (fpv 167), que foi purificado e sequenciado. Esse fragmento de DNA de Avipoxvirus presente nas lesões relevou alta homologia genética (100,0%) com outros poxvírus detectados em diferentes espécies de aves em vários países, mas nenhum deles estava relacionado a lesões tumorais ou carcinoma espinocelular. Este é o primeiro relato de Avipoxvirus detectado em lesões semelhantes a tumores em uma marreca piadeira com caracterização molecular do vírus.

This study aimed at proposing a new technical criteria for condemnation of turkey carcasses due to fowlpox in turkeys as a contribution for the work of the Brazilian Federal Meat Inspection Service. Skin samples from 30 carcasses of a flock of 840 turkeys (Meleagris gallopavo), previously vaccinated for fowlpox and slaughtered in June 2013, were collected. Samples were submitted to histological examination under light microscopy. The virus was identified using standard PCR techniques. The main histological findings were hyperplasia and hydropic degeneration of the epithelium and the presence of intracytoplasmic eosinophilic inclusion bodies. PCR results yielded 83.3% positive and 16.7% negative samples. Fowlpox virus is species specific, and there are no reports of its occurrence in mammals. The macroscopic and microscopic findings of the skin lesions do not justify the total condemnation of carcasses of poultry affected with fowlpox, except in cases of cachexia or repulsive appearance, as established by SIF regulation.

SUMMARY Layer chickens with severe respiratory disease belonging to farms with chickens infected by gallid herpesvirus (GaHV-1) were evaluated. Samples of nasal turbinates/sinuses and trachea from 31 chickens were subjected to histopathological analysis, which showed that 22.6% of chickens had lesions suggestive of co-infection, most frequently by GaHV-1 or Mycoplasma spp. PCR analysis showed the presence of at least two respiratory pathogens in 61.2% of chickens. Partial sequencing was performed for identification of the intergenic spacer region (IGSR) and cytadhesin 2 (mgc2) gene of the Mycoplasma gallisepticum genome. Analysis of IGSR revealed the involvement of two field and one vaccine strains. However, analysis of mgc2 from the same samples revealed five different strains of M. gallisepticum, four of which were field strains and one of which was a vaccine strain. These results demonstrate that combined molecular and histopathological analysis is a reliable and conclusive method for diagnosis of respiratory diseases in chickens since some etiologic agents can remain latent or maintain chickens as carriers. The results further confirm the importance and feasibility of the molecular characterization of M. gallisepticum genes to differentiate between vaccine and field strains involved in cases of respiratory disease in chickens.

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises. RESEARCH HIGHLIGHTS Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A. Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis. Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows. Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).

Seventy-eight chickens from a very high poultry density (approximately eight million) region and twelve backyard chickens from neighboring areas were analyzed by histopathology and additional techniques for the presence of the infectious laryngotracheitis virus. The virus distribution was determined in different tissues using immunohistochemistry (IHC) and polymerase chain reaction (PCR). The disease was histopathologically diagnosed in 41.0% (32/78) of the commercial layers. Lesions were mainly characterized by syncytial cells with eosinophilic intranuclear inclusion body formed from the hyperplastic epithelium of the upper respiratory tract, primary and secondary bronchi, and conjunctiva. IHC showed 70% (21/30) positive signal in the larynx/trachea and, 53.8% (14/26) in the lungs, either in epithelial cells or syncytia. In the turbinates and paranasal sinuses, 29.6% (8/27) of samples showed positive signal. PCR detected the following gallid herpesvirus 1-positive percentages: conjunctiva 63.2% (31/49), lungs 57.6% (30/52), turbinates and paranasal sinuses 56% (28/50), and larynx/trachea 50% (39/78). IHC showed to be a useful additional tool for definitive ILT diagnosis, especially during the subacute phase of the disease when syncytial cells with intranuclear inclusion bodies are no longer observed. PCR using specific primers from ICP4 gene, generating a product of 237 base pairs, was sensitive for ILT diagnosis, and very useful for rapid detection of GaHV-1 in chickens. Fixed tissues allowing histopatological examination and detection of GaHV-1 by PCR, are a good option in areas where farms are located several hundred kilometers away from a diagnostic center, reducing problems with conservation of fresh samples and the risk of virus spread.

