An atypical clinicopathological manifestation of fowlpox virus associated with reticuloendotheliosis virus in commercial laying hen flocks in Brazil.

Fowlpox (FP) is a common epitheliotropic disease in chickens that is usually controlled by live attenuated vaccines. However, there have been some reports of outbreaks of FP in recent years, even in vaccinated flocks, presenting as atypical lesions and feathering abnormalities in chickens. These findings can be associated with fowlpox virus (FPV) with the reticuloendotheliosis virus (REV) integrated into its genome. In the present study, outbreaks of atypical FP were explored in vaccinated commercial laying hen flocks to determine the nature of the causative agent by histopathologic and molecular approaches. FPV and REV were detected and classified into subclade A1 of the genus Avipoxvirus and subtype 3 of REV (REV3), respectively. Additionally, heterogeneous populations of FPV with partial (containing only a remnant long terminal repeat-LTR) or total (all functional genes) integration of REV were identified by heterologous PCRs and detected considering reference integration sites. These results indicate the mechanism of chimeric genome FPV-REV associated with outbreaks and atypical clinicopathological manifestations in commercial laying hens for the first time in Brazil and in South America. In addition, this study demonstrates the emergence of REV integrated in the FPV genome in Brazilian chicken flocks.

Detection of Fowlpox virus carrying distinct genome segments of Reticuloendotheliosis virus.

Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.

Spotlight on avian pathology: fowlpox virus.

Fowlpox virus is the type species of an extensive and poorly-defined group of viruses isolated from more than 200 species of birds, together comprising the avipoxvirus genus of the poxvirus family. Long known as a significant poultry pathogen, vaccines developed in the early and middle years of the twentieth century led to its effective eradication as a problem to commercial production in temperate climes in developed western countries (such that vaccination there is now far less common). Transmitted mechanically by biting insects, it remains problematic, causing significant losses to all forms of production (from backyard, through extensive to intensive commercial flocks), in tropical climes where control of biting insects is difficult. In these regions, vaccination (via intradermal or subcutaneous, and increasingly in ovo, routes) remains necessary. Although there is no evidence that more than a single serotype exists, there are poorly-described reports of outbreaks in vaccinated flocks. Whether this is due to inadequate vaccination or penetrance of novel variants remains unclear. Some such outbreaks have been associated with strains carrying endogenous, infectious proviral copies of the retrovirus reticuloendotheliosis virus (REV), which might represent a pathotypic (if not newly emerging) variant in the field. Until more is known about the phylogenetic structure of the avipoxvirus genus (by more widespread genome sequencing of isolates from different species of birds) it remains difficult to ascertain the risk of novel avipoxviruses emerging from wild birds (and/or by recombination/mutation) to infect farmed poultry.

Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox.

The present report documents the occurrence of a poxvirus infection in commercial meat turkeys. The affected farm had six flocks, with a total of 11,680 birds at different ages; birds from two of these flocks were affected. The clinical picture was characterized by severe epithelial lesions and proliferations on the head and neck regions as reported for the cutaneous form of poxvirus infection. Except for these lesions, no adverse clinical signs or gross pathologic lesions were observed. Only a low number of birds was affected (n = 20) and no increase of mortality could be seen. Bacteriologic investigations from the lesions revealed multiresistant Staphylococcus aureus. Eosinophilic inclusions (Bollinger bodies) in histologic examinations in the cytoplasm of keratinocytes were noticeable. Typical pox virions were demonstrated by electron microscopy, and poxvirus was isolated on the chorioallantoic membrane of specific-pathogen-free chicken eggs. Further identification of the poxvirus species was carried out by PCR and sequencing, revealing an infection with the species fowlpox. Layers in vicinity of the turkey farm that also were affected by fowlpox were considered as potential source of infection. Although it is assumed that avian poxviruses are strongly species specific, the present case report reinforces the changing picture of poxvirus infections in turkeys. Furthermore, it supports the assumption of previous data that fowlpox virus has to be seen as recently emerging pathogen in turkeys.

Atypical distribution of fowl pox lesions in broilers.

