Antigenic and genetic characterization of an avian poxvirus isolated from an endangered Hawaiian goose (Branta sandvicensis)

An avian poxvirus from cutaneous lesions in a Hawaiian goose (Branta sandvicensis) was characterized in this study. The virus was isolated by inoculation onto the chorioallantoic membranes (CAMs) of developing chicken embryos. Cytoplasmic inclusion bodies were observed on histopathological examination of CAM lesions. Western blotting analysis using polyclonal antiserum against fowl poxvirus (FWPV) showed differences from FWPV, but a similar antigenic profile between Hawaiian goosepox (HGP) isolate and two previous Hawaiian poxvirus isolates were observed. Still three avian poxviruses from Hawaiian birds showed distinguishable reaction in approximately 27, 34, 35, and 81 kDa proteins when polyclonal antibodies against the Hawaiian poxvirus isolate (Alala/lanakila) were used. Restriction fragment length polymorphisms (RFLP) of DNA of this isolate also showed differences from those of FWPV and previous avianpox isolates from Hawaiian forest birds. While nucleotide sequences of a 5.3-kb PstI-HindIII fragment of the genome of HGP isolate revealed very high homology (99% identities) with Canary poxvirus (CNPV) ORF266-274, and like CNPV, homologs of three FWPV ORFs (199, 200, and 202) including any reticuloendotheliosis virus (REV) sequences are not present in the genome of HGP isolate.     

Avipoxvirus infection in peregrine falcons (Falco peregrinus) from a reintroduction programme in Germany

Poxvirus infections are common in domestic birds in Germany, but they are rare in birds of prey. Only species of falconidae imported from Arabian or Asian countries have so far tested positive for poxvirus, and, among these, only raptors kept for falconry. As part of a reintroduction programme in the northern county of Mecklenburg-Western Pomerania, which is adjacent to the Baltic Sea, 21 young peregrine falcons were released into the wild; six of them died and one was examined postmortem, its tissues being examined by light and electron microscopy. In addition, an ELISA for fowlpox, pigeonpox and canarypox was applied. No virus could be isolated and propagation in culture failed, but virus particles were detected by electron microscopy in lesions from its skin and tongue.     

Molecular characterization of a poxvirus isolated from an American flamingo (Phoeniconais ruber rubber)

An avian poxvirus from the beak scab of an American flamingo (Phoeniconais ruber rubber) was isolated by inoculation on the chorioallantoic membrane (CAM) of specific-pathogen-free (SPF) chicken embryos. The virus produced multifocal areas of epithelial hyperplasia along with foci of inflammation in the CAM, and rare cells contained small eosinophilic intracytoplasmic bodies. Chickens inoculated with the isolated virus in the feather follicle of the leg did not develop significant lesions. Nucleotide sequence comparison of a PCR-amplified 4.5 kb HindIII fragment of the genome of flamingo poxvirus (FlPV) revealed very high homology (99.7%) with condor poxvirus (CPV), followed by approximately 92% similarity with canary poxvirus (CNPV) and Hawaiian goose poxvirus (HGPV), but less similarity (approximately 69%) to fowl poxvirus (FPV), the type species of the genus Avipoxvirus of family Poxviridae. As in the cases with CPV, CNPV, and HGPV, genetic analysis of FlPV revealed an absence of three corresponding FPV open reading frames (ORF199, 200, and 202) and an absence of any reticuloendotheliosis virus (REV) sequences in this region. There are only nine nucleotide substitutions observed between FlPV and CPV in the 4.5 kb fragment; those were clustered in the ORF201 region, which in FPV genome is a site for integration of REV sequences. Phylogenetic analysis of the predicted amino acid sequences of the ORF201-coded hypothetical protein demonstrated FlPV to be more closely related to CPV, as well as to CNPV and HGPV, than to FPV.     

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.     

Evaluation of pathogenicity of avian poxvirus isolates from endangered Hawaiian wild birds in chickens

Pathogenicity of two avian poxviruses isolated from endangered Hawaiian wild birds, the Hawaiian Goose and the Palila, was compared with fowl poxvirus in chickens. Immune responses were measured by ELISA pre- and postimmunization with Hawaiian poxviruses and after challenge with fowl poxvirus. Both isolates from Hawaiian birds developed only a localized lesion of short duration at the site of inoculation in specific-pathogen-free chickens and did not provide protection against subsequent challenge with virulent fowl poxvirus. On the other hand, birds inoculated with virulent fowl poxvirus developed severe lesions. In contrast to high antibody response in chickens immunized with fowl poxvirus, birds immunized with either of the two Hawaiian isolates developed low to moderate antibody responses against viral antigens. The level of immune responses, however, increased in birds of all groups following subsequent challenge.     

