Detection of Theileria orientalis genotypes in Haemaphysalis longicornis ticks from southern Australia

© 2015 Hammer et al.; licensee BioMed Central. Background: Theileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries. This paper provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite. Methods: To identify possible vectors for disease, ticks, biting flies and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife, and mosquitoes and biting flies were collected in traps in close proximity to outbreak herds. Ticks were identified via DNA barcoding of the mitochondrial cytochrome oxidase I gene. Barcoded ticks were pooled according to species or phylogenetic group and tested for the presence of T. orientalis and the genotypes Ikeda, Chitose and Buffeli using real-time PCR. Results: DNA barcoding and phylogenetic analysis identified ticks from the following species: Haemaphysalis longicornis, Ixodes holocyclus, Ixodes cornuatus, Ixodes hirsti, and Bothriocroton concolor. Additional Haemaphysalis, Ixodes and Bothriocroton spp. were also identified. Of the ticks investigated, only H. longicornis ticks from cattle carried theilerial DNA, with the genotypes Ikeda, Chitose and Buffeli represented. Mosquitoes collected in close proximity to outbreak herds included; Aedes camptorhynchus, Aedes notoscriptus, Coquillettidia linealis, Culex australicus, and Culex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquitoes. The haematophagous flies tested negative. Conclusions: This is the first demonstration of a potential vector for T. orientalis in the current Australasian disease outbreak

Baseline susceptibility and cross-resistance in aphis gossypii glover (Aphididae: Hemiptera) to phorate and sulfoxaflor

© 2013 State of New South Wales. Susceptible discriminating doses of phorate (0.2 g/L) and sulfoxaflor (0.01 g/L) against cotton aphid Aphis gossypii Glover were determined by laboratory bioassay where aphids were sprayed with insecticide with the aid of a Potter spray tower. All of the populations tested were susceptible to sulfoxaflor, and only a pirimicarb resistant strain had cross-resistance to phorate. If phorate is used as a side dressing in Australian cotton for insect control, neither pirimicarb, or any other chemical associated with insensitive acetylcholinesterase type one resistance, should be used as the first foliar spray for any subsequent aphid control

Quantification of the pirimicarb resistance allele frequency in pooled cotton aphid (Aphis gossypii glover) samples by TaqMan SNP genotyping assay

Background: Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance. Methodology/Principal Findings: We demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k′ at the inflexion point based on a four parameter sigmoid curve. Our results show that k′ is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k′ has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k′, allowing the combination of samples across multiple runs in a single analysis. Conclusions/Significance: The transformed fluorescence ratio (k′) method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally © 2014 Chen et al

A TaqMan qPCR method for detecting kdr resistance in Aphis gossypii demonstrates improved sensitivity compared to conventional PCR–RFLP

© 2015, Springer-Verlag Berlin Heidelberg. Cotton aphid, Aphis gossypii Glover, has emerged as a prominent pest in Australian cotton production, and monitoring pesticide resistance including pyrethroids in field populations is crucial for its sustainable management. We examined the distribution of kdr resistance in 35 field-collected A. gossypii populations and used TaqMan qPCR assays with pooled samples. The study demonstrated proof of concept that pooled insect qPCR methodology provided effective detection with better sensitivity than individual PCR–RFLP genotyping techniques for the kdr resistance allele. The practical outcome is that routine resistance monitoring can examine more sites while increasing the likelihood of detecting incipient resistance at those sites. More importantly, the method is adaptable to any genetically caused resistance and so not limited to A. gossypii or even insect control. It cannot be overstressed that the ability to detected resistance at very low frequencies is critical to all sustainable resistance management. Early detection of resistance provides critical time for the modification of chemical use prior to potential insecticide control failure

Draft Genome Sequence of a Novel "<i>Candidatus</i> Liberibacter" Species Detected in a <i>Zanthoxylum</i> Species from Bhutan.

The draft genome sequence of a novel "Candidatus Liberibacter" species detected in an unidentified species of Zanthoxylum (Rutaceae) collected in Bhutan is reported. The total length is 1,408,989 bp with 1,169 coding sequences in 96 contigs, a GC content of 37.3%, and 76 to 77% average nucleotide identity with several other "Ca Liberibacter" species

Whole-genome analysis of extraintestinal Escherichia coli sequence type 73 from a single hospital over a 2 year period identified different circulating clonal groups

© 2020 The Authors. Sequence type (ST)73 has emerged as one of the most frequently isolated extraintestinal pathogenic Escherichia coli. To examine the localized diversity of ST73 clonal groups, including their mobile genetic element profile, we sequenced the genomes of 16 multiple-drug resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia, between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccurred, over a 2 year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 clone D i2 reference genome shows high bootstrap support and clusters into four major groups that correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes, but the presence of iss, pic and several iron-acquisition operons was notable

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

© 2016 The Authors. Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endopro-teolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity

Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle

© 2015 Elsevier B.V. In Australia, outbreaks of clinical theileriosis caused by Theileria orientalis have been largely associated with the Ikeda genotype which can occur as a sole infection, or more commonly, as a mixture of genotypes. The most prevalent genotype, Chitose, frequently co-occurs with type Ikeda, however the role of this genotype in clinical disease has not been clearly established. Furthermore, the dynamics of individual genotypes in field infection of cattle have not been examined. In this study we developed quantitative PCR (qPCR) and genotyping methods to examine the role of the Chitose genotype in clinical disease and to investigate the temporal dynamics of T. orientalis Ikeda, Chitose and Buffeli genotypes in naïve animals introduced to a T. orientalis-endemic area. Analysis of the major piroplasm surface protein (MPSP) genes of Chitose isolates revealed the presence of two distinct phylogenetic clusters, Chitose A and Chitose B. A genotyping assay aimed at determining Chitose A/B allele frequency revealed that the Chitose A phylogenetic cluster is strongly associated with clinical disease but nearly always co-occurs with the Ikeda genotype. qPCR revealed that the Chitose genotype (particularly Chitose A), undergoes temporal switching in conjunction with the Ikeda genotype and contributes substantially to the overall parasite burden. The benign Buffeli genotype can also undergo temporal switching but levels of this genotype appear to remain low relative to the Ikeda and Chitose types. Interplay between vector and host immunological factors is presumed to be critical to the population dynamics observed in this study. Genotypic switching likely contributes to the persistence of T. orientalis in the host

Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes.

Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines