[show abstract][hide abstract] ABSTRACT: Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2-8 dpi. Viral ribonucleic acid was detected from 1-15 days post inoculation from the oral route and 1-24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection.
PLoS ONE 01/2014; 9(1):e83417. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Veterinarians have few tools to predict the rate of disease progression in FIV-infected cats. In contrast, in HIV infection plasma viral RNA load and acute phase protein concentrations are commonly used as predictors of disease progression. This study evaluated these predictors in cats naturally infected with FIV. In older cats (> 5 years), log10 FIV RNA load was higher in the terminal stages of disease compared to the asymptomatic stage. There was a significant association between log10 FIV RNA load and both log10 serum amyloid A concentration and age in unwell FIV-infected cats. This study suggests that viral RNA load and serum amyloid A warrant further investigation as predictors of disease status and prognosis in FIV-infected cats.
[show abstract][hide abstract] ABSTRACT: The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.
[show abstract][hide abstract] ABSTRACT: A cost-benefit analysis using deterministic and stochastic modelling was conducted to identify the net benefits for households that adopt (1) vaccination of individual birds against Newcastle disease (ND) or (2) improved management of chick rearing by providing coops for the protection of chicks from predation and chick starter feed inside a creep feeder to support chicks' nutrition in village chicken flocks in Myanmar. Partial budgeting was used to assess the additional costs and benefits associated with each of the two interventions tested relative to neither strategy. In the deterministic model, over the first 3 years after the introduction of the interventions, the cumulative sum of the net differences from neither strategy was 13,189Kyat for ND vaccination and 77,645Kyat for improved chick management (effective exchange rate in 2005: 1000Kyat=1$US). Both interventions were also profitable after discounting over a 10-year period; Net Present Values for ND vaccination and improved chick management were 30,791 and 167,825Kyat, respectively. The Benefit-Cost Ratio for ND vaccination was very high (28.8). This was lower for improved chick management, due to greater costs of the intervention, but still favourable at 4.7. Using both interventions concurrently yielded a Net Present Value of 470,543Kyat and a Benefit-Cost Ratio of 11.2 over the 10-year period in the deterministic model. Using the stochastic model, for the first 3 years following the introduction of the interventions, the mean cumulative sums of the net difference were similar to those values obtained from the deterministic model. Sensitivity analysis indicated that the cumulative net differences were strongly influenced by grower bird sale income, particularly under improved chick management. The effects of the strategies on odds of households selling and consuming birds after 7 months, and numbers of birds being sold or consumed after this period also influenced profitability. Cost variations for equipment used under improved chick management were not markedly associated with profitability. Net Present Values and Benefit-Cost Ratios discounted over a 10-year period were also similar to the deterministic model when mean values obtained through stochastic modelling were used. In summary, the study showed that ND vaccination and improved chick management can improve the viability and profitability of village chicken production in Myanmar.
Preventive Veterinary Medicine 02/2013; · 2.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine the pathobiology of Indonesian H5N1 highly pathogenic avian influenza, two viruses representing clades 2.1.1 and 2.1.3 were inoculated into broiler chickens and Pekin ducks via the eyes, nostrils and oropharynx. In chickens, both viruses produced fulminant disease; tissue tropism was broad but predominantly endothelial and viral loads in tissues were high. Except for one case of meningoencephalitis, the infection in ducks was sub-clinical, leading only to seroconversion. In these ducks, virus and viral antigen occurred in lower amounts, mainly in the respiratory tract (airsac and sinuses), prior to day 7 after inoculation. During clinical disease, chickens shed high virus titres orally and cloacally. Ducks intermittently shed low virus titres from the oral route for up to 8 days post-inoculation. We discuss the significance of the data for understanding the pathogenesis and pathobiology of Indonesian H5N1 in chickens and ducks.
[show abstract][hide abstract] ABSTRACT: Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia's closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace's Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace's Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.
