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.

Issues and advances in the integrated control of sheep lice.

Ongoing pressure to minimise costs of production, growing markets for low residue and organic wool and meat, resistance to chemicals in louse populations, and the deregistration of diazinon for dipping and jetting have contributed to a move away from routine annual application of lousicides to more integrated approaches to controlling lice. Advances including improved methods for monitoring and detection of lice, an expanded range of louse control products and the availability of a web-accessible suite of decision support tools for wool growers (LiceBossTM) will aid this transition. Possibilities for the future include an on-farm detection test and non-chemical control methods. The design and extension of well-constructed resistance management programs to preserve the effectiveness of recently available new product groups should be a priority.

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix.

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and E.necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P < 0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P < 0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

In vitro infection of sheep lice (Bovicola ovis Schrank) by Steinernematid and Heterorhabditid nematodes.

Control of sheep lice with conventional pesticides can be compromised by difficulty in contacting lice in the dense water repellent fleeces of sheep, particularly when sheep have not been recently shorn. Entomopathogenic nematodes (ENs) are motile and are able to actively seek out insect hosts. They have particular advantages for the control of pests in cryptic habitats, such as the fleeces of sheep and avoid many of the problems frequently associated with chemical controls. This study investigated whether ENs were able infect and kill Bovicola ovis and compared the effectiveness of different species at different temperatures and when applied to wool. Four species of nematodes, Steinernema carpocapsae, Steinernema riobrave, Steinernema feltiae and Heterorhabditis bacteriophora were tested. All were shown to infect and kill lice in Petri dish assays at 30C. At 35C, the percent infection for S. carpocapsae and S. riobrave was significantly higher than for the other two species and percent infection by S. feltiae was significantly greater than for H. bacteriophora (P<0.05). At 37C the percent mortality induced by S. riobrave was significantly greater than for S. carpocapsae (P<0.05). All species were able to locate and infect lice in wool when formulated in water with 8% Tween 80. In wool assays the percent lice infected with nematodes was significantly greater for S. riobrave than H.bacteriophora at 25C, but there were no other differences between species (P=0.05). S. carpocapsae, S. riobrave and S. feltiae caused significantly higher lice mortality than H. bacteriophora at both 25 and 35C in wool assays, but mortality induced by the three steinernematid species did not differ significantly (P>0.05). It is concluded that of the ENs studied S. riobrave is likely to be most effective against B. ovis when applied to live sheep because of its greater tolerance to high temperatures and 'cruiser' foraging strategy .

Controlling fly strike and louse infections in sheep with tea tree oil

This study aimed to assess the effect of tea tree oil based formulations against two major ectoparasitic diseases in the sheep industry, flystrike and louse infestation, and to provide data to assist the assessment of the commercial feasibility of development of tea tree oil based ectoparasiticides. The results demonstrate insecticidal effects against both sheep lice and blowflies and repellent effects against adult flies and maggots. Dipping sheep in a Tea Tree Oil based formulation appeared to completely eradicate lice and suggests its potential use in sheep dipping formulations. Repellent and insecticidal effects against sheep blowflies, together with previously reported anti-microbial and wound healing properties, suggest significant benefits from the inclusion of tea tree oil in flystrike and wound treatment formulations. These effects occurred at concentrations of Tea Tree Oil that suggest the commercial viability of development of Tea Tree Oil based formulations for sheep parasite control and wound treatment and a potential new market for Tea Tree Oil.

Haemophilus parasuis treatment

The aim of the project is to prove that live vaccination on the farm will protect the piglets from heterologous challenge with H. parasuis. The steps to achieve the aim of the project are to find a dose rate on the farm which guarantees colonisation of the vaccine strain and is safe On farm vaccinated and unvaccinated pigs are then shifted to CAAS at three weeks of age and challenged with a heterologous strains. The method is then applied on a large piggery for a period of nearly 12 months. We will also develop a freeze drying method and a technical manual of procedures to identify serovars prevailing on pig farms and which serovar to include into the vaccine.

Fungal Biopesticide for cattle tick and buffalo fly control

Cattle ticks and buffalo flies impose significant economic burdens on the Northern Australian cattle and dairy industries. With the increased temperatures expected under climate change the range of parasites such as these is likely to extend. Current control options for these ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides offer a sustainable and promising alternative method of control. Laboratory and animal studies have established the potential for the fungus Metarhizium in tick control and provided data that suggests a secondary effect of buffalo fly control is possible. Small field trials are required to obtain a proof of concept for the control of ticks and buffalo flies on animals.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Comparison of bioassay responses to the potential fungal biopesticide Metarhizium anisopliae in Rhipicephalus(Boophilus) microplus and Lucilia cuprina.

