Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity

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.

Australian bat lyssavirus infection in two horses

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain.

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses

Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses

SUMMARY Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Pesticide residues in food - acute dietary exposure

Consumer risk assessment is a crucial step in the regulatory approval of pesticide use on food crops. Recently, an additional hurdle has been added to the formal consumer risk assessment process with the introduction of short-term intake or exposure assessment and a comparable short-term toxicity reference, the acute reference dose. Exposure to residues during one meal or over one day is important for short-term or acute intake. Exposure in the short term can be substantially higher than average because the consumption of a food on a single occasion can be very large compared with typical long-term or mean consumption and the food may have a much larger residue than average. Furthermore, the residue level in a single unit of a fruit or vegetable may be higher by a factor (defined as the variability factor, which we have shown to be typically ×3 for the 97.5th percentile unit) than the average residue in the lot. Available marketplace data and supervised residue trial data are examined in an investigation of the variability of residues in units of fruit and vegetables. A method is described for estimating the 97.5th percentile value from sets of unit residue data. Variability appears to be generally independent of the pesticide, the crop, crop unit size and the residue level. The deposition of pesticide on the individual unit during application is probably the most significant factor. The diets used in the calculations ideally come from individual and household surveys with enough consumers of each specific food to determine large portion sizes. The diets should distinguish the different forms of a food consumed, eg canned, frozen or fresh, because the residue levels associated with the different forms may be quite different. Dietary intakes may be calculated by a deterministic method or a probabilistic method. In the deterministic method the intake is estimated with the assumptions of large portion consumption of a ‘high residue’ food (high residue in the sense that the pesticide was used at the highest recommended label rate, the crop was harvested at the smallest interval after treatment and the residue in the edible portion was the highest found in any of the supervised trials in line with these use conditions). The deterministic calculation also includes a variability factor for those foods consumed as units (eg apples, carrots) to allow for the elevated residue in some single units which may not be seen in composited samples. In the probabilistic method the distribution of dietary consumption and the distribution of possible residues are combined in repeated probabilistic calculations to yield a distribution of possible residue intakes. Additional information such as percentage commodity treated and combination of residues from multiple commodities may be incorporated into probabilistic calculations. The IUPAC Advisory Committee on Crop Protection Chemistry has made 11 recommendations relating to acute dietary exposure.

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.

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).

Hendra virus infection in a veterinarian

A veterinarian became infected with Hendra virus (HeV) after managing a terminally ill horse and performing a limited autopsy with inadequate precautions. Although she was initially only mildly ill, serological tests suggested latent HeV infection. Nevertheless, she remains well 2 years after her initial illness. Recently emerged zoonotic viruses, such as HeV, necessitate appropriate working procedures and personal protective equipment in veterinary practice.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Procedure: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results: Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Infection, colonisation and sporulation by Pseudocercospora macadamiae on macadamia fruit

The structures and manner with which Pseudocercospora macadamiae penetrates, colonises and proliferates from the pericarp of macadamia fruit was studied using scanning electron microscopy and fluorescence light microscopy. Germ tubes arising from conidia penetrated open stomata within 20 h of inoculation, without observation of specialised infection structures such as appressoria. Colonisation of the pericarp was intercellular, without observation of specialised intracellular infection structures such as haustoria, and was complete from the epidermis to the mesocarp. The fungus proliferated at the epidermis by the formation of conidiophores and conidia on substomatal and protuberant subepidermal stromata. These structures were not observed on the mesocarp surface. The onset of visual husk spot symptoms coincided with an increase in pathogen biomass on the pericarp surface. The progression of symptoms from tan-coloured spots to larger red-brown lesions coincided with the production of conidiophores from substomatal and protuberant subepidermal stromata. The darker the colour of the husk spot lesion, the more frequently protuberant subepidermal stromata were observed. These findings are discussed in the context of observation of other cercosporoid fungi.

Cucumber mosaic virus infection of kava (Piper methysticum) and implications for cultural control of kava dieback disease

Cucumber mosaic virus (CMV) was found by reverse transcription polymerase chain reaction (RT-PCR) to be not fully systemic in naturally infected kava (Piper methysticum) plants in Fiji. Twenty-six of 48 samples (54%) from various tissues of three recently infected plants were CMV-positive compared with 7/51 samples (14%) from three long-term infections (plants affected by dieback for more than 1 year). The virus was also found to have a limited ability to move into newly formed stems. CMV was detected in only 2/23 samples taken from re-growth stems arising from known CMV infected/dieback affected plants. Mechanical inoculation experiments conducted in Fiji indicate that the known kava intercrop plants banana (Musa spp.), pineapple (Ananas comosus), peanut (Arachis hypogaea) and the common weed Mikania micrantha are potential hosts for a dieback-causing strain of CMV It was not possible to transmit the virus mechanically to the common kava intercrop plants taro (Colocasia esculenta), Xanthosoma sp., sweet potato (Ipomoea batatas), yam (Dioscorea alata), papaya (Carica papaya) or the weed Momordica charantia. Implications of the results of this research on a possible integrated disease management strategy are discussed.

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus, caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10(7) copies mu g(-1) DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10(6)-10(8) copies of CyHV-2 mu g(-1) DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22-10 degrees C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (+/- SE) of 7.3 +/- 11 to 394 +/- 55 mu g(-1) DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.

Bats and Emerging Zoonoses: Henipaviruses and SARS.

Nearly 75% of all emerging infectious diseases (EIDs) that impact or threaten human health are zoonotic. The majority have spilled from wildlife reservoirs, either directly to humans or via domestic animals. The emergence of many can be attributed to predisposing factors such as global travel, trade, agricultural expansion, deforestation habitat fragmentation, and urbanization; such factors increase the interface and or the rate of contact between human, domestic animal, and wildlife populations, thereby creating increased opportunities for spillover events to occur. Infectious disease emergence can be regarded as primarily an ecological process. The epidemiological investigation of EIDs associated with wildlife requires a trans-disciplinary approach that includes an understanding of the ecology of the wildlife species, and an understanding of human behaviours that increase risk of exposure. Investigations of the emergence of Nipah virus in Malaysia in 1999 and severe acute respiratory syndrome (SARS) in China in 2003 provide useful case studies. The emergence of Nipah virus was associated with the increased size and density of commercial pig farms and their encroachment into forested areas. The movement of pigs for sale and slaughter in turn led to the rapid spread of infection to southern peninsular Malaysia, where the high-density, largely urban pig populations facilitated transmission to humans. Identifying the factors associated with the emergence of SARS in southern China requires an understanding of the ecology of infection both in the natural reservoir and in secondary market reservoir species. A necessary extension of understanding the ecology of the reservoir is an understanding of the trade, and of the social and cultural context of wildlife consumption. Emerging infectious diseases originating from wildlife populations will continue to threaten public health. Mitigating and managing the risk requires an appreciation of the connectedness between human, livestock and wildlife health, and of the factors and processes that disrupt the balance.