The virus responsible for an outbreak of infectious laryngotracheitis (ILT) in a multi-age flock of egg layer chickens under quarantine in Brazil was characterized. Layer chickens from this area with circulating gallid herpesvirus 1 (GaHV 1) were evaluated using histopathology and molecular characterization techniques based on sequences of infected-cell polypeptide 4 (ICP4) and thymidine kinase (TK) genes. The infected chickens that were analyzed were PCR-positive for GaHV-1 in the trachea and negative in most trigeminal ganglia. The lack of ILT lesions in the conjunctiva and respiratory tissues, combined with detection of viral DNA in the trachea, was found to be associated with latent infection. The sequences from five farms obtained in the present study were identical, and there were no deletions within the 272- to 283-bp region of the ICP4 gene, as observed in the sequences of vaccine strains (CEO and TCO). The lack of a deletion in the ICP4 fragment analyzed in this study indicates that the chickens were infected with a field virus. The absence of the T252M mutation in a fragment of the TK gene, in addition to the low mortality rate observed, suggests that the outbreak in the state of Minas Gerais was not caused by a highly virulent strain but rather by a field virus of lower virulence. In addition, using phylogenetic reconstructions, it was found that this field strain was grouped together in a separate branch, apart from the previously characterized Brazilian strains. The introduction of vectored vaccines apparently has been effective in reducing clinical disease and lesions, and preventing new outbreaks of disease.

A recent (November 2010) outbreak of infectious laryngotracheitis (ILT) in a multi-age laying hen facility in Minas Gerais state, Brazil, is described. Previous ILT outbreak in laying hens was only notified in São Paulo state, Brazil, in 2002. In the outbreak described here, the affected population was approximately eight million hens, with flock sizes ranging from 100,000 to 2,900,000 chickens. The average mortality ranged from 1 to 6%, and morbidity was around 90% (most of the twenty seven farms of the area were positive for ILT virus). Three multi-age laying farms from one company were selected for this report. Clinical signs included prostration, dyspnea, conjunctivitis, occasional swelling of the paranasal sinuses and bloody mucous nasal discharge. Severely affected chickens presented with dyspnea, gasping and became cyanotic before death. At necropsy, these chickens had fibrinous exudate blocking the larynx and the lumen of cranial part of the trachea. In addition, conjunctivitis with intense hyperemia, edema and sinuses with caseous exudate were present. On histopathology, there were marked necrosis and desquamation of respiratory ephitelium and conjunctiva with numerous syncytial cells formation and fibrinous exudate. Moderate to marked non suppurative (especially lymphocytes and plasma cells) infiltration in the lamina propria also was observed. Sixteen out of 20 examined chickens, eosinophilic intranuclear inclusion bodies were observed in the syncytial cells. The DNA extracted from larynx and trachea produced positive PCR results for ILT virus (ILTV) DNA using formalin-fixed, paraffin embedded (FFPE) samples. Amplicons from a small region of ICP4 gene were submitted to sequencing and showed 100% identity with ILTV EU104910.1 (USA strain), 99% with ILTV JN596963.1 (Australian strain) and 91% with ILTV JN580316.1 (Gallid herpesvirus 1 CEO vaccine strain) and JN580315.1 (Gallid herpesvirus 1 TCO vaccine strain).

