Identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed Nile crocodiles, Crocodylus niloticus

When large numbers of crocodile skins were downgraded because of the presence of small pin prick-like holes, collapsed epidermal cysts were found deep in the dermis of juvenile crocodiles while forming cysts were observed in hatchlings. Histopathology of these forming cysts showed the presence of intracytoplasmic inclusions in proliferating and ballooning epidermal cells. Pox virions were seen in electron microscope preparations made from the scabs of such early lesions. The partial sequencing of virus material from scrapings of these lesions and comparison of it with the published sequence of crocodile poxvirus showed the virus associated with the deep lesions to be closely related, but different. To differentiate between the two forms of crocodile pox infection it is suggested that the previously known form should be called "classical crocodile pox" and the newly discovered form "atypical crocodile pox". The application of strict hygiene measures brought about a decline in the percentage of downgraded skins.     

Isolation of surface tubules of fowlpox virus

Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.     

Vaccinating chickens against avian influenza with fowlpox recombinants expressing the H7 haemagglutinin

OBJECTIVE: To evaluate the vaccine efficacy of a fowlpox virus recombinant expressing the H7 haemagglutinin of avian influenza virus in poultry. PROCEDURE: Specific-pathogen-free poultry were vaccinated with fowlpox recombinants expressing H7 or H1 haemagglutinins of influenza virus. Chickens were vaccinated at 2 or 7 days of age and challenged with virulent Australian avian influenza virus at 10 and 21 days later, respectively. Morbidity and mortality, body weight change and the development of immune responses to influenza haemagglutinin and nucleoprotein were recorded. RESULTS: Vaccination of poultry with fowlpox H7 avian influenza virus recombinants induced protective immune responses. All chickens vaccinated at 7 days of age and challenged 21 days later were protected from death. Few clinical signs of infection developed. In contrast, unvaccinated or chickens vaccinated with a non-recombinant fowlpox or a fowlpox expressing the H1 haemagglutinin of human influenza were highly susceptible to avian influenza. All those chickens died within 72 h of challenge. In younger chickens, vaccinated at 2 days of age and challenged 10 days later the protection was lower with 80% of chickens protected from death. Chickens surviving vaccination and challenge had high antibody responses to haemagglutinin and primary antibody responses to nucleoprotein suggesting that although vaccination protected substantially against disease it failed to completely prevent replication of the challenge avian influenza virus. CONCLUSION: Vaccination of chickens with fowlpox virus expressing the avian influenza H7 haemagglutinin provided good protection against experimental challenge with virulent avian influenza of H7 type. Although eradication will remain the method of first choice for control of avian influenza, in the circumstances of a continuing and widespread outbreak the availability of vaccines based upon fowlpox recombinants provides an additional method for disease control.     

Poxvirus infection in Nile crocodiles (Crocodylus niloticus)

An outbreak was encountered of numerous yellowish cutaneous nodules in one- to two-year-old farmed Nile crocodiles (Crocodylus niloticus) in Kasaba Bay Crocodile Farm at Lake Tanganyika in Zambia during 1988. Out of 4000 crocodiles of different age groups, 300 yearlings were affected and 82 of those affected died. The lesions were prominent on the head, especially around the eyelids, the nostrils, both sides of the mouth, ventral neck, ventral pale belly, limbs and the root of the tail. Histologically, the epidermal lesions revealed large focal areas of marked acanthosis accompanied by hyperkeratosis and parakeratosis. In the prickle cells, there were multiple small neutrophilic granular inclusions and large eosinophilic homogeneous cytoplasmic inclusions. Electron microscopically, there were numerous poxvirus particles and matrices in the cytoplasm of many prickle cells. Dark blue homogeneous cytoplasmic inclusions in toluidine blue sections consisted of an electron opaque matrix with mature viral particles (160 x 200 x 230 nm). Furthermore, there were clumps of granular matrix containing immature viral particles (200 x 399 nm) in the cytoplasm. Various features of viral developing processes were observed.     

A novel DNA virus associated with feather inclusions in psittacine beak and feather disease

The nature of feather inclusions was characterized in 32 psittacine birds (30 cockatoos, one peach-faced lovebird (Agapornis roseicollis), and one red-lored Amazon parrot (Amazona autumnalis autumnalis] with naturally-acquired psittacine beak and feather disease. Intranuclear inclusions within feather epithelial cells and intracytoplasmic inclusions within macrophages in the feather epithelium and pulp cavity contained psittacine beak and feather disease viral antigen when stained by the avidin-biotin complex immunoperoxidase technique. Ultrastructurally, inclusions were observed primarily within macrophages and to a lesser extent within epithelial cell nuclei. Macrophage inclusions appeared as paracrystalline arrays of viral particles. Intranuclear inclusions were less well defined, although scattered viral particles were present. Intracytoplasmic and intranuclear particles in ultrastructural preparations were identified by colloidal gold labeling as psittacine beak and feather disease virus. Feather epithelium was more frequently and severely involved in the disease process than was adjacent follicular epithelium. Plucked feathers with an intact epidermal collar and feather epithelium were preferred to follicular biopsies for histopathologic examination.     

Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.     

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.     

Detection of fowlpox in chickens and turkeys in Germany

The present work outlines molecular diagnostic examinations for detection of poxvirus infection in chickens and turkeys in Germany over a time period of twelve years. Diagnostic samples suspected for fowlpox were investigated using the polymerase chain reaction (PCR) in combination with restriction enzyme analysis (REA) for presence of fowlpox virus (FPV) specific DNA. For a long period of time fowlpox did not play a role in commercial poultry farms in Germany. Beginning in 1999 an increasing number of new infections was identified. During the whole study period FPV specific DNA was detected in 92 out of 192 investigated samples. Positive samples were derived especially from layer hens but also from broiler breeders, turkey breeders, and meat turkeys. Thereby, a differentiation between isolates of chickens and turkeys by restriction enzyme analysis (REA) was not possible. As possible explanations for this reemergence, especially the lack of prophylactic vaccination in the past as well as an increasing number of alternative rearing systems has to be considered. Beginning in 2003, a downward tendency was observed following intensification of prophylactic vaccination.     

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.     

The pathology of psittacine beak and feather disease

Psittacine beak and feather disease is characterised by loss of feathers, abnormally shaped feathers and overgrowth and irregularity of the surface of the beak. The disease occurs in a number of psittacine species including the Sulphur-crested Cockatoo, Lovebirds , Budgerigars and Galahs . The abnormal appearance of feathers and beak is due to a dystrophic process within the epidermis of the feather and beak. The process consists of epidermal cell necrosis, epidermal hyperplasia and hyperkeratosis. Many of the feather abnormalities are due to retention of a hyperkeratotic feather sheath. A characteristic microscopic finding is the presence of macrophages containing purple intracytoplasmic inclusions in affected epidermis and feather pulp. The inclusions consist of aggregates of particles 17 to 22 nm in diameter. Similar but smaller inclusions occur in epidermal cells. In addition, non-suppurative inflammation occurs in the feather pulp. The findings are suggestive of a viral infection.     

抗鸡传染性喉气管炎重组鸡痘病毒基因工程疫苗同居感染试验

疫苗的接触传播是疫苗免疫接种需要考虑的重要因素,为了检测重组鸡痘病毒载体疫苗水平传播的能力,对隔离条件下饲养的SPF鸡用重组鸡痘病毒基因工程疫苗接种,同时设立非免疫对照鸡,饲养期间特意延长清粪时间以增加感染的机会,1个月之后攻击传染性喉气管炎WG株强毒和鸡痘102株强毒,疫苗免疫鸡全部获得保护,而非免疫鸡则全部发病.在试验动物饲养场的自然条件下,将免疫鸡和试验对照两组鸡饲养在同一个鸡舍内,让疫苗毒的传播更接近自然条件.在每个月的攻毒试验中,对照鸡都没有获得对鸡痘和传染性喉气管炎强毒的保护.在疫苗免疫期间进行连续5个月的跟踪检测,同居未免疫鸡没有检测到抗传染性喉气管炎病毒gB抗体.这些实验结果表明抗鸡传染性喉气管炎重组鸡痘病毒基因工程疫苗不能通过接触传播.     

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.     

The DNA repair enzyme, CPD-photolyase restores the infectivity of UV-damaged fowlpox virus isolated from infected scabs of chickens

Fowlpox virus (FWPV), an important pathogen of poultry, replicates very efficiently in the featherless areas of skin, and persists in dried and desiccated scabs for prolonged periods. Although the molecular mechanisms underlying the stability of the virus are not completely known, we recently identified the presence of a virus-encoded novel DNA repair enzyme, CPD-photolyase, in FWPV. This enzyme repairs the ultraviolet (UV)-induced pyrimidine dimers, converting them to monomers using photons from white light as a renewable source of energy. In this study, we examined the role of photolyase in the pathogenesis of fowlpox. A comparison of pathogenesis of fowlpox in chickens infected with parental FWPV with that in chickens infected with photolyase-deficient FWPV (Phr(-) FWPV) found no significant differences in terms of replication of virus or formation of secondary lesions. When the virions isolated from infected scabs were exposed to UV light, UV-damaged parental FWPV, unlike Phr(-) FWPV, were rescued through the CPD-photolyase-mediated photoreactivation pathway by at least 48%. However, the mutant virus triggered host's immune response and conferred complete protection against subsequent challenge with virus similar to that conferred by the parental virus. Since the mutant virus is less stable than the parental virus in the infected scabs but is as immunogenic, Phr(-) FWPV might be less persistent in the environment. Furthermore, this particular genetic locus can also be used to insert foreign genes for the development of FWPV recombinant vaccines.     

