Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens.

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

Detection of ALV p27 in cloacal swabs and virus isolation medium by sELISA.

BACKGROUND: Avian leukosis (AL), which is caused by avian leukosis virus (ALV), has led to substantial economic losses in the poultry industry. The kit used to detect all ALV-positive chickens in breeder flocks is very important for efficiently controlling AL. However, a new emerging ALV subtype is currently a severe challenge in the poultry industry. RESULTS: In this paper, we compared different enzyme-linked immunosorbent assay (ELISA) kits for detecting p27 of ALV in the same batch of meconium samples. Different positive samples were further analyzed by PCR or virus isolation. The results showed that 36 positive samples among the 1812 chicken meconium samples could be detected by a sandwich ELISA (sELISA) kit, but only 17 positive samples could be identified by a commercial kit. To verify this result, cloacal swabs and viruses isolated from the positive chickens (2 days old) were used to detect the presence of p27. The results showed that the positive rate of p27 was 100% for the swabs and 40% for virus isolation. Surprisingly, PCR and sequence analysis revealed that the env gene of ALV in these positive samples belonged to the novel subgroup K (ALV-K). CONCLUSION: These data not only demonstrate the relatively high sensitivity of the sELISA kit but also highlight the challenge of controlling ALV-K.

A recombination efficiently increases the pathogenesis of the novel K subgroup of avian leukosis virus.

In this study, a recombinant ALV with ALV-K env and ALV-J backbone was generated (designated ALV-K-env-J) and tested in vitro and in vivo. The growth curve in DF1 cells showed that the recombinant virus replicated more efficiently in comparison with the ALV-J and ALV-K. Although all the infected chickens showed growth retardation compared with the non-infected chickens, the viral and serological detection showed that the positive rate and virus load detected in blood and cloaca, and the positive rate and titer of antibody against p27 from the chickens infected with ALV-K-env-J were higher than those from the chickens infected with the ALV-K, but less than those from the chickens infected with the ALV-J. All these data clearly demonstrated that the recombination event in this study increased the pathogenesis of ALV-K, and the potential recombination between different ALV subgroups should be worried when the clinical co-infections occur.

Comparison of Viremia, Cloacal Virus Shedding, Antibody Responses and Pathological Lesions in Adult Chickens, Quails, and Pigeons Infected with ALV-A.

Subgroup A of the avian leukosis virus (ALV-A) can cause severe pathological lesions and death in infected chickens, and its reported hosts have increased recently. To assess the susceptibility of adult chickens, quails, and pigeons to ALV-A, three sets of 250-day-old birds were intraperitoneally inoculated with ALV-A. Viremia and cloacal virus shedding were dynamically detected using an immunofluorescence assay (IFA), ALV-P27 antigen ELISA or RT-PCR; pathological lesions were assessed using tissue sections; ALV-A in tissues was detected by IFA; and ALV-A antibody responses were detected using antibody ELISA kits and an immune diffusion test. The results indicated that persistent viremia occurred in 80% (8/10) of infected chickens, and transient viremia occurred in 17% (2/12) of infected quails, but no viremia occurred in infected pigeons. Cloacal virus shedding occurred intermittently in 80% (8/10) of infected chickens and in 8% (1/12) of infected quails but did not occur in infected pigeons. Severe inflammatory pathological lesions occurred in the visceral tissues of most infected chickens, and mild lesions occurred in a few of the infected quails, but no pathological lesions occurred in the infected pigeons. The ALV-A virus was detected in the visceral tissues of most infected chickens but not in the infected quails and pigeons. Obviously different ALV-A antibody responses occurred in the infected chickens, quails and pigeons. It can be concluded that adult chickens, quails and pigeons have dramatically different susceptibilities to ALV-A. This is the first report on artificial infection by ALV-A in different birds.

Psittacid Adenovirus-2 infection in the critically endangered orange-bellied parrot (Neophema chrysogastor): A key threatening process or an example of a host-adapted virus?