PLoS ONE 01/2013; 8(4):e61316. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ducks are considered to play a major role in the spread of highly pathogenic avian influenza (HPAI) H5N1 in Viet Nam, but detailed information on their management is limited. We distinguished two different systems (1) stationary duck flocks that are not commonly driven to rice fields beyond village boundaries and that are confined overnight on farms and (2) moving duck flocks that are intentionally driven to rice fields beyond village boundaries, that are not returning to home farms for extended periods and that are housed overnight in temporary enclosures in rice paddies. A total of 115 stationary and 22 moving flock farmers were interviewed in 2007 in the Mekong Delta of Viet Nam. Moving duck flocks are larger than stationary flocks, which is indicative of their more commercial production. Moving flock farmers apparently are more aware of HPAI risks than stationary flock farmers, as their flocks are more likely fully vaccinated and have less contact with chickens during scavenging. On the other hand, the spread of HPAI virus between birds might be promoted by moving duck flocks as they repeatedly use transport vehicles and numerous rice paddies for scavenging and are often visited by hatchery owners in the field for purchasing duck eggs. In addition, long distances travelled by moving duck flocks might also result in widespread dissemination of HPAI virus. Further studies are necessary to describe HPAI prevalence and travel patterns of moving duck flocks and to explore the moving duck flock network in detail.
Tropical Animal Health and Production 10/2012; · 1.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Chicken red blood cells (RBCs) are commonly used in hemagglutination inhibition (HI) tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN) test was conducted with the experimental sera. RESULTS: In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86) when results were dichotomised (titre [greater than or equal to] 4 log2), and substantial agreement (weighted kappa = 0.80) for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02), with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK) = 0.88) and for log titres (weighted PABAK = 0.93), respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed. CONCLUSION: In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre [greater than or equal to] 4 log2) than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a recommended assay for avian influenza sero-surveillance in both wild and domestic birds.
BMC Veterinary Research 07/2012; 8(1):117. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: SUMMARYA prospective longitudinal study was conducted on 96 smallholder duck farms in Indonesia over a period of 14 months in 2007 and 2008 to monitor bird- and flock-level incidence rates of H5 highly pathogenic avian influenza (HPAI) infection in duck flocks, and to identify risk factors associated with these flocks becoming H5 seropositive. Flocks that scavenged around neighbouring houses within the village were at increased risk of developing H5 antibodies, as were flocks from which carcases of birds that died during the 2 months between visits were consumed by the family. Duck flock confinement overnight on the farm and sudden deaths of birds between visits were associated with lower risk of the flock developing H5 antibodies. Scavenging around neighbouring houses and non-confinement overnight are likely to be causal risk factors for infection. With this study we have provided insights into farm-level risk factors of HPAI virus introduction into duck flocks. Preventive messages based on these risk factors should be included in HPAI awareness programmes.
Epidemiology and Infection 06/2012; · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.
[show abstract][hide abstract] ABSTRACT: A longitudinal study to monitor prevalence and incidence of antibodies against Newcastle disease (ND) virus and prevalence of antibodies against Avian Influenza (AI) virus in scavenging village chickens was conducted in 20 villages within 4 districts of Timor-Lesté. A total of 3600 blood samples was collected from 1674 individual birds in 300 household chicken flocks during three sampling periods (December 2008-February 2009, March-May 2009, and June-August 2009). The mean interval between household visits was 101.6±1.9 days. None of the birds enrolled in the study was vaccinated against ND or AI. A haemagglutination inhibition (HI) test was used to determine antibody titres against ND virus and a competitive ELISA and HI tests were used to detect antibody against AI virus. The bird-level ND seroprevalence pooled across all samplings (adjusted for clustering by households) was 4.4% (95% CI 3.5-5.2). The bird-level ND seroprevalence in each of the three sampling periods (adjusted for clustering by household) was 3.0% (95% CI 2.0-4.0), 6.6% (95% CI 5.1-8.0) and 3.6 (95% CI 2.5-4.6), respectively. A total of 12.6% individual birds tested ND seropositive at least once over the total study period (95% CI 10.5-14.7). The flock-level ND seroprevalence (at least one bird tested had antibodies against ND virus) pooled across all samplings was 15.9% (95% CI 13.5-18.3). A total of 35.3% flocks had a minimum of one bird being ND seropositive at least once over the study period. The bird-level incidence rate for the period between the first and the second sampling and between the second and the third sampling was 5.6 (95% CI 4.1-7.5) and 0.5 (95% CI 0.5-3.8) per 10,000 bird-years-at-risk, respectively. A total of 1134 serum samples from the last sampling period between June and August 2009 was tested for antibodies against AI virus. Only 4 samples tested Influenza A positive, indicating a bird-level seroprevalence level for Influenza A of 0.4% (CI 0.0-0.7%). These Influenza A positive samples were further tested for HI antibodies against AI virus subtypes of H5N1, H5N3, H7N3 and H9N2, but all tested negative, suggesting that the influenza antibodies in those four birds resulted from exposure to low pathogenic AI viruses of different H subtypes. Our results indicate that village chickens in Timor-Lesté are exposed to ND virus; there was a higher risk of infection during the early months of 2009 than either immediately prior or subsequent to this. No evidence of infection of village chickens with H5, H7 or H9 AI viruses was detected in this study.