Immune responses of swine inoculated with a recombinant fowlpox virus co-expressing P12A and 3C of FMDV and swine IL-18

Two recombinant fowlpox viruses (rFPV-P1 and rFPV-IL18-2AP12A) containing foot-and-mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 were evaluated to determine their abilities to induce humoral and cellular responses in the presence or absence of swine IL-18 as genetic adjuvant. The ability to protect swine against homologous virus challenge was examined. All swine were given booster vaccinations at 21 days after the initial inoculation and were challenged 10 days after the booster vaccination. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-P12A and rFPV-IL18-P12A developed specific anti-FMDV ELISA antibody and neutralizing antibody and T-lymphocyte proliferation was observed. Cellular immune function was evaluated via examination of IFN-gamma production in swine peripheral blood serum. The results demonstrate the potential viability of a fowlpox virus-based recombinant vaccine in the control and prevention of FMDV infections.     

禽痘病毒感染对禽流感重组禽痘病毒疫苗免疫效力的影响

表达禽流感病毒 (AIV)HA和NA基因的重组禽痘病毒rFPV_HA_NA能够诱导鸡体产生 10 0 %抵抗高致病性禽流感病毒 (HPAIV)H5N1的攻击。而当鸡群已进行禽痘疫苗免疫或者感染了禽痘病毒的情况下 ,此重组疫苗的免疫效力如何 ?首先用禽痘病毒S_FPV_0 17人工感染SPF试验鸡 ,既而在感染后的不同间隔时间接种重组疫苗 ,免疫后检测鸡群的HI抗体水平 ,同时用 10 0LD50 的HPAIVH5N1进行攻击。结果重组疫苗免疫与禽痘病毒人工感染时间间隔在 4周 (或以上 )时 ,预先感染禽痘病毒对重组疫苗的免疫效力不构成影响 ,对禽流感的保护力为 10 0 % ,而间隔时间在 1、2、3周时 ,重组疫苗的免疫保护效力则受到不同程度的影响。     

北京油鸡快慢羽母鸡鉴定以及羽毛发育、生长和产蛋性能的对比分析

试验旨在为北京油鸡品系选育、配套利用和科学养殖提供基础数据。选用北京油鸡纯系母雏534只,开展快慢羽群体的鉴定,并对比快慢羽母鸡羽毛发育、生长和繁殖性能的差异。北京油鸡保种群出雏时按照主翼羽与覆主翼羽的相对长度表型特征鉴别,将其分为快慢羽亚群,其中快羽包括K1（主翼羽长于覆主翼羽5 mm以上）和K2（主翼羽长于覆主翼羽2~5 mm）,慢羽包括M1（主翼羽与覆主翼羽等长或主翼羽长于覆主翼羽2 mm以内）、M2（主翼羽短于覆主翼羽）和M3（主翼羽未长出）。6周龄时通过PCR方法进行再次鉴定快慢羽。7 d内每隔1 d测量1次主翼羽与覆主翼羽羽长;7~42 d每隔1周测量1次;1~8周每周测定体重,9~18周每隔1周测定体重;产蛋期根据个体产蛋记录统计群体开产日龄、个体产蛋数以及连产相关性状等产蛋性能。结果表明,北京油鸡初生雏鸡快慢羽表型鉴定结果与通过PCR方法鉴定的结果一致,快羽鸡占总数的25%,慢羽鸡占75%,慢羽鸡又以M2为主,有少量M1和M3。21日龄以内快羽鸡的主翼羽羽长显著高于慢羽鸡（P<0.05）;28日龄时,M1和M2型慢羽鸡的主翼羽长度与快羽鸡差异不显著（P>0.05）,但是M3仍显著短于快羽鸡（P<0.05）。与慢羽鸡相比,快羽鸡70日龄后体重显著增加（P<0.05）,且开产日龄显著提前（P<0.05）,但是43周产蛋数较低（P<0.05）;快羽鸡除蛋形指数显著高于慢羽鸡外（P<0.05）,其他蛋品质指标均无显著差异。综上,快慢羽北京油鸡在生长和产蛋性能上有一定差异,在选育方向和生产管理中应有所差异化,包括加强慢羽品系早熟性即开产日龄的选育和调整快羽品系育成期饲料能蛋水平等。     

重组鸡痘病毒vFV282活疫苗最小免疫剂量及攻毒保护试验

将重组鸡痘病毒vFV282疫苗用生理盐水作10^-1，10^-2，10^-3，10^-4系列稀释，分别免疫7天龄鸡，于免疫后21d，分别用NDV、IBDV和FPV攻毒，观察其保护率，结果除NDV攻毒在10^-4组保护率为40％（4／10），其余各组均为100％（10／10）保护。表明该疫苗的最小免疫剂量≤10^-3TCID50/0.02mL。     

Antibody response to and maternal immunity from an experimental psittacine beak and feather disease vaccine.