Psittacid Adenovirus-2 (PsAdv-2) was identified in captive orange-bellied parrots (Neophema chrysogastor) during a multifactorial cluster of mortalities at the Adelaide Zoo, South Australia, and an outbreak of Pseudomonas aeruginosa septicaemia at the Tasmanian Department of Primary Industries, Parks, Water and Environment captive breeding facility, Taroona, Tasmania. This was the first time that an adenovirus had been identified in orange-bellied parrots and is the first report of PsAdv-2 in Australia. To investigate the status of PsAdv-2 in the captive population of orange-bellied parrots, 102 healthy birds from five breeding facilities were examined for the presence of PsAdv-2 DNA in droppings and/or cloacal swabs using a nested polymerase chain reaction assay. Additionally, eight birds released to the wild for the 2016 breeding season were similarly tested when they were recaptured prior to migration to be held in captivity for the winter. PsAdv-2 was identified in all breeding facilities as well as the birds recaptured from the wild. Prevalence of shedding ranged from 29.7 to 76.5%, demonstrating that PsAdv-2 is endemic in the captive population of orange-bellied parrots and that wild parrots may have been exposed to the virus. PsAdv-2 DNA was detected in both cloacal swabs and faeces of the orange-bellied parrots, but testing both samples from the same birds suggested that testing faeces would be more sensitive than cloacal swabs. PsAdv-2 was not found in other psittacine species housed in nearby aviaries at the Adelaide Zoo. The source of the infection in the orange-bellied parrots remains undetermined. In this study, PsAdv-2 prevalence of shedding was higher in adult birds as compared to birds less than one year old. Preliminary data also suggested a correlation between adenovirus shedding prevalence within the breeding collection and chick survival.

First report of fowl adenovirus 8a from commercial broiler chickens in Egypt: molecular characterization and pathogenicity.

This study was performed to isolate fowl adenovirus (FAdV) circulating in commercial meat-type chicken in Egypt during 2015 and to identify the pathogenicity of the isolated virus. Cloacal swabs were collected from 9 commercial broiler farms from chickens of 3-5 wk of age in Behira province in Egypt during 2015. FAdV was isolated on chicken embryo liver cells. The virus was identified by conventional polymerase chain reaction targeting a conserved region in the hexon gene. Moreover, phylogenetic analysis of the L1 loop of the hexon gene revealed that the isolated viruses clustered with reference strains belonging to FAdV serotype 8a. This is the first record of FAdV from Egypt on the GenBank. The isolated virus is closely related to strains directly associated with inclusion body hepatitis (IBH) causing considerable economic losses. Pathogenicity study of the virus did not show any mortality, although necropsy and histopathological examination displayed severe hepatitis and degenerative changes in the immune system after 5 d from infection, proving that the virus can cause IBH with intermittent shedding.

Development of a novel immuno-PCR for detection of avian leukosis virus.

Avian leukosis virus (ALV) is an important pathogen for various neoplasms, including lymphoid, myeloid, and erythroid neoplasms, and it causes significant economic loss in the poultry industry. Several efficient methods for the detection of ALV have been reported. However, these previously developed approaches are based on either PCR or immunoassays. Here, we used a proximity ligation technique and combined PCR with the immunoassay to develop a novel immuno-PCR (Im-PCR) approach for the detection of ALV. Our data showed that the Im-PCR had high specificity and sensitivity to ALV. The Im-PCR method selectively reacted to ALV but not to the other avian viruses tested. The limit of detection of Im-PCR could reach 0.5 TCID50. Moreover, the results of Im-PCR were in agreement with results from commercial ELISA when the clinical cloaca samples were used for ALV detection. The present results demonstrate that the novel Im-PCR method can be efficiently applied to detect ALV in a clinical setting. Our data also highlight that Im-PCR may have promising applications in the diagnosis of pathogens.

Occurrence of Bacterial and Viral Pathogens in Common and Noninvasive Diagnostic Sampling from Parrots and Racing Pigeons in Slovenia.

Airborne pathogens can cause infections within parrot (Psittaciformes) and pigeon (Columbiformes) holdings and, in the case of zoonoses, can even spread to humans. Air sampling is a useful, noninvasive method which can enhance the common sampling methods for detection of microorganisms in bird flocks. In this study, fecal and air samples were taken from four parrot holdings. Additionally, cloacal and oropharyngeal swabs as well as air samples were taken from 15 racing pigeon holdings. Parrots were examined for psittacine beak and feather disease virus (PBFDV), proventricular dilatation disease virus (PDDV), adenoviruses (AdVs), avian paramyxovirus type-1 (APMV-1), avian influenza virus (AIV), Chlamydia psittaci (CP), and Mycobacterium avium complex (MAC). MAC and AdVs were detected in three parrot holdings, CP was detected in two parrot holdings, and PBFDV and PDDV were each detected in one parrot holding. Pigeons were examined for the pigeon circovirus (PiCV), AdVs, and CP; PiCV and AdVs were detected in all investigated pigeon holdings and CP was detected in five pigeon holdings.

