Phylogenetic Variation of Chelonid Alphaherpesvirus 5 (ChHV5) in Populations of Green Turtles Chelonia mydas along the Queensland Coast, Australia.

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north-south trend. Received October 22, 2016; accepted May 7, 2017.

Avian influenza in Australia: a summary of 5 years of wild bird surveillance.

BACKGROUND: Avian influenza viruses (AIVs) are found worldwide in numerous bird species, causing significant disease in gallinaceous poultry and occasionally other species. Surveillance of wild bird reservoirs provides an opportunity to add to the understanding of the epidemiology of AIVs. METHODS: This study examined key findings from the National Avian Influenza Wild Bird Surveillance Program over a 5-year period (July 2007-June 2012), the main source of information on AIVs circulating in Australia. RESULTS: The overall proportion of birds that tested positive for influenza A via PCR was 1.9 ± 0.1%, with evidence of widespread exposure of Australian wild birds to most low pathogenic avian influenza (LPAI) subtypes (H1-13, H16). LPAI H5 subtypes were found to be dominant and widespread during this 5-year period. CONCLUSION: Given Australia's isolation, both geographically and ecologically, it is important for Australia not to assume that the epidemiology of AIV from other geographic regions applies here. Despite all previous highly pathogenic avian influenza outbreaks in Australian poultry being attributed to H7 subtypes, widespread detection of H5 subtypes in wild birds may represent an ongoing risk to the Australian poultry industry.

Viral and vector zoonotic exploitation of a homo-sociome memetic complex.

As most newly characterized emerging infectious diseases are considered to be zoonotic, a modern pre-eminence ascribed within this classification lies clearly within the viral taxonomic realm. In particular, RNA viruses deserve special concern given their documented impact on conservation biology, veterinary medicine and public health, with an unprecedented ability to promote an evolutionary host-pathogen arms race from the ultimate infection and immunity perspective. However, besides the requisite molecular/gross anatomical and physiological bases for infectious diseases to transmit from one host to another, both viral pathogens and their reservoirs/vectors exploit a complex anthropological, cultural, historical, psychological and social suite that specifically defines the phylodynamics within Homo sapiens, unlike any other species. Some of these variables include the ecological benefits of living in groups, decisions on hunting and foraging behaviours and dietary preferences, myths and religious doctrines, health economics, travel destinations, population planning, political decisions on agricultural product bans and many others, in a homo-sociome memetic complex. Taken to an extreme, such complexities elucidate the underpinnings of explanations as to why certain viral zoonoses reside in neglected people, places and things, whereas others are chosen selectively and prioritized for active mitigation. Canine-transmitted rabies serves as one prime example of how a neglected viral zoonosis may transition to greater attention on the basis of renewed advocacy, social media, local champions and vested international community engagement. In contrast, certain bat-associated and arboviral diseases suffer from basic ignorance and perpetuated misunderstanding of fundamental reservoir and vector ecology tenets, translated into failed control policies that only exacerbate the underlying environmental conditions of concern. Beyond applied biomedical knowledge, epidemiological skills and biotechnical abilities alone, if a homo-sociome memetic complex approach is also entertained in a modern transdisciplinary context, neglected viral zoonosis may be better understood, controlled, prevented and possibly eliminated, in a more holistic One Health context.

Causes of morbidity and mortality of wild aquatic birds at Billabong Sanctuary, Townsville, North Queensland, Australia.

Infectious diseases are common causes of significant morbidity and mortality events of wild aquatic birds (WABs) worldwide. Reports of Australian events are infrequent. A 3-yr passive surveillance program investigating the common causes of morbidity and mortality of WABs was conducted at Billabong Sanctuary near Townsville, North Queensland, from April 2007 to March 2010. Forty-two carcasses were obtained and evaluated by clinico-pathologic, histologic, bacteriologic, and virologic (molecular) examinations. Morbidity and mortality were sporadic and more commonly observed in chicks and juvenile birds in April than other months of the year. Morbid birds were frequently unable to walk. Hemorrhagic lesions and infiltration of lymphocytes in various organs were the most common findings in dead birds. Identified bacterial diseases that could cause bird mortality were colibacillosis, pasteurellosis, and salmonellosis. Salmonella serotypes Virchow and Hvittingfoss were isolated from an Australian white ibis (Threskiornis molucca) chick and two juvenile plumed whistling ducks (Dendrocygna eytoni) in April 2007. These strains have been previously isolated from humans in North Queensland. A multiplex real time reverse transcriptase-PCR (rRT-PCR) detected Newcastle disease viral RNA (class 2 type) in one adult Australian pelican (Pelecanus conspicillatus) and a juvenile plumed whistling duck. No avian influenza viral RNA was detected from any sampled birds by the rRT-PCR for avian influenza. This study identified the public health importance of Salmonella in WABs but did not detect the introduction of the high pathogenicity avian influenza H5N1 virus in the population. A successful network was established between the property owner and the James Cook University research team through which dead birds, with accompanying information, were readily obtained for analysis. There is an opportunity for establishing a long-term passive disease surveillance program for WABs in North Queensland, an important region in Australian biosecurity, thus potentially significantly benefitting public health in the region and the country.

