Improved Post Entry Quarantine Diagnostics

Develop a new diagnostic platform for Post Entry Plant Quarantine to support the detection of Emergency Plant Pests in the Australian Grains and Nursery Industries.

Banana leaf disease diagnostics

Provide a leaf disease diagnostic service for samples received from throughout all banana growing areas of Queensland. This extends to both commercial and non-commercial situations.

Developing molecular diagnostics for the detection of strawberry viruses

Developing molecular diagnostics for the detection of strawberry viruses.

Black Sigatoka disease: new technologies to strengthen eradication strategies in Australia

In 2001, an incursion of Mycosphaerella fijiensis, the causal agent of black Sigatoka, was detected in Australia's largest commercial banana growing region, the Tully Banana Production Area in North Queensland. An intensive surveillance and eradication campaign was undertaken which resulted in the reinstatement of the disease-free status for black Sigatoka in 2005. This was the first time black Sigatoka had ever been eradicated from commercial plantations. The success of the eradication campaign was testament to good working relationships between scientists, growers, crop monitors, quarantine regulatory bodies and industry. A key contributing factor to the success was the deployment of a PCR-based molecular diagnostic assay, developed by the Cooperative Research Centre for Tropical Plant Protection (CRCTPP). This assay complemented morphological identification and allowed high throughput diagnosis of samples facilitating rapid decision-making during the eradication campaign. This paper describes the development and successful deployment of molecular diagnostics for black Sigatoka. Shortcomings in the gel-based assay are discussed and the advantages of highly specific real-time PCR assays, capable of differentiating between Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae are outlined. Real-time assays may provide a powerful diagnostic tool for applications in surveillance, disease forecasting and resistance testing for Sigatoka leaf spot diseases.

The physiological and metabolic impacts on sheep and cattle of feed and water deprivation before and during transport

Sheep and cattle are frequently subjected to feed and water deprivation (FWD) for about 12 h before, and then during, transport to reduce digesta load in the gastrointestinal tract. This FWD is marked by weight loss as urine and faeces mainly in the first 24 h but continuing at a reduced rate subsequently. The weight of rumen contents falls although water loss is to some extent masked by saliva inflow. FWD is associated with some stress, particularly when transportation is added. This is indicated by increased levels of plasma cortisol that may be partly responsible for an observed increase in the output of water and N in urine and faeces. Loss of body water induces dehydration that may induce feelings of thirst by effects on the hypothalamus structures through the renin-angiotensin-aldosterone system. There are suggestions that elevated cortisol levels depress angiotensin activity and prevent sensations of thirst in dehydrated animals, but further research in this area is needed. Dehydration coupled with the discharge of Na in urine challenges the maintenance of homeostasis. In FWD, Na excretion in urine is reduced and, with the reduction in digesta load, Na is gradually returned from the digestive tract to the extracellular fluid space. Control of enteropathogenic bacteria by normal rumen microbes is weakened by FWD and resulting infections may threaten animal health and meat safety. Recovery time is required after transport to restore full feed intake and to ensure that adequate glycogen is present in muscle pre-slaughter to maintain meat quality.

Differences between the in vitro digestibility of extrusa collected from oesophageal fistulated steers and the forage consumed.

In a study that included C-4 tropical grasses, C-3 temperate grasses and C-3 pasture legumes, in vitro dry matter digestibility of extrusa, measured as in vitro dry matter loss (IVDML) during incubation, compared with that of the forage consumed, was greater for grass extrusa but not for legume extrusa. The increase in digestibility was not caused by mastication or by the freezing of extrusa samples during storage but by the action of saliva. Comparable increases in IVDML were achieved merely by mixing bovine saliva with ground forage samples. Differences were greater than could be explained by increases due to completely digestible salivary DM. There was no significant difference between animals in relation to the saliva effect on IVDML and, except for some minor differences, similar saliva effects on IVDML were measured using either the pepsin-cellulase or rumen fluid-pepsin in vitro techniques. For both C-4 and C-3 grasses the magnitude of the differences were inversely related to IVDML of the feed and there was little or no difference between extrusa and feed at high digestibilities (>70%) whereas differences of more than 10 percentage units were measured on low quality grass forages. The data did not suggest that the extrusa or saliva effect on digestibility was different for C-3 grasses than for C-4 grasses but data on C-3 grasses were limited to few species and to high digestibility samples. For legume forages there was no saliva effect when the pepsin-cellulase method was used but there was a small but significant positive effect using the rumen fluid-pepsin method. It was concluded that when samples of extrusa are analysed using in vitro techniques, predicted in vivo digestibility of the feed consumed will often be overestimated, especially for low quality grass diets. The implications of overestimating in vivo digestibility and suggestions for overcoming such errors are discussed.