Reverse genetics based rgH5N2 vaccine provides protection against high dose challenge of H5N1 avian influenza virus in chicken.

An inactivated vaccine was developed using the rgH5N2 virus (6 + 2 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5 ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (n = 10) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (n = 10) on 200(th) dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5 dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titre > 128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28 dpv and 200 dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200 dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses.

Antibody responses induced by recombinant ALV-A gp85 protein vaccine combining with CpG-ODN adjuvant in breeder hens and the protection for their offspring against early infection.

To observe the antibody responses induced by recombinant A subgroup avian leukosis virus (ALV-A) gp85 protein vaccine plus CpG-ODN adjuvant and the protection of maternal antibodies (MAbs) for the hatched chickens against early infection, the gp85 gene was amplified from the proviral cDNA of ALV-A-SDAU09C1 strain using PCR and the recombinant plasmid containing target gene was constructed and expressed in EscherichiaColi. The expressed product was confirmed using SDS-PAGE and western blot that it is about 46KD of recombinant protein. The purified recombinant proteins combining with CpG-ODN adjuvant or Freund's adjuvant were inoculated into the breeder hens, the ALV-A antibodies in serum and in egg-yolk were detected; the fertilized eggs from the vaccinated hens with different titers of egg-yolk antibody were hatched and then challenged with 10(4.2)/0.1mL TCID50 of ALV-A-SDAU09C1 strain, all the hatched chickens were weekly detected for the viremias and the cloacal swab P27 antigen and pathological lesions; the neutralizing test of antisera in vitro was conducted. The results showed that the recombinant gp85 proteins combining with CpG-ODN adjuvant could induce the breeder hens to produce better antibody responses than gp85 protein with Freund's adjuvant or without adjuvant; the MAbs with higher titers induced by CpG-ODN+gp85 proteins could obviously decrease the ratios of viremias (13% vs 33%), cloacal detoxification (20% vs 67%) and death (0% vs 22%) caused by ALV-A infection than those by gp85 protein without adjuvant. The results of the neutralizing test indicated that the antisera from the hatched chickens could neutralize the ALV-A-SDAU09C1 strain in vitro, but which depends on the antibody titers. The results of IFA confirmed that the serum antibody could combine with the ALV in DF1 cells. It can be concluded that the prepared ALV-A gp85 subunit vaccine combining with CpG-ODN adjuvant could induce the breeder hens to produce better neutralizing antibody responses and protect 80% of their offspring chickens against early infection.

[First detection of psittacid herpesvirus 2 in Congo African grey parrots (Psittacus erithacus erithacus) associated with pharyngeal papillomas and cloacal inflammation in Germany]. / Erste Nachweise des Psittacid Herpesvirus 2 bei Kongo-Graupapageien (Psittacus erithacus erithacus) mit papillomatösen Rachenveränderungen und Kloakenentzündungen in Deutschland.

Congo African Grey Parrots (GP; Psittacus erithacus erithacus) from four different avicultures, presented in the Clinic for Exotic Pets, Reptiles and Birds, University of Veterinary Medicine Hannover, Foundation, showed choanal papillomas or hyperemia of the cloacal mucosa. Histologically, the mucosal choanal proliferations were diagnosed as exophytic papillomas and a mild hyperplasia of the cloacal mucosa with lympho-histiocytic inflammation with no visible inclusion bodies was found. Herpesvirus genome was detected by nested PCR in pooled choanal and cloacal swabs from clinically diseased parrots and healthy contact animals. Sequencing of parts of the herpesvirus DNA-polymerase gene indicated 98-100% homology of the detected herpesviruses with the Psittacid Herpesvirus 2 (PsHV-2). In one aviculture with cloacal inflammation papillomavirus-DNA was concurrently found to a PsHV-2 infection. In addition to the four avicultures with clinical symptoms 25 more flocks of grey parrots, in total 57 Congo-GP and 13 Timneh-GP, were examined for a herpesvirus infection. A total of six out of 29 studied parrot avicultures were tested positive for PsHV-2. The detection of this virus also in flocks of GP, which were bred in Europe, shows the establishment of this infection in the GP population in captivity. As indicated in the literature as well as in our study PsHV-2 could be only detected in Congo-GP, independently if they were kept either alone or in mixed avicultures with amazon and macaw species. These findings suggest that PsHV-2 is adapted to this Psittacus species.

