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.

Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment.

BACKGROUND: Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes. METHODS: The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays. RESULTS: A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15). CONCLUSION: This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.

Draft Genome Sequences of Campylobacter fetus subsp. venerealis bv. venerealis Strain B6 and bv. intermedius Strain 642-21.

Campylobacter fetus subsp. venerealis is an important venereal pathogen. We sequenced the genomes of Campylobacter fetus subsp. venerealis bv. venerealis strain B6 and bv. intermedius strain 642-21. The genetic variability of these Australian strains will facilitate the study of mechanisms of geographical adaptation of these pathogens that impact livestock.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5-6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30mg semi-engorged ticks fed more reliably, ticks as small as 15mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Observation of a novel Babesia spp. in Eastern Grey Kangaroos (Macropus giganteus) in Australia.

The roles and epidemiological features of tick-borne protozoans are not well elicited in wildlife. Babesia spp. are documented in many domestic animals, including cattle, horses, pigs, dogs and cats. Three cases affecting eastern grey kangaroos are described. The kangaroos exhibited neurological signs, depression and marked anaemia, and microscopic examination of blood smears revealed intraerythrocytic piroplasms. One to seven intraerythrocytic spherical, oval, pyriform and irregularly-shaped parasites consistent with Babesia spp. were seen in the blood smears and the percentage of infected erythrocytes was estimated to be approximately 7% in each case. Data suggest that the tick vector for this kangaroo Babesia sp. is a Haemaphysalis species. For Case 2, ultrastructural examination of the erythrocytes of the renal capillaries showed parasites resembling Babesia spp. and 18 of 33 erythrocytes were infected. DNA sequencing of the amplified 18S rDNA confirmed that the observed intraerythrocytic piroplasms belong to the genus Babesia. The phylogenetic position of this new kangaroo Babesia sp. (de novo Babesia macropus), as a sister species to the new Australian woylie Babesia sp., suggests a close affinity to the described Afro-Eurasian species Babesia orientalis and Babesia occultans suggesting perhaps a common ancestor for the Babesia in kangaroos.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3).

Rhipicephalus microplus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZαA vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1). qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle.

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

The Rhipicephalus microplus 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. microplus 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.

The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones.

BACKGROUND: Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the de-novo assembly of two R. microplus BAC sequences from the understudied R microplus genome. Based on available R. microplus sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction. RESULTS: In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region.In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb papilin gene was a helicase gene. This helicase overlapped in two exonic regions with the papilin. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also determined for the papilin gene and the protein binding sites of the 18S subunit in a comparison to Bos taurus. CONCLUSION: In the absence of a sequenced reference genome we have assembled two complex BAC sequences, characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first to describe Rhipicephalinae papilin, and the first to report the complete ribosomal DNA repeated unit sequence structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin future efforts for genome sequencing and assembly of the R. microplus genome.

Suppressive subtractive hybridization analysis of Rhipicephalus (Boophilus) microplus larval and adult transcript expression during attachment and feeding.

Ticks, as blood-feeding ectoparasites, affect their hosts both directly and as vectors of viral, bacterial and protozoal diseases. The tick's mode of feeding means it must maintain intimate contact with the host in the face of host defensive responses for a prolonged time. The parasite-host interactions are characterized by the host response and parasite counter-response which result in a highly complex biological system that is barely understood. We conducted transcriptomic analyses utilizing suppressive subtractive hybridization (SSH) to identify transcripts associated with host attachment and feeding of larval, adult female and adult male ticks. Five SSH libraries resulted in 511 clones (assembled into 36 contigs and 90 singletons) from differentially expressed transcripts isolated from unattached frustrated larvae (95), feeding larvae (159), unattached frustrated adult female ticks (68), feeding adult female ticks (95) and male adult ticks (94 clones). Unattached 'frustrated' ticks were held in fabric bags affixed to cattle for up to 24h to identify genes up-regulated prior to host penetration. Sequence analysis was based on BLAST, Panther, KOG and domain (CDD) analyses to assign functional groups for proteins including: cuticle proteins, enzymes (ATPases), ligand binding (histamine binding), molecular chaperone (prefoldin), nucleic acid binding (ribosomal proteins), putative salivary proteins, serine proteases, stress response (heat shock, glycine rich) and transporters. An additional 63% of all contigs and singletons were novel R. microplus transcripts or predicted proteins of unknown function. Expression was confirmed using quantitative real time PCR analysis of selected transcripts. This is the first comprehensive analysis of the R. microplus transcriptome from multiple stages of ticks and assists to elucidate the molecular events during tick attachment and development.

Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle.

Cattle demonstrate divergent and heritable phenotypes of resistance and susceptibility to infestation with the cattle tick Rhipicephalus (Boophilus) microplus. Bos indicus cattle are generally more resistant to tick infestation than Bos taurus breeds although large variations in resistance can occur within subspecies and within breed. Increased tick resistance has been previously associated with an intense hypersensitivity response in B. taurus breeds; however, the mechanism by which highly resistant B. indicus cattle acquire and sustain high levels of tick resistance remains to be elucidated. Using the commercially available Affymetrix microarray gene expression platform, together with histological examination of the larval attachment site, this study aimed to describe those processes responsible for high levels of tick resistance in Brahman (B. indicus) cattle that differ from those in low-resistance Holstein-Friesian (B. taurus) cattle. We found that genes involved in inflammatory processes and immune responsiveness to infestation by ticks, although up-regulated in tick-infested Holstein-Friesian cattle, were not up-regulated in Brahman cattle. In contrast, genes encoding constituents of the extracellular matrix were up-regulated in Brahmans. Furthermore, the susceptible Holstein-Friesian animals displayed a much greater cellular inflammatory response at the site of larval R. microplus attachment compared with the tick-resistant Brahman cattle.

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets.

BACKGROUND: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (approximately 75-80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. RESULTS: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. CONCLUSION: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

Immunological profiles of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus, is a major threat to the improvement of cattle production in tropical and subtropical countries worldwide. Bos indicus cattle are naturally more resistant to infestation with the cattle tick than are Bos taurus breeds, although considerable variation in resistance occurs within and between breeds. It is not known which genes contribute to the resistant phenotype, nor have immune parameters involved in resistance to R. microplus been fully described for the bovine host. This study was undertaken to determine whether selected cellular and antibody parameters of the peripheral circulation differed between tick-resistant Bos indicus and tick-susceptible Bos taurus cattle following a period of tick infestations. This study demonstrated significant differences between the two breeds with respect to the percentage of cellular subsets comprising the peripheral blood mononuclear cell population, cytokine expression by peripheral blood leukocytes, and levels of tick-specific immunoglobulin G1 (IgG1) antibodies measured in the peripheral circulation. In addition to these parameters, the Affymetrix bovine genome microarray was used to analyze gene expression by peripheral blood leukocytes of these animals. The results demonstrate that the Bos indicus cattle developed a stabilized T-cell-mediated response to tick infestation evidenced by their cellular profile and leukocyte cytokine spectrum. The Bos taurus cattle demonstrated cellular and gene expression profiles consistent with a sustained innate, inflammatory response to infestation, although high tick-specific IgG1 titers suggest that these animals have also developed a T-cell response to infestation.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides.

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman minor groove-binding(MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.

BACKGROUND: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. RESULTS: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. CONCLUSION: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.