High-resolution melt curve analysis to confirm the presence of co-circulating isolates of avian nephritis virus in commercial chicken flocks.

Avian Nephritis Virus (ANV) has been implicated in poor growth and renal disease of young chickens. This paper describes the development of a reverse-transcriptase polymerase chain reaction for the detection of ANV in commercial meat chickens and the use of high-resolution melt curves to detect the presence of genetically different ANVs. Pooled cloacal swabs from both healthy and ill commercial chicken broiler flocks were tested for the presence of ANV using a combination of polymerase chain reaction, molecular cloning, high-resolution melt curve analysis and sequencing. Except for one, all specimens were found to contain two genetically different ANVs. Phylogenetic analysis of the capsid amino acid sequences revealed the presence of four of six groups of ANV identified previously in other countries as well as in two novel groups of ANV. Phylogenetic analysis of nucleotide sequences of partial polymerase, capsid and 3' untranslated regions reveal that the genes of individual ANV virus isolates have different ancestors. This was shown to be due to a template-switching event in the capsid gene that resulted in the 3' end of the capsid gene and the 3' untranslated region of one ANV isolate being transferred to another ANV. These results reveal that infection of chicken flocks with multiple ANV isolates is common and this needs to be taken into consideration in diagnosis of ANV using molecular techniques and in future epidemiological investigations.

Survey of captive parrot populations around Port Phillip Bay, Victoria, Australia, for psittacine beak and feather disease virus, avian polyomavirus and psittacine adenovirus.

OBJECTIVE: This study investigated the prevalence of psittacine beak and feather disease virus (BFDV), avian polyomavirus (APV) and psittacine adenovirus (PsAdV) in captive psittacine birds around Port Phillip Bay, Victoria, Australia. METHODS: Samples of fresh droppings were collected from 118 psittacine birds (109 clinically normal and 9 with feather abnormalities) from 11 avaries in different locations and were used for detection of BFDV, APV and PsAdV using PCR. RESULTS: BFDV, APV and PsAdV were detected in 31%, 13% and 4%, respectively, of the specimens tested. One budgerigar was found to be co-infected with BFDV and PsAdV. At least one sample tested positive for BFDV at each location. CONCLUSION: This is the first report of the prevalence of BFDV, APV and PsAdV in Victoria and provides a foundation for future studies examining the influence of these viruses on the health of aviary birds in Victoria.

The C-terminal end of the capsid protein of Avian Nephritis Virus is antigenic and induces broadly cross-reactive antibodies.

Avian nephritis virus (ANV) has been isolated frequently from commercial broilers in many countries. The prevalence and economic impact of ANV however has been difficult to ascertain due to the lack of convenient serological techniques. In this study the full-length and fragments of the ANV capsid protein were expressed in Baculovirus and affinity purified recombinant proteins used for the detection of ANV antibodies in ELISA. The crystal structure of Human Astrovirus (HAstV) was used as a model to determine potential homologous C-terminal antigenic regions in ANV. The rp37 fragment from three ANV strains NSW_3, ANV-1 and ANV-2, and a shorter NSW_3 fragment (rp33) were compared for their ability to detect ANV antibodies in seven reference chicken sera. The ANV-1 rp37 antigen was the most strain specific whereas the NSW_3 rp37 and rp33 antigens detected antibodies in all heterologous sera, including ANV-1 serum. Irrespective of the strain used, the two NSW_3 protein fragments rp37 and rp33 were found to be superior as antigens for ELISA when compared to the full-length capsid protein rp75. An ELISA designed using the NSW_3 rp33 could reliably differentiate between uninfected and infected commercial broiler flocks, as demonstrated by statistically significant differences between the OD values. This study identified an ANV immunogenic region and successfully used recombinant protein expression of this region to detect cross-reactive ANV antibodies. The results of this study facilitate future studies into the epidemiology and importance of ANV infections in commercial poultry.

Assessment of the potential relationship between egg quality and infectious bronchitis virus infection in Australian layer flocks.

OBJECTIVE: This investigation aimed to determine if there was a relationship between the production of eggs with poor internal quality, as measured by poor Haugh units, by Australian layer flocks and the detection of infectious bronchitis virus (IBV) in the hens. Other risk factors including flock size, flock type, flock age, chicken breed and vaccination frequency were also assessed. METHODS: The study group comprised 17 flocks from 14 farms. Data relating to the factors investigated were requested on a regular basis. The Haugh unit data were used to grade eggs as good or poor based on the age and flock at the time of data collection. Cloacal swabs were collected from 20 chickens in each flock approximately every 6 weeks. RESULTS: IBV was detected from a majority of the flocks and in 68% of cases the IBV strain detected was an A-vaccine-related field strain. Three variant strains were detected. Detection of IBV in a flock, the farm type and flock size were identified as potential risk factors for the production of eggs with poor Haugh units. CONCLUSION: IBV is prevalent in Australian layer flocks, but infection was primarily subclinical. The results complement previous reports indicating that there are many potential risk factors for the production of eggs with poor Haugh units.

Application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens.

OBJECTIVE: A real-time polymerase chain reaction (PCR)/high-resolution melt (HRM) curve analysis protocol was developed in our laboratory to differentiate infectious bronchitis (IB) virus reference strains. In the current study, this method was used to detect and classify IB viruses in field submissions. PROCEDURE: Over an 11-month period samples from 40 cases of suspected IB virus were received and 17 submissions were positive for IB virus by polymerase chain reaction. HRM curve analysis classified each strain as subgroup 1, 2 or 3 strain (12 submissions) or a strain that was unable to be classified (5 submissions). The 3' untranslated region (UTR) and partial S1 gene nucleotide sequences for the 17 IB virus strains were determined and their identity with those of the relative reference strains compared to confirm the classifications generated using the HRM curve analysis. RESULTS: Of the 12 IB field viruses classified as subgroup 1, 2, or 3 using HRM curve analysis, the 3'UTR and S1 gene nucleotide sequences had identities ≥99% with the respective subgroup reference strain. Analysis of the 3' UTR and S1 gene nucleotide sequences for the five IB virus strains that could not be classified indicated that four belonged to one of the subgroups, and one was a potential recombinant strain (between strains from subgroups 2 and 3). A novel recombinant strain was also detected. CONCLUSION: HRM curve analysis can rapidly assign the majority of IB viruses present in field submissions to known subgroups. Importantly, HRM curve analysis also identified variant genotypes that require further investigation.