Characterization of genotype IX Newcastle disease virus strains isolated from wild birds in the northern Qinling Mountains, China.

This study was conducted to evaluate the virulence and evolution of genotype IX Newcastle disease virus (NDV) isolates obtained from wild birds in the northern Qinling Mountains of China. Five isolates were obtained from 374 larynx and cloacae swabs, which were collected from multiple asymptomatic wild bird species from August 2008 to July 2011, and were subsequently characterized by pathotype and genotype. Deduced amino acid sequences revealed that all five NDV isolates exhibited velogenic fusion protein cleavage sites motif (112)R-R-Q-R-R-F(117), shared as high as 99.8-99.9 % homology with each other, and varied in pathotype by intracerebral pathogenicity indices (ICPI) of 0.425-1.638. Phylogenetic analysis showed that all five isolates were clustered to genotype IX NDV. This is the first study to confirm multiple asymptomatic wild bird species as natural carriers of virulent genotype IX NDV. A novel NDV isolate from the Spotted-necked Dove (family Columbidae) exhibited discordance between its lentogenic ICPI and its virulent proteolytic cleavage site motif (112)R-R-Q-R-R-F(117). Although the five isolates underwent several amino acid mutations in the fusion protein, evidence of continuous evolutionary divergence did exist in the genotype IX NDV, which was always regarded as a conservative genotype.

Preparation and immunoprotection of subgroup B avian leukosis virus inactivated vaccine.

OBJECTIVES: To develop an inactivated vaccine against subgroup B avian leukosis virus (ALV-B) and determine if vaccination of chicken breeders could protect young chicks from ALV-B horizontal infection at early stage and accelerate eradication progress. METHODS: Chicken embryo fibroblast (CEF) cells were inoculated with SDAU09C2 strain of ALV-B and ALV-CEF was inactivated for preparation of oil-adjuvant vaccine. Eggs were collected from un-vaccinated and 9 vaccinated great parent female chickens for incubation. 1-day-old chicks were bled for testing their maternal antibodies to ALV-A/B and then inoculated with ALV-B. Viremia and cloaca p27 detection dynamics were tested and compared between chick groups with or without maternal antibody to ALV. RESULTS: In 3 weeks after 3 vaccination with the inactivated vaccine, all 9 vaccinated breeders developed high antibody titers against ALV-A/B with ELISA read values of 1.69-1.89 (the positive base line was 0.4) and kept at the high titers for at least another 4 weeks. Maternal antibody was detected in 70% (12/17) of chicks from breeders with high antibody titers to ALV-A/B. Only 4 of 12 chickens with maternal antibodies developed temporary viremia and no viremia was detected in the left 8 maternal antibody positive chickens during the whole 14 week after inoculation of ALV-B at 1 day of age. But the persistent viremia was detected in 2-8 weeks in all 9 maternal antibody negative chickens and the viremia persisted in the whole tested period of 14 weeks after inoculation of ALV-B. CONCLUSIONS: The inactivated ALV-B vaccine could induce high titer antibody reaction to ALV-B, it could provide maternal antibodies to 1-day-old chickens and protect chickens from early infection of ALV-B.

Protection of red-legged partridges (Alectoris rufa) against West Nile virus (WNV) infection after immunization with WNV recombinant envelope protein E (rE).

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread.

Identification and characterization of a novel adenovirus in the cloacal bursa of gulls.

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

Tissue tropism and pathology of natural influenza virus infection in black-headed gulls (Chroicocephalus ridibundus).