An unusual cutaneous fowl pox outbreak occurred in 8-wk-old broilers in California. Rounded and longitudinal, proliferative scratch-associated lesions were found only in feathered areas of the body. Both sides of the hip, the lower abdomen, pericloacal area, and lateral lower neck area were involved. The head, legs, feet, and toes did not have lesions. Birds in only one section of one of five houses were affected. Fifteen percent condemnations occurred in birds from the affected house due to the skin lesions. A diagnosis of fowl pox was achieved by histopathology, viral isolation, and direct electron microscopy. The unusual distribution of pox lesions was assumed to be associated with skin scratches. There was no evidence that mosquitoes or other types of insects were involved in this outbreak. To the knowledge of the authors, this is the first report of this kind of unusual fowl pox in the United States.

Pathology of cutaneous fowlpox with amyloidosis in layer hens inoculated with fowlpox vaccine.

Cutaneous fowlpox occurring in vaccinated layer hens was investigated pathologically and microbiologically. Anorexia, decrease of egg production, increased mortality, yellow scabs on faces, and alopecia of feathered skins with yellow scabs were observed in affected hens. Histologically, proliferative and necrotic dermatitis with eosinophilic ring-shaped cytoplasmic inclusions (Bollinger bodies) and clumps of gram-positive cocci (Staphylococcus hyicus) were noted in the affected birds. Fowlpox lesions were primarily observed in the feathered skins. Proliferation of feather follicle epidermal cells, with cytoplasmic inclusions and degeneration of the feather, and bacterial clumps in the feather follicles were noted in the affected skins. Ultrastructurally, characteristic fowlpox viral particles were observed in the cytoplasmic inclusions of hyperplastic epidermal cells. Amyloid deposition was observed in the Disse space of the liver, splenic sinus, and lamina propria of the bronchiolar, bronchial, and tracheal areas. Amyloidosis could be one factor inducing the fowlpox infection in vaccinated chickens.

Squamous cell carcinoma-like and pox lesions occurring simultaneously in chorioallantoic membranes of chicken embryos inoculated with materials from squamous cell carcinoma and pox lesions in broiler chickens.

The finding of closely associated squamous cell carcinoma (SCC)-like lesions and pox lesions in chorioallantoic membranes (CAMs) inoculated with skin and palate samples taken from broilers is described. The samples were obtained from two broilers coming from different flocks that were not vaccinated against fowl pox. Both birds presented skin lesions, which were diagnosed in one bird as fowl pox, and in the other as SCC. After inoculation of CAMs with fresh tissues from both birds, histologic examination revealed, in all CAMs, lesions that were characteristic of fowl pox together with lesions consistent with those seen in the skin of broilers affected with SCC. This finding was unexpected and may shed some light on the etiology of SCC.

Debilitating cutaneous poxvirus infection in a Hodgson's grandala (Grandala coelicolor).

A case of cutaneous avian pox infection in a Hodgson's grandala (Grandala coelicolor) is described. The bird was emaciated and had nodules on the eyelids, bill, neck, legs, and toes. Eosinophilic intracytoplasmic inclusion bodies were visualized by light microscopy in epithelial cells of the cutaneous nodules. Electron microscopy revealed numerous pox virions in the inclusion bodies. This is the first report of cutaneous poxvirus infection in a Hodgson's grandala.

Application of the polymerase chain reaction for the diagnosis of fowl poxvirus infection.

The polymerase chain reaction (PCR) was used to amplify a 578-bp fragment of the fowl poxvirus (FPV) genome and with a set of primers framed a region within the gene coding for 4b core protein. An amplified product was detected with six strains of FPV, whereas none was obtained from uninfected cell cultures, skin tissue or four unrelated avian pathogens. The sensitivity of PCR was tested with nucleic acids from the FPV-infected cell cultures. The detection limit was 10(-1) TCID50 in an ethidium bromide-stained gel. In addition, this assay system was used to detect FPV in tissue specimens of skin and respiratory swabs collected from commercially reared chickens. The identity of the amplification products from the tissue specimen preparations was determined further by using a simple, rapid procedure in which an internally nested, end-labeled probe was used.

Debilitating cutaneous poxvirus lesions on two captive houbara bustards (Chlamydotis undulata).

