Screening and breeding for genetic resistance to anthracnose in mango

Postharvest diseases remain a significant constraint to the transport, storage and marketing of mangoes. The two main ones are anthracnose and stem end rot. Anthracnose caused by Colletotrichum gloeosporioides is the more wide-spread of the two. Varieties within Mangifera indica are known to vary in their level of reactions to anthracnose; however, the best tolerance in current commercial cultivars is not sufficient to eliminate the need for pre- and postharvest fungicides treatments. A screening program was initiated in mango accessions in the Australian National Mango Genebank to look for any significant resistance to C. gloeosporioides in fruit as they ripened. Screening was conducted by rating reactions to natural infection of anthracnose and reactions to artificially inoculating fruit with virulent isolates of C. gloeosporioides. A range of reactions to the pathogen were identified, with strong resistance found in one accession of the species M. laurina. This accession was used as the pollen parent in a controlled crossing program with a M. indica hybrid from the Australian Mango Breeding Program (AMBP). Sixty successful hybrids between the species have been generated. The hybrid population will be screened for resistance to anthracnose and used for gene discovery investigations to identify markers for anthracnose resistance.

Australian Bat Lyssavirus: Observations of Natural and Experimental Infection in Bats

This conference abstract gives data and conclusions arising from targeted surveillance of wild bats for naturally occuring Australian bat lyssavirus (ABLV) infection and other central nervous system diseases. It also provides data and conclusions arising from experimental infection of 10 Greyheaded flying foxes (Pteropus poliocephalus).

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety-three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Ruppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S.agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S.agalactiae; genotyping of selected S.agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S.agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and Pconspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Timing of infection of macadamia fruit by Pseudocercospora macadamiae and climatic effects on growth and spore germination.

Pseudocercospora macadamiae causes husk spot of macadamia. Husk spot control would be improved by verifying the stages in fruit development susceptible to infection, and determine some of the climatic conditions likely to lead to high disease pressure periods in the field. Our results showed that the percent conidia germination and growth of germ tubes and mycelia of P. macadamiae were greatest at 26 degrees C, with better conidia germination associated with high relative humidity and free water. The exposure of match-head-sized and pea-sized fruit stages to natural P. macadamiae inoculum in the field led to 2 5-fold increases in husk spot incidence, and up to 8.5-fold increases in premature abscission, compared with unexposed fruit. Exposure of fruit stages later than match-head-sized and pea-sized fruit generally caused no further increases in disease incidence or premature abscission. Climatic conditions were found to have a strong influence on the behaviour of P. macadamiae, the host, oil accumulation, and the subsequent impact of husk spot on premature abscission. Our findings suggest that fungicide application should target fruit at the match-head-sized stage of development in order to best reduce yield losses, particularly in seasons where oil accumulation in fruit is prolonged and climatic conditions are optimal for P. macadamiae.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Flying-Fox Species Density-A Spatial Risk Factor for Hendra Virus Infection in Horses in Eastern Australia

Hendra virus causes sporadic but typically fatal infection in horses and humans in eastern Australia. Fruit-bats of the genus Pteropus (commonly known as flying-foxes) are the natural host of the virus, and the putative source of infection in horses; infected horses are the source of human infection. Effective treatment is lacking in both horses and humans, and notwithstanding the recent availability of a vaccine for horses, exposure risk mitigation remains an important infection control strategy. This study sought to inform risk mitigation by identifying spatial and environmental risk factors for equine infection using multiple analytical approaches to investigate the relationship between plausible variables and reported Hendra virus infection in horses. Spatial autocorrelation (Global Moran’s I) showed significant clustering of equine cases at a distance of 40 km, a distance consistent with the foraging ‘footprint’ of a flying-fox roost, suggesting the latter as a biologically plausible basis for the clustering. Getis-Ord Gi* analysis identified multiple equine infection hot spots along the eastern Australia coast from far north Queensland to central New South Wales, with the largest extending for nearly 300 km from southern Queensland to northern New South Wales. Geographically weighted regression (GWR) showed the density of P. alecto and P. conspicillatus to have the strongest positive correlation with equine case locations, suggesting these species are more likely a source of infection of Hendra virus for horses than P. poliocephalus or P. scapulatus. The density of horses, climate variables and vegetation variables were not found to be a significant risk factors, but the residuals from the GWR suggest that additional unidentified risk factors exist at the property level. Further investigations and comparisons between case and control properties are needed to identify these local risk factors.

Natural host range, thrips and seed transmission of distinct Tobacco streak virus strains in Queensland, Australia

Diseases caused by Tobacco streak virus (TSV) have resulted in significant crop losses in sunflower and mung bean crops in Australia. Two genetically distinct strains from central Queensland, TSV-parthenium and TSV-crownbeard, have been previously described. They share only 81% total-genome nucleotide sequence identity and have distinct major alternative hosts, Parthenium hysterophorus (parthenium) and Verbesina encelioides (crownbeard). We developed and used strain-specific multiplex Polymerase chain reactions (PCRs) for the three RNA segments of TSV-parthenium and TSV-crownbeard to accurately characterise the strains naturally infecting 41 hosts species. Hosts included species from 11 plant families, including 12 species endemic to Australia. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was both a natural host of, and experimentally infected by TSV-parthenium but this infection combination resulted in non-viable seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. TSV-crownbeard was seed transmitted from naturally infected crownbeard at a rate of between 5% and 50% and was closely associated with the geographical distribution of crownbeard in central Queensland. TSV-parthenium and TSV-crownbeard were also seed transmitted in experimentally infected ageratum (Ageratum houstonianum) at rates of up to 40% and 27%, respectively. The related subgroup 1 ilarvirus, Ageratum latent virus, was also seed transmitted at a rate of 18% in ageratum which is its major alternative host. Thrips species Frankliniella schultzei and Microcephalothrips abdominalis were commonly found in flowers of TSV-affected crops and nearby weed hosts. Both species readily transmitted TSV-parthenium and TSV-crownbeard. The results are discussed in terms of how two genetically and biologically distinct TSV strains have similar life cycle strategies in the same environment.