A review of Australian classical biological control of weeds programs and research activities over the past 12 years

Considerable progress has been made towards the successful classical biological control of many of Australia’s exotic weeds over the past decade. Some 43 new arthropod or pathogen agents were released in 19 projects. Effective biological control was achieved in several projects with the outstanding successes being the control of rubber vine, Cryptostegia grandiflora, and bridal creeper, Asparagus asparagoides. Significant developments also occurred in target prioritization, procedures for target and agent approval, funding, infrastructure and cooperation between agencies. Scientific developments included greater emphasis on climate matching, plant and agent phylogeny, molecular diagnostics, agent prioritization and agent evaluation.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Pests and Diseases in Field Crops

Insect pest diagnostics.

CRC20093 Increasing Diagnostic Capacity in Thailand

Accurate identification of pests is essential for practically all aspects of agricultural development and is critical to the operations of biosecurity that safeguard agricultural integrity and facilitate trade. Diagnostic capability is at the forefront of and complementary to, activities such as border protection, incursion management, surveillance and pest and disease certification. The efficiency of a biosecurity system therefore depends largely on the feedback between these activities and diagnostics. Australian scientists will train Thai scientists in diagnostics and surveillance to provide the Thai DOA with skills that will aid in the development of a Thai Diagnostic Network. The skills will be taught using a range of pests, including some which have particular biosecurity importance for both Australia and Thailand such as citrus canker, potato viruses and fruit flies.

The diagnosis and management of a case of leishmaniosis in a dog imported to Australia

This case study discusses in detail for the first time the diagnosis and management of a case of leishmaniosis in a dog imported to Australia. The dog presented with epistaxis and a non-regenerative anaemia five years after being imported from Europe. Protozoa were identified within macrophages in bone marrow and splenic cytology. A Leishmania indirect fluorescent antibody test was performed and was positive while an Ehrlichia canis antibody test was negative. Polymerase chain reaction of the ITS-1 and ITS-2 regions of skin, lymph node, spleen and bone marrow were all positive for Leishmania infantum. The dog was treated with amphotericin B with a strong clinical response. The importance of thorough diagnostics in non-endemic areas, particularly Australia, is discussed. Treatment with amphotericin B is discussed. Vigilance, disease reporting and response frameworks are recommended for non-endemic areas. © 2014 Elsevier B.V.

Nipah Virus in the Fruit Bat Pteropus vampyrus in Sumatera, Indonesia

Nipah virus causes periodic livestock and human disease with high case fatality rate, and consequent major economic, social and psychological impacts. Fruit bats of the genus Pteropus are the natural reservoir. In this study, we used real time PCR to screen the saliva and urine of P. vampyrus from North Sumatera for Nipah virus genome. A conventional reverse transcriptase (RT-PCR) assay was used on provisionally positive samples to corroborate findings. This is the first report of Nipah virus detection in P. vampyrus in Sumatera, Indonesia.

Status, challenges and tools for identification and diagnosis of Peronosclerospora and Sclerophthora of regulatory concern for graminicolous crops

Graminicolous Downy Mildew (GDM) diseases caused by the genera Peronosclerospora (13 spp.) and Sclerophthora (6 spp. and 1 variety) are poorly studied but destructive diseases of major crops such as corn, sorghum, sugarcane and other graminoids. Eight of the 13 described Peronosclerospora spp. are able to infect corn. In particular, P. philippinensis (= P. sacchari), P. maydis, P. heteropogonis, and S. rayssiae var. zeae cause major losses in corn yields in tropical Asia. In 2012 a new species, P. australiensis, was described based on isolates previously identified as P. maydis in Australia; this species is now a pathogen of major concern. Despite the strong impact of GDM diseases, there are presently no reliable molecular methods available for their detection. GDM pathogens are among the most difficult Oomycetes to identify using molecular tools, as their taxonomy is very challenging, and little genetic sequence data are available for development of molecular tools to detect GDM pathogens to species level. For example, from over 15 genes used in identification, diagnostics or phylogeny of Phytophthora, only ITS1 and cox2 show promise for use with GDM pathogens. Multiplex/multigene conventional and qPCR assays are currently under evaluation for the detection of economically important GDM spp. Scientists from the USA, Germany, Canada, Australia, and the Philippines are collaborating on the development and testing of diagnostic tools for these pathogens of concern.

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.