Adult umbrella cockatoos, Moluccan cockatoos, African grey parrots, and a yellow-headed Amazon parrot were inoculated IM or SC with beta-propiolactone-treated psittacine beak and feather disease (PBFD) virus. Thirty- to 45-day-old African grey parrot, umbrella cockatoo, and sulphur-crested cockatoo chicks also were vaccinated with the same inoculum. The hemagglutination inhibition (HI) and agar-gel diffusion tests were used to assay for post-vaccination development of anti-PBFD virus antibodies. All adult vaccinates seroconverted and had increases in HI and precipitating antibodies. The vaccinated chicks had increased concentrations of HI antibodies, but precipitating antibodies could not be detected. To demonstrate that chicks from vaccinated hens are protected from PBFD virus challenge, 3 African grey parrot chicks and 2 umbrella cockatoo chicks from vaccinated hens and 1 African grey parrot chick and 1 umbrella cockatoo chick from nonvaccinated hens were exposed to purified PBFD virus. Chicks from the vaccinated hens remained clinically normal during the 50-day test period. Chicks from the nonvaccinated hens developed clinical and histologic lesions of PBFD. Infected tissues from these birds were confirmed to contain viral antigen, using immunohistochemical staining techniques. The PBFD virus was recovered from the affected birds. These findings indicate that adult and 30- to 45-day-old psittacine birds will seroconvert following vaccination with beta-propiolactone-treated PBFD virus. Also, hens inoculated with beta-propiolactone-treated PBFD virus produce chicks that are, at least temporarily, resistant to virus challenge.     

Skin microbiota of quaker parrots (Myiopsitta monachus) with normal feathering or feather loss via next-generation sequencing technology

BackgroundFeather damaging behavior (feather picking) is a common malady of captive parrots. Diagnosis is challenging as a primary disadvantage to clinicians faced with diagnosis of parrot skin disease remains the lack of normative data for the healthy parrot epidermal microbiology. The objective of this pilot study was to establish bacterial and fungal cutaneous microbiota baselines for quaker parrots with and without feather loss.MethodsHealthy quaker parrots (Myiopsitta monachus n = 12) with intact feathers were compared to 12 quaker parrots with evidence of feather loss via next-generation DNA sequencing of bacteria and fungus swabbed from skin.ResultsOf 351 bacterial and 97 fungal species identified, no statistical differences occurred in bacteria or fungal alpha-diversity or beta-diversity based on age, sex, or feather loss. However, enclosure affected bacterial beta-diversity. The most abundant bacterial species for fully feathered quaker parrots were Exiguobacterium indicum, Sphingomonas yabuuchiae, and Corynebacterium spp.,. Fragaria/Populus euphratica, S. yabuuchiae, and E. indicum for quaker parrots with feather loss. Lactobacillales, Acinetobacter, Kocuria sp., and Streptococcus epidermidis were significantly enriched in fully feathered quaker parrots, while Streptococcaceae, Methylobacterium, Clostridiales, Nocardioidaceae, Nesterenkonia, and Actinomycetospora were significantly enriched in quaker parrots with feather loss. Both groups had Cladosporium halotolerans/sphaerospermum as the predominant fungus, while Capnodiales was significantly enriched in quaker parrots with feather loss.Conclusions and clinical relevanceUse of next generation microbial sequencing as a preliminary diagnostic tool of bacterial and fungal communities of captive birds allows veterinarians to confirm cytologic diagnoses and circumvent the limitations of culture testing. Diagnostic implications include the potential transmission of pathogenic bacteria and/or fungi and pathogen resistance between cohoused birds.     

Vaccination of chickens with a recombinant fowlpox virus containing the hemagglutinin-neuraminidase gene of Newcastle disease virus under the control of the fowlpox virus thymidine kinase promoter.

When chickens were vaccinated with a recombinant fowlpox virus (FPV) containing the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) cDNA under the control of the thymidine kinase (TK) promoter and inserted into the FPV TK gene, the FPV antibody response to the recombinant virus was similar to the response to vaccination with standard FPV, and the recombinant virus protected chickens against challenge with virulent FPV. While the presence of the NDV HN cDNA was demonstrated in the recombinant virus, which was stable on serial passage, expression of HN was not detected by hemagglutination, Western blot analysis or immunoprecipitation of infected cell lysate. Chickens vaccinated with the recombinant virus failed to mount an NDV hemagglutination-inhibition antibody response, and they did not resist challenge with velogenic NDV. It was concluded that the TK promoter was too weak to drive the HN gene, but that the insertion into the FPV TK gene did not reduce the immunogenicity of the virus.