Preventive Veterinary Medicine 01/2012; 104(3-4):301-8. · 2.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serum acute phase protein concentrations are used as diagnostic, therapeutic and prognostic markers in human and, less frequently, in animal medicine. The aim of this study was to determine how the health status and signalment of the cat are associated with concentrations of acute phase proteins. Generally, medians of the positive acute phase proteins appeared to be higher in sick cats compared to healthy cats. In multivariable regression models, log-transformed serum amyloid A concentration was higher in older cats, in sick and in female cats, while log-transformed α1-acid glycoprotein and haptoglobin concentrations were higher in older cats and were associated with interactions of health status (sick/healthy) and gender (male/female). The data from healthy cats in this study contribute to the limited knowledge of normal reference ranges for this species. This study highlights the potential of acute phase proteins as diagnostic markers in sick cats, but also emphasises that the signalment of the cat needs to be taken into consideration.
Research in Veterinary Science 12/2011; 93(2):649-54. · 1.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: The objectives of the current study were to estimate the sensitivity and specificity of three real-time polymerase chain reaction (PCR) tests for diagnosis of feline immunodeficiency virus (FIV) infection in domestic cats, both individually and when interpreted in series with one of two serological tests, separately in populations of cats at low and high risk of being infected with FIV. One PCR test targeted the pol gene and two targeted the gag gene of FIV. For comparison, sensitivities and specificities of the individual serological tests (IDEXX SNAP(®) test and AGEN Simplify(®) test) were also estimated. The study populations consisted of domestic cats thought to be not vaccinated against FIV. Low-risk (males aged 4 years or less and females; n=128) and high-risk (males over 4 years; n=128) cats were selected from those where blood samples were submitted to a commercial clinical pathology service. Bayesian latent class models were used to obtain posterior probability distributions for sensitivity and specificity for each test, based on prior distributions obtained from three experts. Medians of the posterior sensitivity distributions for the PCR tests based on the pol gene and two regions of the gag gene tests ranged from 0.85 to 0.89, compared to 0.89-0.97 for the two serological tests. The medians of posterior specificity distributions for these PCR tests were 0.94-0.96, and 0.95-0.97 for the serological tests. In contrast, the PCR based on one region of the gag gene had lower median sensitivity. Sensitivities of combinations of these serological and PCR tests interpreted in series were low; medians of posterior sensitivity distributions ranged from 0.75 to 0.83. Relative to the low-risk population, median sensitivities in the high-risk population were lower for all tests other than the AGEN Simplify(®) test; specificities were similar in both populations. We conclude that the sensitivities of the two PCR tests based on the pol gene and two regions of the gag gene, respectively, in non-vaccinated cats are probably lower than the sensitivities of the two serological tests we assessed. We do not recommend screening cats whose FIV vaccination status is uncertain with one of these serological tests and then testing positives with one of these PCR tests because in non-vaccinates, the sensitivities of combinations of these serological and PCR tests interpreted in series are low. Assessment of the validity of these PCR assays in FIV-vaccinated cats is required.
Preventive Veterinary Medicine 11/2011; 104(1-2):136-48. · 2.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI) virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed.
All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1), clade 2.1.3 (80), and IDN/6/05-like viruses (3) that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA) and neuraminidase (NA) sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens.
The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to chickens over the study period suggests that ducks are more likely to survive infection and they may better suit the role of long-term maintenance host for H5N1. As some viruses were isolated from dead birds, there was no clear correlation between genetic variations and pathogenicity of these viruses.
