Theileria parasites cause a benign infection of cattle in parts of Australia where they are endemic, but have, in recent years, been suspected of being responsible for a number of outbreaks of disease in cattle near the coast of New South Wales. The objective of this study was to identify and characterize the species of Theileria in cattle on six farms in New South Wales where disease outbreaks have occurred, and compare with Theileria from three disease-free farms in Queensland that is endemic for Theileria. Special reference was made to sub-typing of T. orientalis by type-specific PCR and sequencing of the small subunit (SSU) rRNA gene, and sequence analysis of the gene encoding a polymorphic merozoite/piroplasm surface protein (MPSP) that may be under immune selection. Nucleotide sequencing of SSU rRNA and MPSP genes revealed the presence of four Theileria genotypes: T. orientalis (buffeli), T. orientalis (ikeda), T. orientalis (chitose) and T. orientalis type 4 (MPSP) or type C (SSU rRNA). The majority of animals showed mixed infections while a few showed single infection. When MPSP nucleotide sequences were translated into amino acids, base transition did not change amino acid composition of the protein product, suggesting possible silent polymorphism. The occurrence of ikeda and type 4 (type C) previously not reported to occur and silent mutation is thought to have enhanced parasite evasion of the host immune response causing the outbreak.

Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic. The aim of this study was to identify diverse tick microbiomes, which may contain as-yet unidentified pathogens, using a metagenomic approach. DNA prepared from bacteria/archaea-enriched fractions obtained from seven tick species, namely Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus and Ixodes ricinus, was subjected to pyrosequencing after whole-genome amplification. The resulting sequence reads were phylotyped using a Batch Learning Self-Organizing Map (BLSOM) program, which allowed phylogenetic estimation based on similarity of oligonucleotide frequencies, and functional annotation by BLASTX similarity searches. In addition to bacteria previously associated with human/animal diseases, such as Anaplasma, Bartonella, Borrelia, Ehrlichia, Francisella and Rickettsia, BLSOM analysis detected microorganisms belonging to the phylum Chlamydiae in some tick species. This was confirmed by pan-Chlamydia PCR and sequencing analysis. Gene sequences associated with bacterial pathogenesis were also identified, some of which were suspected to originate from horizontal gene transfer. These efforts to construct a database of tick microbes may lead to the ability to predict emerging tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding tick biology, including vector competency and interactions with pathogens and symbionts.

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.

We sequenced the genome of Theileria orientalis , a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T . orientalis relative to other highly pathogenic Theileria species, T . parva and T . annulata . T . parva and T . annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T . orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T . orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T . parva and T . annulata and not in T . orientalis , Babesia bovis , or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T . orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/ . IMPORTANCE Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis , and compared it to the published sequences representative of two malignant, transforming species, T . parva and T . annulata . The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.

Internal transcribed spacer one (ITS1) of the ribosomal DNA is known to be a suitable target for PCR-based detection of trypanosomes. The analysis of this region provides a multi-species-specific diagnosis by a single PCR. Using ITS1 primer-based PCR, a cross sectional study was carried out in the period from September to November 2009 on samples collected from 687 camels from geographically distinct zones in the Sudan to detect all possible African trypanosomes, which can infect camels.

Background: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium. Results: Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. Conclusions: Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.