Characterization of avipoxviruses from wild birds in Norway

Avipoxviruses from different geographic regions of the world have been characterized to study their genetic and biological properties, but so far, no such work has been performed on Norwegian isolates. Lesions suggestive of avian pox, found on a Norwegian wild sparrow (Passer domesticus) and wood pigeon (Palumbus palumbus), were obtained in 1972 and 1996, respectively. Histologically, these lesions were demonstrated to be characteristic of poxvirus infections and the poxvirus was observed using an electron microscope. The resulting viruses were propagated in chicken embryo fibroblast cells. Restriction fragment length polymorphism of genomes from 2 Norwegian isolates and fowl pox vaccine strain, generated by BamHI, revealed a high degree of heterogeneity among the isolates. The profiles of avipoxviruses isolated from wild birds were clearly distinct from each other and also to the fowl poxvirus strain. Furthermore, chickens experimentally infected with pigeon poxvirus had higher antibody titers and extensive lesions compared to other isolates. This may suggest that pigeon poxvirus is more virulent than the other isolates.     

Genetic and antigenic characterization of a poxvirus isolate from ostriches

Avian poxvirus was isolated from nodules on the heads and conjunctiva of two 3-to-4-wk-old ostrich chicks. The ostriches from which poxvirus was isolated had been placed on premises where turkeys that had shown evidence of poxvirus infection had been raised earlier. Microscopically, the nodules from the ostriches were composed of proliferating and hypertrophic epithelial cells that formed large fronds. Most of the hypertrophic epithelial cells contained large eosinophilic intracytoplasmic inclusion bodies characteristic of poxvirus. Characterization of the avian poxvirus isolated from the cutaneous lesions in ostriches was based on western blotting of virus antigen, restriction fragment length polymorphism of genomic DNA, pathogenesis, and cross-protection studies in chickens. Antigenic and genetic studies did not reveal any significant difference between the poxvirus isolated from ostriches (PVO) and fowl poxvirus (FPV). Further, susceptible chickens immunized with the PVO were protected when challenged with a virulent strain of FPV. Thus, the poxvirus isolated from ostriches had similar antigenic, genetic, and biological properties to FPV.     

Diagnosis of avian viral diseass by electron microscopy.

Clinical material from avian species was examined directly by electron microscopy for the presence of viruses. Mycoplasma-like and coronavirus-like particles were found in chicken feces. These particles did not appear to be associated with disease and were not propagated in the laboratory. Infectious bursal disease virus was readily detected in impression smears of bursas from experimentally infected birds. Poxviruses were demonstrated in smears made from canarypox lesions. Difficulty in distinguishing intact particles of Newcastle disease virus from mycoplasmas and orthomyxoviruses was resolved by treating viral preparations with deoxycholate. After treatment, Newcastle disease virus was lysed, rendering the nucleocapsid visible, whereas influenza virus was mainly unaffected. Viral particles that were recognized only with difficulty by direct elecron microscopic examination were more easily identified using immunoelectron microscopy.     

Avian pox in blue-fronted Amazon parrots

During a 1-month period at a quarantine station, an epornitic of avian pox occurred in blue-fronted Amazon parrots (Amazona aestiva). Clinical signs included conjunctivitis, blepharitis, and varying degrees of anorexia and respiratory distress. Lesions included periocular ulcerations and scabs and necrotic plaques in the oral cavity. Histologically, the lesions consisted of epithelial hyperplasia, secondary inflammatory changes, and eosinophilic inclusions which, by electron microscopy, were shown to contain poxvirus. When chicken embryos were inoculated with material from eyelid scabs and pharyngeal plaques, lesions of avian pox developed on the chorioallantoic membrane. The death rate of infected birds was high because of secondary bacterial and fungal infections, but uncomplicated cases were usually self-limiting. Periocular lesions also developed in 2 other species of psittacine birds housed in the same facility.     

Isolation of a poxvirus from a sparrow (Passer domesticus)

From liver, lung, and kidney of a dead sparrow with signs of conjunctivitis a cytopathic agent was isolated in chicken embryo cell culture which was identified as a poxvirus by electron microscopy. From its growth characteristics in avian and mammalian cell culture it was concluded to belong to the genus avipox virus.     