Black-headed gulls (Chroicocephalus ridibundus) are a suitable host species to study the epidemiology of low-pathogenic avian influenza virus (LPAIV) infection in wild waterbirds because they are a common colony-breeding species in which LPAIV infection is detected frequently, limited mainly to the H13 and H16 subtypes. However, the sites of virus replication and associated lesions are poorly understood. We therefore performed virological and pathological analyses on tissues of black-headed gulls naturally infected with LPAIV. We found that 24 of 111 black-headed gulls collected from breeding colonies were infected with LPAIV (10 birds with H16N3, one bird with H13N8, 13 birds undetermined), based on virus and viral genome detection in pharyngeal and cloacal swabs. Of these 24 gulls, 15 expressed virus antigen in their tissues. Virus antigen expression was limited to epithelial cells of intestine and cloacal bursa. No histological lesions were detected in association with virus antigen expression. Our findings show that LPAIV replication in the intestinal tract of black-headed gulls is mainly a superficial infection in absence of detectable lesions, as determined recently for natural LPAIV infection in free-living mallards (Anas platyrhynchos). These findings imply that LPAIV in black-headed gulls has adapted to minimal pathogenicity to its host and that potentially the primary transmission route is faecal-oral.

Response of white leghorn chickens to infection with avian leukosis virus subgroup J and infectious bursal disease virus.

The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hcl, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J-induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J-infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.

Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E).

The present study describes the development of a SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of all fowl adenovirus (FAdV) species. Primers were designed based on conserved nucleotide sequences within the 52K gene. Ten-fold serial dilutions of a vector DNA were used as standard for quantitation. The real-time PCR had an efficiency of 98%, a regression squared value of 0.999 and showed a range of 6.73-6.73×10(8) copies of FAdV DNA per reaction. The assay was highly specific for FAdVs and an exact quantitation of all 5 FAdV species (FAdV-A to FAdV-E) could be demonstrated. It was shown, that twelve FAdV serotypes (FAdV-1 to 8a, and 8b to 11) were detectable and quantifiable. Other viral genomes as well as uninfected chicken embryo liver (CEL) cells did not produce positive signal. Cloacal swabs were taken during the animal experiment, which was performed with all FAdV species. Shedding of FAdVs was investigated in cell culture, by conventional PCR and by the developed real-time PCR. The real-time PCR was found more sensitive than cell culture and conventional PCR. Detection and quantitation of FAdVs in different type of samples was possible by the new real-time PCR.

Monitoring of wild birds for Newcastle disease virus in north Queensland, Australia.

Wild aquatic birds (WABs) are considered as reservoir hosts for Newcastle disease viruses (NDVs) and may act as vectors for transferring these viruses to poultry, causing outbreaks of disease. A 3-year epidemiological study was conducted on WABs of north Queensland from April 2007 to March 2010. Swab and fresh moist faecal samples of WABs were screened to detect Newcastle disease viral (NDV) RNA by one-step real time reverse transcriptase polymerase chain reaction (rRT-PCR) in multiplex primers, targeting the matrix gene. The potential reactor samples in rRT-PCR were processed for sequencing of the different NDV genes using conventional PCR. The overall NDV RNA prevalence was 3.5% for live bird samples (N=1461) and 0.4% for faecal samples (N=1157). Plumed whistling ducks (PWDs) had a higher prevalence (4.2%) than Pacific black ducks (PBDs) (0.9%) (χ(2) test, p=0.001). Univariate and multivariate logistic regression analyses were used to estimate the association between the proportion of reactor and non-reactor NDV RNA samples of PWDs and potential risk factors. The odds of reactor samples were 2.7 (95% Confidence Interval 1.5-4.9) times more likely in younger than older ducks (p=0.001) (data set B, multivariate analysis). Both NDV RNA class-one and class-two types were identified in samples of WABs (12 and 59, respectively) (Supplementary Table 1). Phylogenetic analysis of the matrix gene identified two reactor sequences of class-one type NDV RNA (PWD-48 and 55) which were closely related to the sequences of Australian Ibis and duck isolates (Fig. 2). Another reactor sample sequence was determined as class-two type NDV RNA (PWD-46, avirulent) based on analysis of the matrix and fusion genes which was more similar to the sequences of Australian I-2 progenitor virus and vaccine strain virus (Figs. 3 and 4). Our findings of higher prevalence in PWDs along with confirmation of class-one and class-two type NDV RNAs will significantly contribute to the design of surveillance programs for NDVs in northern Australia.

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus.

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 µg of HA antigens and complete heterologous protection required over 5.0 µg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 µg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.