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.

Adaptations of a competitive enzyme-linked immunosorbent assays for the detection of antibodies to influenza a virus in horse sera for use in wild aquatic birds.

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

Host preferences of tabanid flies based on identification of blood meals by ELISA.

Tabanid flies in Australia are potential vectors of the parasite Trypanosoma evansi which causes the animal disease surra. It is endemic to most of south-east Asia and could enter Australia, but evaluation of the potential impact of a surra incursion requires identification of the major hosts of Australian tabanids. This study investigated the natural pattern of feeding and host preference by tabanid flies of Townsville, north Queensland by identification of ingested blood in trap-caught tabanids using ELISA. The assays were developed for identification of horse, cow, macropod and pig blood meals. Macropods were the most frequent food source for each of six major tabanid species in the area. This did not vary with location for one species, Tabanus pallipennis, despite macropod densities being lower than other hosts such as cattle and horses in some locations. Feeding patterns on other hosts generally depended on availability and density of animals. All tabanid species fed on at least three of the host species tested and mixed meals were also commonly encountered, suggesting a level of opportunistic feeding in addition to a preference for macropods. Some of the blood meals detected were possibly from previous gonotrophic cycles. The results indicate that all tabanid species examined could potentially transmit surra and all the host types investigated could be affected, but macropods face the highest transmission risk.

Development of solid phase antigen for indirect ELISA for the detection of specific antibody responses to infection with Newcastle disease virus.

A simple and inexpensive method of antigen preparation by ultrafiltration was investigated using the V4 strain of Newcastle disease virus. The antigen designated XM300 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Newcastle disease virus in chicken serum. The assay was evaluated using both experimental and field sera, as well as reference control reactor and non-reactor sera. Antigen prepared by the ultrafiltration method was compared with antigen prepared by ultracentrifugation and the ultrafiltration antigen was found to react specifically with Newcastle disease virus antiserum in this ELISA system. This antigen preparation technique is also suitable for use in developing countries. The ELISA provides an excellent method for measuring antibodies in the early stages of infection in serum samples from experimentally infected chickens. More than 14.58 % of the total serum samples which failed to be recognized as reactors by the conventional haemagglutination inhibition test were detected in the ELISA.

Production and characterization of monoclonal antibodies to fowl adenoviruses.

A panel of 10 monoclonal antibodies to a hypervirulent fowl adenovirus (FAdV) strain 398A was produced. Monoclonal antibodies (mAbs) were characterized for their isotype, neutralizing activity, ability to capture viral antigens, immunoblotting of viral polypeptides, competitive inhibition with chicken antisera and their reaction pattern with other FAdVs in indirect immunoperoxidase. Eight out of 10 mAbs reacted strongly in indirect immunoperoxidase staining with most of the FAdVs isolated from inclusion body hepatitis in Australia. One of these mAbs (6E1) was found to specifically react with the strains presumably characterized as hypervirulent FAdVs. IgG(2a) was the predominant sub-isotype. Two out of 10 mAbs neutralized the homologous strain of virus and six captured their target antigen onto a plastic surface. Chicken anti-serum to FAdV strain 398A inhibited the reaction of the seven mAbs that bound with high affinity in a blocking competitive enzyme-linked immunosorbent assay. This panel of mAbs can be used to improve diagnostic assays, study pathogenesis and carry out strain identification.

An economic assessment of porcine parvovirus vaccination.

A decision analysis model was designed to evaluate the cost effectiveness of a vaccination program for preventing endemic or epidemic porcine parvovirus (PPV) induced reproductive failure in a 100-sow pig herd. The results showed that the cost of vaccination was less than the cost incurred by continuing endemic PPV infection, or the cost of a severe epidemic. A long term vaccination program is a cost effective method for controlling PPV-induced reproductive failure in pig herds suffering endemic and epidemic PPV infection.

Monoclonal antibodies to Kunjin and Kokobera viruses.

Five monoclonal antibodies (MoAb) were produced to the envelope (E) protein of Kunjin (KUN) virus. Three of these were specific for the KUN/West Nile complex, and two reacted with epitopes that were common to the flavivirus family. These MoAb defined at least four distinct epitopes on the E protein of KUN virus. Eight MoAb were also produced to unidentified proteins of Kokobera (KOK) virus. Seven were specific for KOK, while the remaining antibody cross-reacted with Stratford virus and an unregistered flavivirus CS946. The application of these MoAb to arboviral surveillance is discussed.

