• Bertram Harmon a publié une note il y a 6 jours et 1 heure

    AcknowledgementsTick-borne haemoprotozoan parasites of the genus cause infections in a wide range of mammals including the Bovidae. In Australasia, is emerging as a significant pathogen of beef and dairy cattle, causing anaemia, jaundice, ill-thrift, mortalities and late term abortion ().Eleven genotypes of the organism have been identified from cattle and ticks based on the gene for the major piroplasm surface protein (MPSP) (); however clinical disease has been linked to the presence of a specific cyclooxygenase-2 inhibitors of the organism, Type 2 (Ikeda genotype). Furthermore, there is some evidence that Type 7, a phylogenetic relative of Ikeda, may be related to recent clinical outbreaks in India (). A phylogenetic subgroup of Type 1 (Chitose A) is strongly associated with clinical cases in Australia but nearly always co-occurs with the Ikeda genotype (); therefore the pathogenic potential of this genotype is unclear. Type 3 (Buffeli) and its phylogenetic sister group, Type 5 are considered benign () and the clinical relevance of the remaining genotypes has not been fully elucidated. infection often presents as a mixture of genotypes () which may contribute to the persistence of the organism by allowing the parasite to evade the host immune response ().Clinical diagnosis of theileriosis involves eliminating alternative causes of similar symptoms in combination with blood film and/or PCR-based methods for detection of the parasite. PCR-based methods are more sensitive than blood film examinations () and can provide genotype discrimination () although conventional and semi-quantitative PCR methods cannot accurately assess total parasite load, a parameter that has been shown to correlate with clinical disease (). However, a recently developed multiplex hydrolysis probe quantitative PCR (qPCR) assay allows for both accurate quantification of parasite load and discrimination of clinically-associated genotypes (). Despite advances in PCR-based methodologies for the diagnosis of bovine theileriosis, blood films are often relied upon due to the expense associated with molecular techniques. A large proportion of the cost associated with molecular methods relates to the extraction of DNA for downstream PCR testing. Here we describe a simple and efficient method for extracting DNA from bovine blood which substantially reduces the cost of multiplex qPCR testing.The bovine blood samples analysed in this study (=434) were derived from a total of 92 separate herds across five states of Australia and comprised samples PCR positive and negative for (). Blood samples were collected into either EDTA (=425) or lithium-heparin (=9) and either extracted without freezing (=70) or frozen and extracted using both methods at a later date (=364). DNA was extracted from 100μL of each blood sample using both a commercial DNA extraction kit (DNeasy Blood and Tissue Kit; Qiagen, USA) and an in-house method. The commercial DNA extraction method has been extensively used in prior PCR studies on () and was performed according to the manufacturer’s instructions with elution in a 100μL volume as described previously (). The commercial method was considered a gold standard in this study. The in-house method involved mild hypotonic erythrocyte lysis, centrifugation to remove contaminating PCR inhibitors such as haemoglobin, and a detergent-proteinase K treatment (DPK method). The DPK method was adapted from a technique originally designed for cattle genotyping from hair roots (), and was modified here for use on blood samples. For this method, 100μL of bovine blood sample was added to a 1.5mL tube containing 900μL TE buffer (10mM Tris, 1mM EDTA; pH 8.0) and mixed by vortexing. The tubes were centrifuged for 10s at 16000× in a Sorvall Biofuge microcentrifuge and the supernatant discarded. The pellets were resuspended by pipetting in 1mL TE buffer and centrifuged as above. This step was repeated one further time. Each pellet was then resuspended in 200μL of DPK digest reagent (50mM KCl, 10mM Tris, pH 8.3, 1.5mM MgCl, 100μg/mL proteinase K and 0.5% Tween 20) and incubated for 30min at 60°C followed by 10min at 100°C. All DNA extracts were tested using multiplex qPCR as previously described (), except that only the Chitose A probe, rather than a mix of Chitose A/B probes, was included with the Ikeda and Universal probes in the PCR reaction. All probes in the reaction were used at a concentration of 250nM. Because extracts from the DPK method are relatively crude, both 10-fold diluted and undiluted DPK extracts were tested to determine whether inhibitors from blood had an effect on qPCR detection. Gene copy data from the quantitative (Universal) component of the assay were doubled for the DPK extraction to account for the larger elution volume used with this method and so that all concentrations could be expressed as gene copies/μL blood.

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