STUDIES ON POXVIRUS ISOLATED FROM A MAGPIE IN QUEENSLAND

An avian poxvirus was isolated from a black-backed magpie in Queensland. The virus produced pocks on the chorio-allantoic membrane of chicken embryos and lesions on the skin of chickens. Comparison by passive haemagglutination test and neutralisation test indicated that the virus was related more closely to pigeonpox virus than to fowlpox virus.     

Survey of peafowl (Pavo cristatus) for potential pathogens at three Michigan zoos.

Blood samples collected from 31 free-roaming peafowl from three zoos in Michigan were tested serologically. Antibody titers were present against avian adenovirus and Bordetella avium in 19.3% and 61.3% of the samples, respectively. Serum plate agglutination tests were positive for Mycoplasma meleagridis and Mycoplasma synoviae in 3.2% and 38.7% of the samples, respectively. All birds were seronegative for avian influenza, Newcastle disease virus, West Nile virus, Mycoplasma gallisepticum, Salmonella pullorum, Salmonella typhimurium, and Giardia sp. No parasites were seen in blood smears. Cloacal swabs were cultured for anaerobic, aerobic, and microaerophilic bacteria. Clostridium perfringens type A and Escherichia coli were cultured most frequently from 64.5% and 29% of the samples, respectively, whereas Salmonella sp. and Campylobacter sp. were not isolated. Fecal samples contained moderate numbers of ascarid and Capillaria sp. ova and coccidian oocysts. Female biting lice (Goniodes gigas) were identified on three birds.     

Canary pox causing high mortality in an aviary

In an aviary housing 200 six-month-old canaries, 165 became ill and 145 died over a 6-week period from a disease initially characterized by lethargy, ruffled feathers, open-mouth breathing, and death in 2 to 3 days. Proliferative "pox-like" lesions around the eyes and mouth were not seen until the 4th week. At necropsy, initially affected birds had cloudy air sacs and patchy pneumonia. Histologically, the lungs had proliferative necrotizing bronchitis. Birds necropsied later had proliferative skin lesions and intracytoplasmic inclusions typical of poxvirus in the epidermis and airway epithelium. A virus was isolated from an organ pool of lung, air sac, liver, and skin of affected birds and was identified by electron microscopy as poxvirus.     

Preliminary molecular characterization of a fowl poxvirus isolate in Grenada

Two 1-mo-old local breed chickens, with gross lesions in the skin of the head region suspected to be fowl poxvirus infection, were submitted to the Diagnostic Laboratory of the School of Veterinary Medicine, Grenada, West Indies. Cutaneous lesions were collected from these birds for virus isolation, histopathologic diagnosis, and molecular analysis. Fowl poxvirus infection was confirmed by virus isolation in chicken embryo and by histopathology. Molecular characterization of the fowl poxvirus was conducted by PCR amplification of selected genomic fragments and by nucleotide sequencing. Integration of reticuloendotheliosis virus fragments into the fowl poxvirus genome was confirmed by PCR and DNA sequencing. This is the first report from the Caribbean region on the preliminary molecular characterization of a fowl poxvirus isolate.     

Surveillance for highly pathogenic avian influenza in migratory shorebirds at the terminus of the East Asian-Australasian Flyway

AIM: To determine if migratory birds arriving in New Zealand in the Southern Hemisphere spring of 2004 were infected with the highly pathogenic avian influenza (AI) virus, H5N1.

METHODS: Cloacal and faecal samples were collected from migratory red knots following their arrival in New Zealand in October 2004. Two species of resident sympatric birds, wrybill and mallard duck, were sampled prior to, and following, the arrival of migratory birds.

RESULTS: No AI viruses were isolated from migratory or resident shorebirds. Non-pathogenic AI viruses were isolated from six resident mallard ducks, comprising the endemic subtypes H4 (n=2), H7 (non-pathogenic), H10, and H11 (n=2).

CONCLUSIONS: Highly pathogenic AI H5N1 virus was not detected in migratory shorebirds or sympatric water birds in the Firth of Thames, New Zealand, in 2004-2005, despite the possible proximity of migratory birds to outbreaks of the disease in East Asia in 2004.     

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.     

No detection of avian influenza A viruses of the subtypes H5 and H7 and isolation of lentogenic avian paramyxovirus serotype 1 in passerine birds during stopover in the year 2001 on the island Helgoland (North Sea)

A total of 543 migrating passerines were captured during their stopover on the island of Helgoland (North Sea) in spring and autumn 2001. They were sampled for the detection of avian influenza A viruses (AIV) subtypes H5 and H7, and for avian paramyxoviruses serotype 1 (APMV-1). The goal of the study was to examine the role of migrating birds as potential vectors for these zoonotic viral diseases. For virus detection samples were taken from a) short-distance migrants such as chaffinches (Fringilla coelebs, n = 131) and song trushes (Turdus philomelos, n = 169), and b) long-distance migrants such as garden warbler (Sylvia borin, n = 142) and common redstarts (Phoenicurus phoenicurus, n = 101). Virus detection was done on conjunctival, choanal cleft and cloacal swabs. Embryonated SPF chicken eggs were used to isolate and propagate virus followed by virus identification in a hemagglutination test, hemagglutination inhibition test and in an agar gel diffusion test. In none of the tested samples AIV was detected. Therefore, we conclude that the tested four species of passerines were infected by these pathogens. Six out of 543 birds (1.1 %) were found to carry non-pathogenic and lentogenic strains of APMV-1. This indicates that the passerine species examined in this study may play only a minor role as potential vectors of APMV-1.     

Phylogenetic analysis of avian poxviruses among free-ranging birds of Virginia

Polymerase chain reaction was used to amplify a portion of the avian poxvirus core 4b gene of infected free-ranging birds that presented at the Wildlife Center of Virginia during the 2003 and early 2004 years. The species of bird infected were a great blue heron (Ardea herodias), two American crows (Corvus brachyrhyncos), two American robins (Turdus migratorius), two mourning doves (Zenaida macroura), a red-tailed hawk (Buteo jamaicensis), a blue-gray gnatcatcher (Polioptila caerulea), a northern mockingbird (Mimus polyglottos), a house finch (Carpodacus mexicanus), and a northern cardinal (Cardinalis cardinalis). Phylogenetic analysis was performed using the consensus sequences determined for each avian case in Virginia in combination with avian poxvirus core 4b gene sequence from isolates previously described in Europe and that of vaccinia virus. Alignment of DNA sequences identified areas of point mutations and, in the case of a single mourning dove, the incorporation of a triplet of nucleotides. Maximum-likelihood analysis grouped the 2003-2004 Virginia avian poxviruses into a clade distinct from those reported in European free-ranging birds, with the exception of a single case in a mourning dove that clustered within one European clade. The cladogram that resulted from our analysis of the European isolates is in agreement with those previously published. This study identified a distinct clade of avian poxvirus unique from four clades previously described and associated with epornitics in free-ranging birds, where the core 4b gene DNA sequence has been the basis of comparison.     

Evaluation of plasma (1-->3) beta-D-glucan concentrations in birds naturally and experimentally infected with aspergillus fumigatus

Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1-->3)-beta-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M = 3098.7 pg/dl, SD = 5022.6, n = 22) followed by companion avian species and raptors with confirmed aspergillosis (M = 1033.8 pg/dl, SD = 1531.6, n = 19) and experimentally infected Japanese quail (Coturnix japonica; M = 1066.5 pg/dl, SD = 1348.2, n = 17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-offvalue of > or = 461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection.     

Neurotropism of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica)

This investigation assessed the susceptibility of experimentally infected pigeons to the highly pathogenic avian influenza virus (HPAIV) H5N1 that caused recent outbreaks of avian influenza in birds and humans in several countries of Asia. For this purpose 14 pigeons were infected ocularly and nasally with 10(8) EID50 and clinical signs were recorded and compared with five chickens infected simultaneously as positive controls. The chickens demonstrated anorexia, depression, and 100% mortality within 2 days postinoculation. Three of the pigeons died after a history of depression and severe neurological signs consisting of paresis to paralysis, mild enteric hemorrhage, resulting in a mortality of 21%. Gross lesions in these pigeons were mild and inconsistent. Occasionally subcutaneous hyperemia and hemorrhage and cerebral malacia were observed. Microscopic lesions and detection of viral antigen were confined to the central nervous system of these pigeons. In the cerebrum and to a minor extent in the brain stem a lymphohistiocytic meningoencephalitis with disseminated neuronal and glial cell necrosis, perivascular cuffing, glial nodules, and in one bird focally extensive liquefactive necrosis could be observed. The remaining nine pigeons showed neither clinical signs nor gross or histological lesions associated with avian influenza, although seroconversion against H5 indicated that they had been infected. These results confirm that pigeons are susceptible to HPAIV A/chicken/Indonesia/2003 (H5N1) and that the disease is associated with the neurotropism of this virus. Although sentinel chickens and most pigeons did not develop disease, further experiments have to elucidate whether or not Columbiformes are involved in transmission and spread of highly pathogenic avian influenza.