Epitope analysis of the envelope and non-structural glycoproteins of Murray Valley encephalitis virus.

Previous studies have shown that antibodies produced against strategic flavivirus epitopes play an important role in recovery and immunity. Definition of the conformation and location of these epitopes and the degree of their conservation among flaviviruses is important to understanding the humoral response to flavivirus infection. In this study we have examined epitopes recognized by 14 monoclonal antibodies (MAbs) produced to the envelope (E) and non-structural (NS1) proteins of Murray Valley encephalitis virus (MVE). These antibodies were analysed for specificity, neutralization, haemagglutination inhibition (HI) and competitive binding. We have identified six distinct epitopes on the E protein which are located in four non-overlapping domains. MAbs to epitopes in one domain neutralized virus, were specific for MVE and Japanese encephalitis virus, and reacted with epitopes resistant to reduction. Two other E domains, one specific to MVE and the other shared by all flaviviruses, also contained neutralization sites and were stabilized by disulphide bonds. The fourth domain on E was conserved among the flaviviruses, sensitive to SDS denaturation and did not induce neutralizing antibody. Studies with MVE NS1 MAbs revealed that they were mostly type-specific, unreactive with conserved epitopes, and unreactive in HI and neutralization tests. The six epitopes identified on NS1 did not overlap and represent antigenic domains either resistant or sensitive to reduction. Immunoblotting of viral proteins in MVE-infected C6/36 cells revealed two distinct forms of NS1 and high Mr proteins of 97K and 108K that represented disulphide-linked heterodimers of E and NS1.

Epitopes on 14S subunits, natural empty capsids and virions of bovine enteroviruses.

Eighteen monoclonal antibodies were produced against three Australian bovine enterovirus serogroups. Based on their binding patterns to 14S subunits, natural empty capsids and virions of 4 selected viral isolates (K2577, SL305, 66/27, J2129) in two types of quantitative ELISAs, the 18 antibodies were shown to belong to four groups. Due to the specific reaction of monoclonal antibodies, the four groups likely react with four epitopes. The first epitope was shown mainly on the surface of isolate K2577. The second and the third epitopes were common epitopes shared by all four isolates. These epitopes presented equally well on 14S subunits, natural empty capsids and virions. The fourth epitope was present mainly on the natural empty capsids and shared by most, but not all isolates. The four epitopes seem to be conformation dependent epitopes.

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.

A capture antibody ELISA for detection of antibodies against bovine enterovirus.

A capture antibody ELISA was developed for detecting antibodies against bovine enterovirus. Chicken IgG F(ab')2 fragments against strain J2129 captured virus effectively. The captured virus was used to detect specific antibodies against the virus in bovine sera. The specificity of the ELISA is high. The factors affecting the specificity are discussed. The assay is more sensitive and economic than traditional serum neutralisation.

Type-specific monoclonal antibodies produced to proteins of Murray Valley encephalitis virus.

Two monoclonal antibodies have been produced to the flavivirus, Murray Valley encephalitis (MVE). Immunofluorescent antibody and ELISA tests revealed that one antibody was specific for MVE while the other cross-reacted weakly with Alfuy and Kunjin viruses. These antibodies have neither haemagglutination inhibition nor neutralising activity. One of the monoclonal antibodies reacted with the envelope glycoprotein (E) and a 45,000 MW protein, while the other reacted with a protein of 23,000 daltons. Competitive binding assays confirmed that these antibodies reacted with unrelated epitopes.

Detection of immunological tolerance to bovine spumavirus (BSV) with evidence for salivary excretion and spread of BSV from the tolerant animal.

A group of 17 Friesian-Holstein steers held in individual pens was examined for evidence of infection with bovine spumavirus (BSV). Serum was examined for specific antibody by 2 serological procedures, and circulatory leucocytes and throat swabs were examined for the presence of circulatory leucocyte-associated BSV (CLAB) and saliva-associated BSV (SAB). Initial tests showed that 7 of the 17 steers had specific antibody to BSV by both serological procedures, and a further steer developed such antibody during the first 3 months of holding the animals in single contiguous pens. All 8 of these specific antibody-positive steers were CLAB positive and SAB negative. Nine steers showed no specific antibody to BSV by either of the 2 serological procedures; 8 of these 9 steers showed no evidence of CLAB or SAB. The exception was one steer which was CLAB- and SAB-positive at each of 30 samplings taken over a period of 9 months observation, whilst remaining specific-antibody free. This steer was classed as immunologically tolerant of BSV, and epidemiological data suggested that lateral spread of infection had originated from this animal through the agency of saliva.