Impact of the invasive rust Puccinia psidii (myrtle rust) on native Myrtaceae in natural ecosystems in Australia

The invasive rust Puccinia psidii (myrtle rust) was detected in Australia in 2010 and is now established along the east coast from southern New South Wales to far north Queensland. Prior to reaching Australia, severe damage from P. psidii was mainly restricted to exotic eucalypt plantations in South America, guava plantations in Brazil, allspice plantations in Jamaica, and exotic Myrtaceous tree species in the USA; the only previous record of widespread damage in native environments is of endangered Eugenia koolauensis in Hawai’i. Using two rainforest tree species as indicators of the impact of P. psidii, we report for the first time severe damage to endemic Myrtaceae in native forests in Australia, after only 4 years’ exposure to P. psidii. A 3-year disease exclusion trial in a natural stand of Rhodamnia rubescens unequivocally showed that repeated, severe infection leads to gradual crown loss and ultimately tree mortality; trees were killed in less than 4 years. Significant (p < 0.001) correlations were found between both incidence (r = 0.36) and severity (r = 0.38) of P. psidii and subsequent crown loss (crown transparency). This provided supporting evidence to conclude a causal association between P. psidii and crown loss and tree mortality in our field assessments of R. rubescens and Rhodomyrtus psidioides across their native range. Assessments revealed high levels of damage by P. psidii to immature leaves, shoots and tree crowns—averaging 76 % (R. rubescens) and 95 % (R. psidioides) crown transparency—as well as tree mortality. For R. psidioides, we saw exceptionally high levels of tree mortality, with over half the trees surveyed dead and 40 % of stands with greater than 50 % tree mortality, including two stands where all trees were dead. Tree mortality was less prevalent for R. rubescens, with only 12 % of trees surveyed dead and two sites with greater than 50 % mortality. Any alternative causal agents for this tree mortality have been discounted. The ecological implications of this are unclear, but our work clearly illustrates the potential for P. psidii to negatively affect Australia’s biodiversity.

Spatiotemporal aspects of Hendra Virus infection in Pteropid Bats (Flying-Foxes) in Eastern Australia

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.

Coronavirus Infection and Diversity in Bats in the Australasian Region

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

Australian Bat Lyssavirus Infection in a Captive Juvenile Black Flying Fox

The newly emerging Australian bat lyssavirus causes rabies like disease in bats and humans. A captive juvenile black flying fox exhibited progressive neurologic signs, including sudden aggression, vocalization, dysphagia, and paresis over 9 days and then died. At necropsy, lyssavirus infection was diagnosed by fluorescent antibody test, immunoperoxidase staining, polymerase chain reaction, and virus isolation. Eight human contacts received postexposure vaccination.

Impact of mirid (Creontiades spp.) (Hemiptera: Miridae) pest management on Helicoverpa spp. (Lepidoptera: Noctuidae) outbreaks: The case for conserving natural enemies

Creontiades spp. (Hemiptera: Miridae) are sucking pests that attack buds, flowers and young pods in mungbeans, Vigna radiata (L.), causing these structures subsequently to abort. If left uncontrolled, mirids can cause 25-50% yield loss. Traditional industry practice has involved prophylactic applications of dimethoate to control mirids at budding and again a week later. The present trial was initiated to highlight the dangers of such a practice, in particular the risk of a subsequent Helicoverpa spp. lepidopteran pest outbreak. A single application of dimethoate halved the population of important natural enemies of Helicoverpa spp., and caused an above-threshold outbreak of Helicoverpa spp. within 11 days. This shows that even a moderate (e.g. 50%) reduction in natural enemies may be sufficient to increase Helicoverpa spp. populations in mungbeans. As a result, prophylactic sprays should not be used for the control of mirids in mungbeans, and dimethoate should be applied only when mirids are above the economic threshold. Indoxacarb was also tested to establish its effect on Helicoverpa spp., mirids and natural enemies. Indoxacarb showed potential for Helicoverpa spp. control and suppression of mirids and had little impact on natural enemies.

Henipaviruses: Emerging Paramyxoviruses Associated with Fruit Bats

Two related, novel, zoonotic paramyxoviruses have been described recently. Hendra virus was first reported in horses and thence humans in Australia in 1994; Nipah virus was first reported in pigs and thence humans in Malaysia in 1998. Human cases of Nipah virus infection, apparently unassociated with infection in livestock, have been reported in Bangladesh since 2001. Species of fruit bats (genus Pteropus ) have been identified as natural hosts of both agents. Anthropogenic changes (habitat loss, hunting) that have impacted the population dynamics of Pteropus species across much of their range are hypothesised to have facilitated emergence. Current strategies for the management of henipaviruses are directed at minimising contact with the natural hosts, monitoring identified intermediate hosts, improving biosecurity on farms, and better disease recognition and diagnosis. Investigation of the emergence and ecology of henipaviruses warrants a broad, cross-disciplinary ecosystem health approach that recognises the critical linkages between human activity, ecological change, and livestock and human health.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.