This study describes an outbreak of avian poxvirus disease in previously pox-vaccinated turkeys in Brazil. The turkeys had suggestive gross lesions of cutaneous avian poxvirus in the skin of the head and cervical area without changes in the flock mortality rates. In the slaughterhouse, 30 carcasses were removed from the slaughter line to collect tissue from cutaneous lesions for histological analyses and characterization of the virus. The virus was identified by conventional polymerase chain reaction (PCR) and subsequent gene sequencing. Acanthosis, hyperkeratosis, and hydropic degeneration were seen on skin histopathology. Eosinophilic intracytoplasmic inclusion bodies (Bollinger) on keratinocytes were observed in 46.6% of the samples. Avian poxvirus DNA was detected on PCR in 83.3% of the total samples. PCR associated with histopathology had 93.3% of positivity for avian poxvirus. In the phylogenetic study, samples show 100% matching suggesting that the outbreak occurred by a single viral strain and was different from those strains affecting other wild birds such as canaries and sparrows. A single mutation (Adenine for Guanine) was detected in our study’s strain and in the strains of turkey, chickens, and vaccine strains published in GenBank. Also, when the sequence strain of the present study and sequences from GenBank of canarypox and sparrowpox strains were aligned, a Thymine was found replacing the Adenine or Guanine. The in ovo vaccination method as single-use in turkeys of this study apparently did not provide adequate protection against avianpox disease, but additional vaccination administered by wing-web when turkeys were 45-60 days old in the new flocks controlled the disease. In the subsequent year, new cases of this disease were not found. It was not possible to confirm the source of the virus strain, but infection with a field strain derived from chickens is one possibility, considering the poultry farm population in the area and biosecurity aspects. For wide characterization of avipoxvirus and differentiation among strains, the complete sequence of the viral genome is required.

A 20-year-old unneutered male poodle presented prostration, apathy, staggering gait, lack of appetite and tick infestation. The dog was diagnosed with a Sertoli cell tumor in an undescended testicle by cytological, histopathological and immunohistochemical tests. Pancytopenia with moderate nonregenerative anemia, leukopenia and severe thrombocytopenia were detected in the complete blood count. Cytological and histopathological evaluation of the bone marrow revealed a cellularity of 30%, with erythroid (59%), lymphoid (40%) and mast cells (1%), and an absence of granulocytic, monocytic and megakaryocytic lineage cells. In post-mortem examinations, changes related to hemostatic disorders were found. The absence of microorganisms in molecular tests and an estrogen serum concentration over reference values confirmed hyperestrogenism as a possible cause of pancytopenia. The literature describes a Sertoli cell tumor hyperestrogenism that induced pancytopenia, along with bone marrow hypoplasia of all hematopoietic lineages. In contrast, in the present case, the erythroid precursor cells were preserved in the bone marrow, although there were no reticulocytes circulating in the blood. This case, therefore, should be considered in future investigations of pancytopenia induced by Sertoli cell tumor hyperestrogenism. RESUMO Um cão Poodle, macho, de 20 anos, não castrado, apresentou prostração, apatia, andar cambaleante, falta de apetite e infestação por carrapatos. Nesse animal, foi diagnosticado tumor de células de Sertoli em um testículo não descendente, utilizando-se citologia, histopatologia e imuno-histoquímica. Pancitopenia com anemia moderada não regenerativa, leucopenia e trombocitopenia intensas foram detectadas no hemograma. Na avaliação citológica e histopatológica da medula óssea, havia celularidade de 30%, constituída pelas linhagens eritroide (59%) e linfoide (40%) e por mastócitos (1%), com ausência de células das linhagens granulocítica, monocítica e megacariocítica. Em exames post mortem, mudanças relacionadas à hemostasia foram encontradas. A ausência de micro-organismos nos testes moleculares e a concentração sérica de estrogênio acima dos valores de referência confirmaram hiperestrogenismo como a possível causa da pancitopenia. A literatura descreve hiperestrogenismo em tumores de células de Sertoli induzindo pancitopenia associada com hipoplasia da medula óssea de todas as linhagens hematopoiéticas. Em contraste, no presente caso, as células precursoras eritróides estavam preservadas na medula óssea, embora não houvesse reticulócitos no sangue. Assim, o relato apresentado deve ser considerado em futuras investigações de pancitopenia induzida por hiperestrogenismo em tumor de células de Sertoli.