Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

Merozoites of Theileria orientalis buffeli reduce the parasitaemia of T. orientalis ikeda following tick challenge.

Theileria orientalis is a tick-borne protozoal parasite causing anaemia and death in susceptible cattle. This investigation aimed to confirm whether immunisation with the "benign" buffeli genotype of T. orientalis could reduce the parasitaemia of the virulent ikeda genotype. Calves were inoculated intravenously or subcutaneously with bovine blood containing merozoites of T. orientalis buffeli and when recipients became positive by PCR, they and control calves were challenged with unfed nymphs of Haemaphysalis longicornis ticks infected as larvae with T. orientalis ikeda. All calves became positive for the challenge within 12 days after tick application. In the immunised calves, the first wave of parasitaemia with T. orientalis ikeda from 4 to 6 weeks was reduced significantly by >80 % before the parasite burden declined into the carrier state by 9 weeks. The parasitaemias in two calves which exhibited low infections with T. orientalis ikeda shortly after arrival, were also significantly reduced after tick challenge. The results confirm the previous studies on immunity to T. sergenti in Japan, and field experience with theileriosis in endemic zones where the carrier state appears to prevent clinical disease despite repeated, seasonal tick infestations with virulent genotypes of the parasite. This method offers a means to reduce the severity of the first wave of theilerial parasitaemia after tick challenge and possibly recover associated production losses.

Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis.

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Haemaphysalis longicornis: the life-cycle on dogs and cattle, with confirmation of its vector status for Theileria orientalis in Australia.

The intracellular protozoal parasite Theileria orientalis ikeda has rapidly spread across South-eastern Australia since 2006, causing deaths and production losses in cattle. The 3-host "bush tick" Haemaphysalis longicornis (Neumann) appears the principal biological vector in the endemic regions. To generate sufficient numbers of ticks to produce stabilate for infection to confirm vector competency and for acaricide trials, the optimal conditions and stage-specific intervals for the generational life-cycle of H.longicornis was defined on two dogs and two steers. To determine whether H.longicornis was a definitive host for Theileria orientalis, nymphal stages were fed on a steer infected with T.orientalis and moulted adults were permitted to feed for 3 days on an uninfected calf prior to harvest. Subsequent detection of infection after inoculation of four naïve calves with stabilate produced from ground-up adult ticks or dissected salivary glands confirmed H.longicornis as one final (definitive) host for T.orientalis in Australia.

Haemaphysalis longicornis: the life-cycle on dogs and cattle, with confirmation of its vector status for Theileria orientalis in Australia.

The intracellular protozoal parasite Theileria orientalis ikeda has rapidly spread across South-eastern Australia since 2006, causing deaths and production losses in cattle. The 3-host "bush tick" Haemaphysalis longicornis (Neumann) appears the principal biological vector in the endemic regions. To generate sufficient numbers of ticks to produce stabilate for infection to confirm vector competency and for acaricide trials, the optimal conditions and stage-specific intervals for the generational life-cycle of H.longicornis was defined on two dogs and two steers. To determine whether H.longicornis was a definitive host for Theileria orientalis, nymphal stages were fed on a steer infected with T.orientalis and moulted adults were permitted to feed for 3 days on an uninfected calf prior to harvest. Subsequent detection of infection after inoculation of four naïve calves with stabilate produced from ground-up adult ticks or dissected salivary glands confirmed H.longicornis as one final (definitive) host for T.orientalis in Australia.

An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green) for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates.

Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation) showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

Genetic Diversity of Tick-Borne Rickettsial Pathogens; Insights Gained from Distant Strains.

The ability to capture genetic variation with unprecedented resolution improves our understanding of bacterial populations and their ability to cause disease. The goal of the pathogenomics era is to define genetic diversity that results in disease. Despite the economic losses caused by vector-borne bacteria in the Order Rickettsiales, little is known about the genetic variants responsible for observed phenotypes. The tick-transmitted rickettsial pathogen Anaplasma marginale infects cattle in tropical and subtropical regions worldwide, including Australia. Genomic analysis of North American A. marginale strains reveals a closed core genome defined by high levels of Single Nucleotide Polymorphisms (SNPs). Here we report the first genome sequences and comparative analysis for Australian strains that differ in virulence and transmissibility. A list of genetic differences that segregate with phenotype was evaluated for the ability to distinguish the attenuated strain from virulent field strains. Phylogenetic analyses of the Australian strains revealed a marked evolutionary distance from all previously sequenced strains. SNP analysis showed a strikingly reduced genetic diversity between these strains, with the smallest number of SNPs detected between any two A. marginale strains. The low diversity between these phenotypically distinct bacteria presents a unique opportunity to identify the genetic determinants of virulence and transmission.

Viability assays of intra-erythrocytic organisms using fluorescent dyes.

Three intra-erythrocytic tick fever organisms of cattle (Babesia bovis, Babesia bigemina and Anaplasma centrale) were subjected to a range of stressors, including heat, storage over time, specific chemotherapy and cryopreservation. Various stains, both alone and in combination, were used in an attempt to assess viability of these organisms before and after the stressors were applied. Carboxyfluorescein diacetate succinimidyl ester (CFSE) stained live Babesia spp. very well while fluorescein diacetate (FDA) stained A. centrale successfully. Propidium iodide (PI) and ethidium-homodimer-1 (Eth-D) were used as counter stains to identify dead organisms. Stain combinations allowed differentiation between living and dead Babesia organisms after exposure to heat and after chemotherapy. PI and Eth-D as counter stains were of little value after deglycerolisation of cryopreserved organisms. Possible reasons for this limited success in determining death or viability of tick fever organisms after some treatments include the impermeability of red blood cells to PI and Eth-D counter stains or the loss of live and/or dead organisms during sample processing.