Poxvirus was isolated from cutaneous nodules on two young Houbara bustards (Chlamydotis undulata) bred in captivity in Saudi Arabia. Birds were emaciated and presented nodules on tibiotarso-tarsometatarsal joints, toes, and the carpal joint. Diagnosis was confirmed by histopathology, virus isolation on inoculated chorioallantoic membranes of embryonated chicken's eggs, and electron microscopy. Progressive leg lesions were extensive and interfered with walking, significantly debilitating the birds. Successful excisions of these lesions were performed.

An atypical fowl pox outbreak in broilers in southern Brazil.

An unusual fowl pox outbreak occurred in two integrated broiler operations. The uncommon characteristic of this outbreak was that the pox lesions were manifested in the feathered parts of the body, mainly in the posterior dorsal area and external part of the thigh. Diagnosis was made by means of histopathology and virus isolation. Severe losses were incurred at the processing plant as a consequence of condemnation due to dermatitis. Vaccination of day-old chicks with mild fowl pox vaccine combined with Marek's disease (HVT) vaccine in the area of the outbreak appeared to be important in controlling the disease.

Avian pox in birds of prey (order Falconiformes) in Bahrain.

Avian pox is an important disease in birds of prey in Bahrain. A live pigeon pox vaccine was administered to hunting falcons (Falco species) together with other therapeutic methods to arrest the development of primary stage pox lesions and for the treatment of well established secondary stage pox lesions. Quarantine and general hygiene procedures were also used as an integral part of the management and control of the disease.

Isolation of poxvirus from debilitating cutaneous lesions on four immature grackles (Quiscalus sp.).

Poxvirus was isolated from nodules on four immature grackles (Quiscalus sp.) collected in two residential areas of Victoria, Texas. All of the birds were emaciated and had nodules on the eyelids, bill, legs, toes, and areas of the skin on the wings, neck, and ventral abdomen. These pox nodules were extensive and probably interfered with both sight and flight. The preliminary diagnosis was confirmed by virus isolation, histopathology, and electron microscopy. Poxvirus was isolated on the chorioallantoic membrane of embryonated hen's eggs and in Muscovy duck embryo fibroblast cell culture. Phaenicia calliphoridae (blowfly) larvae were found in one of the pox nodules, raising the possibility of mechanical transmission of the virus by contaminated adult blowflies.

Pathologic changes in chickens caused by intravenous inoculation with fowlpox virus.

Twenty chickens were inoculated intravenously with fowlpox (FP) virus, and clinical and pathological examinations were carried out chronologically. Upon gross examination, miliary nodules scattered in the kidneys were observed from 10 to 18 days postinoculation (PI), as were papules on the skin and diphtheritic lesions on the mucous membrane of the upper respiratory tract. Microscopically, characteristic FP lesions, composed of swelling and proliferation of cells with formation of Bollinger bodies, were observed in the epithelial cells of renal tubules from 4 to 14 days PI and in the epithelial reticular cells of the thymic medulla from 4 to 10 days PI, as well as in the skin and mucous membrane. Immunofluorescent and electron microscopic observations confirmed the presence of viral antigen and virus particles in the characteristic lesions of FP.

Isolation of Erysipelothrix rhusiopathiae from a red-tailed hawk (Buteo jamaicensis) with a concurrent pox virus infection.

Erysipelothrix rhusiopathiae was isolated from the spleen, liver, lung, heart, kidney, and skin of a red-tailed hawk (Buteo jamaicensis) which had a concurrent avian pox virus infection. The hawk had been housed on a farm with domestic turkeys, providing a possible source of the E. rhusiopathiae.

Pathological changes of tracheal mucosa in chickens infected with fowl pox virus.

Five-week-old chickens were inoculated with fowl pox (FP) virus and killed on various days through day 30 postinoculation (PI). The trachea was examined with a scanning electron microscope (SEM), a transmission electron microscope (TEM), and a light microscope (LM). From day 3 PI, small focal lesions of the mucosa were detected. On day 7 PI, upon formation of cytoplasmic inclusion bodies, epithelial cells proliferated profusely, enlarged, and formed clusters like papillomata. The disease proceeded to the gradual disruption of the lesions owing to the collapse of individual degenerating epithelial cells. Total desquamation of the lesions was observed. Ultrastructural examination revealed that the surface degenerating epithelial cells of the lesions ruptured and had virus particles inside. These changes were accompanied by severe inflammatory reaction. Thereafter, epithelial cells regenerated actively and the mucosa recovered by day 27 PI.