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.

Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease PCR using a 3' minor groove binder fluorogenic probe.

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqManMGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5' Taq nuclease assay.

Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strain.

Phenotypic criteria for the identification of erythrocytic ruminant Anaplasma species has relied on subjective identification methods such as host pathogenicity (virulence for cattle or sheep) and/or the location of Anaplasma inclusion bodies within the host's red cells. Sequence comparisons of new and available GenBank Accessions were investigated to elucidate the relationships among these closely related Anaplasma species. Twenty-one 16S rDNA and GroEL (HSP60) sequences from 13 Anaplasma marginale (South Africa, Namibia, Zimbabwe, Israel, USA, Australia and Uruguay), three A. centrale (South Africa and Japan), two A. ovis (USA and South Africa), and two unknown Anaplasma species isolated from wild ruminants (South Africa), were compared. 16S rDNA maximum-likelihood and distance trees separated all A. marginale (and the two wild ruminant isolates) from the two South African A. centrale (including original vaccine strain, Theiler, 1911). The Japanese A. centrale (Aomori) demonstrated the lowest sequence identity to the remaining erythrocytic Anaplasma species. A. ovis inter-species relationships could not be resolved through the 16S rDNA analyses, whereas strong bootstrap branch support is demonstrated in the GroEL distance tree using A. ovis OVI strain. All erythrocytic Anaplasma species and isolates were confirmed to belong to the same cluster showing strong branch support to Anaplasma (Ehrlichia) phagocytophilum with Ehrlichia (Cowdria) ruminantium and Rickettsia rickettsii serving as appropriate out-groups. Based on groEL sequences, a specific PCR method was developed which amplified A. centrale vaccine (Theiler, 1911) specifically. This study confirms the suitability of 16S rDNA sequences to define genera and demonstrates the usefulness of GroEL sequences for defining species of erythrocytic Anaplasma.