[show abstract][hide abstract] ABSTRACT: Scavenging ducks are thought to play an important role in the maintenance and transmission of highly pathogenic avian influenza (HPAI) H5N1 virus among domesticated and wild bird populations in South East Asia, but detailed field epidemiological results describing the infection status of domestic ducks and in-contact chickens have not been published. We conducted a longitudinal study, monitoring ducks and in-contact chickens in 80 flocks in the Mekong Delta of Viet Nam with bi-monthly testing from May 2007 until May 2008. Because H5 vaccination campaigns are conducted at regular intervals in poultry flocks in Viet Nam, both unvaccinated sentinel and H5 vaccinates were monitored. On each farm, a total of 10 birds were selected: 7 ducks (4 unvaccinated and 3 vaccinated) and 3 chickens (2 unvaccinated and 1 vaccinated) that were in close contact with the ducks. Blood samples were tested for H5 antibodies using the hemagglutination inhibition test, with H5 antibody titers ≥2(4) considered to indicate past exposure to H5 field or vaccine virus. Titers of vaccinated birds were analyzed for samples collected more than 3 weeks post-vaccination. Pooled oropharyngeal and cloacal swabs were assessed for H5 viral RNA using real-time PCR. Bird- and flock-level prevalences were estimated accounting for sampling fractions and clustering under the multi-stage sampling design with birds being sampled within flocks within villages in four different provinces. In total, serum and swab samples from 5409 birds-samplings were analyzed. Bird-level seroprevalence was 17.5% (95% CI: 14.1, 20.9) amongst unvaccinated ducks and 10.7% (95% CI: 7.4, 14.4) amongst unvaccinated in-contact chickens. Flock-level seroprevalence (proportion of flock-visits with at least one unvaccinated bird test positive) was 42.6% (95% CI: 38.0, 47.2) for ducks and 19.0% (95% CI: 13.6, 24.4) for chickens. Only 54.3% (95% CI: 39.2, 69.3) of vaccinated ducks and 55.5% (95% CI: 46.8, 64.2) of vaccinated in-contact chickens had H5 antibodies at more than 3 weeks post-vaccination. At about 40% and 48% of flock-visits, less than 50% of sampled vaccinated ducks and chickens, respectively, had positive titers. The flock-level virus prevalence (proportion of flocks with at least one bird positive for H5 virus of the vaccinated and unvaccinated birds tested) was 0.7% (95% CI: 0.0, 2.1). No HPAI outbreaks or mortality suspected to be due to HPAI occurred in study flocks during the observation period. Our results indicate that a substantial proportion of ducks and in-contact chickens were exposed to H5 virus during the study period. In the face of this widespread exposure to H5 virus, and despite only moderate proportions of birds developing positive titers post-vaccination, flocks were not affected by HPAI outbreaks during our study period. The higher bird-level seroprevalence in ducks compared to in-contact chickens may be due to greater durations of antibody persistence in ducks or greater rates of H5 virus exposure. These findings indicate that ducks are potentially an important source of H5 virus for other bird species.
Preventive Veterinary Medicine 09/2011; 101(3-4):229-40. · 2.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study investigated the seroepidemiology of Hendra virus in a spectacled flying-fox (Pteropus conspicillatus) population in northern Australia, near the location of an equine and associated human Hendra virus infection in late 2004. The pattern of infection in the population was investigated using a serial cross-sectional serological study over a 25-month period, with blood sampled from 521 individuals over six sampling sessions. Antibody titres to the virus were determined by virus neutralisation test. In contrast to the expected episodic infection pattern, we observed that seroprevalence gradually increased over the two years suggesting infection was endemic in the population over the study period. Our results suggested age, pregnancy and lactation were significant risk factors for a detectable neutralizing antibody response. Antibody titres were significantly higher in females than males, with the highest titres occurring in pregnant animals. Temporal variation in antibody titres suggests that herd immunity to the virus may wax and wane on a seasonal basis. These findings support an endemic infection pattern of henipaviruses in bat populations suggesting their infection dynamics may differ significantly from the acute, self limiting episodic pattern observed with related viruses (e.g. measles virus, phocine distemper virus, rinderpest virus) hence requiring a much smaller critical host population size to sustain the virus. These findings help inform predictive modelling of henipavirus infection in bat populations, and indicate that the life cycle of the reservoir species should be taken into account when developing risk management strategies for henipaviruses.
PLoS ONE 01/2011; 6(12):e28816. · 3.73 Impact Factor