Quantal response bioassays were conducted with cattle ticks and sheep blowflies with three different isolates of Metarhizium anisopliae and different methods of inoculation. Ticks were either topically dosed with 2 mu l or immersed in the conidial preparations. Blowflies were either topically dosed with 2 mu l of the conidial preparation or fed on conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95% of ticks after 2 days and 88 (+/- 2)% of blowflies after 4 days. Strikingly different mortality patterns indicated that virulence is dependent on different mechanisms in ticks and blowflies. The pattern of mortality seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose.

Bovine cysticercosis-Development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection

Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 +/- 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Bioactivity of tea tree oil from Melaleuca alternifolia against sheep lice (Bovicola ovis Schrank) in vitro

Tea tree oil (TTO) from the Australian native plant Melaleuca alternifolia has wide ranging bio-active properties, including insecticidal and repellent activity against arthropods. Furthermore, composition of commercially available Australian TTO is specified under an International Organization for Standardization standard (ISO 4730), reducing the potential for variable effects often noted with botanical pesticides. The effect of TTO, meeting the ISO standard for terpinen-4-ol chemotype, was tested against sheep lice (Bovicola ovis Schrank) in a series of laboratory studies. Immersion of wool for 60s in formulations containing concentrations of 1% TTO and above caused 100% mortality of adult lice and eggs. Exposure to vapours from TTO, delivered as droplets in fumigation chambers and when applied to wool also caused high mortality in both lice and eggs. The main active component of TTO in the fumigant tests was terpinen-4-ol. Treated surface assays and tests with wool where the formulation was allowed to dry before exposure of lice indicated low persistence. These studies demonstrate that TTO is highly toxic to sheep lice and active at concentrations that suggest potential for the development of TTO-based ovine lousicides. (C) 2012 Elsevier B.V. All rights reserved.

Dipping and jetting with tea tree (Melaleuca alternifolia) oil formulations control lice (Bovicola ovis) on sheep

The in vivo pediculicidal effectiveness of 1% and 2% formulations of tea tree (Melaleuca alternifolia) oil (TTO) against sheep chewing lice (Bovicola ovis) was tested in two pen studies. Immersion dipping of sheep shorn two weeks before treatment in both 1% and 2% formulations reduced lice to non detectable levels. No lice were found on any of the treated sheep despite careful inspection of at least 40 fleece partings per animal at 2, 6, 12 and 20 weeks after treatment. In the untreated sheep louse numbers increased from a mean (+/- SE) of 2.4 (+/- 0.7) per 10 cm fleece part at 2 weeks to 12.3 (+/- 4.2) per part at 20 weeks. Treatment of sheep with 6 months wool by jetting (high pressure spraying into the fleece) reduced louse numbers by 94% in comparison to controls at two weeks after treatment with both 1% and 2% TTO formulations. At 6 and 12 weeks after treatment reductions were 94% and 91% respectively with the 1% formulation and 78% and 84% respectively with the 2% formulation. TTO treatment also appeared to reduce wool damage in infested sheep. Laboratory studies indicated that tea tree oil 'stripped' from solution with a progressive reduction in concentration as well as volume as more wool was dipped, indicating that reinforcement of active ingredient would be required to maintain effectiveness when large numbers of sheep are treated. The results of these studies suggest significant potential for the development of ovine lousicides incorporating TTO. (C) 2012 Elsevier B.V. All rights reserved.

Comparative analysis of Tritrichomonas foetus (Riedmuller, 1928) cat genotype, T. foetus (Riedmuller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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.

CattleTickBase: An integrated Internet-based bioinformatics resource for Rhipicephalus (Boophilus) microplus

The Rhipicephalus micro plus genome is large and complex in structure, making it difficult to assemble a genome sequence and costly to resource the required bioinformatics. In light of this, a consortium of international collaborators was formed to pool resources to begin sequencing this genome. We have acquired and assembled genomic DNA into contigs that represent over 1.8 Gigabase pairs of DNA from gene-enriched regions of the R. micro plus genome. We also have several datasets containing transcript sequences from a number of gene expression experiments conducted by the consortium. A web-based resource was developed to enable the scientific community to access our datasets and conduct analysis through a web-based bioinformatics environment called YABI. The collective bioinformatics resource is termed CattleTickBase. Our consortium has acquired genomic and transcriptomic sequence data at approximately 0.9X coverage of the gene-coding regions of the R. microplus genome. The YABI tool will facilitate access and manipulation of cattle tick genome sequence data as the genome sequencing of R. microplus proceeds. During this process the CattleTickBase resource will continue to be updated. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.