Search

Key Points Multimerization of p67C Ag as nanoparticle increases its immunogenicity. Vaccine efficacy of p67C Ag increases delivered as nanoparticles., Visual Abstract, East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 μg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)–p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

Intracellular pathogens have evolved intricate mechanisms to subvert host cell signaling pathways and ensure their own propagation. A lineage of the protozoan parasite genus Theileria infects bovine leukocytes and induces their uncontrolled proliferation causing a leukemia-like disease. Given the importance of E2F transcription factors in mammalian cell cycle regulation, we investigated the role of E2F signaling in Theileria-induced host cell proliferation. Using comparative genomics and surface plasmon resonance, we identified parasite-derived peptides that have the sequence-specific ability to increase E2F signaling by binding E2F negative regulator Retinoblastoma-1 (RB). Using these peptides as a tool to probe host E2F signaling, we show that the disruption of RB complexes ex vivo leads to activation of E2F-driven transcription and increased leukocyte proliferation in an infection-dependent manner. This result is consistent with existing models and, together, they support a critical role of E2F signaling for Theileria-induced host cell proliferation, and its potential direct manipulation by one or more parasite proteins.

Summary Despite having different cell tropism, the pathogenesis and immunobiology of the diseases caused by Theileria parva and T. annulata are remarkably similar. Live vaccines have been available for both parasites for over 40 years, but although they provide strong protection, practical disadvantages have limited their widespread application. Efforts to develop alternative vaccines using defined parasite antigens have focused on the sporozoite and intracellular schizont stages of the parasites. Experimental vaccination studies using viral vectors expressing T. parva schizont antigens and T. parva and T. annulata sporozoite antigens incorporated in adjuvant have, in each case, demonstrated protection against parasite challenge in a proportion of vaccinated animals. Current work is investigating alternative antigen delivery systems in an attempt to improve the levels of protection. The genome architecture and protein coding capacity of T. parva and T. annulata are remarkably similar. The major sporozoite surface antigen in both species and most of the schizont antigens are encoded by orthologous genes. The former have been shown to induce species cross-reactive neutralising antibodies and comparison of the schizont antigen orthologues has demonstrated that some of them display high levels of sequence conservation. Hence, advances in development of subunit vaccines against one parasite species are likely to be readily applicable to the other. This article is protected by copyright. All rights reserved.

Background The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome. Results The re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized. Conclusions The process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translational modification in apicomplexan parasites.

Background Wildebeest associated malignant catarrhal fever (WA-MCF) is a fatal disease of cattle. Outbreaks are seasonal and associated with close interaction between cattle and calving wildebeest. In Kenya, WA-MCF has a dramatic effect on cattle-keepers who lose up to 10% of their cattle herds per year. The objective of this study was to report the impact of WA-MCF on a commercial ranch and assess the performance of clinical diagnosis compared to laboratory diagnosis as a disease management tool. A retrospective study of WA-MCF in cattle was conducted from 2014 to 2016 at Kapiti Plains Ranch Ltd., Kenya. During this period, 325 animals showed clinical signs of WA-MCF and of these, 123 were opportunistically sampled. In addition, 51 clinically healthy animals were sampled. Nested polymerase chain reaction (PCR) and indirect enzyme linked immunosorbent assay (ELISA) were used to confirm clinically diagnosed cases of WA-MCF. A latent class model (LCM) was used to evaluate the diagnostic parameters of clinical diagnosis and the tests in the absence of a gold standard. Results By PCR, 94% (95% C.I. 89–97%) of clinically affected animals were positive to WA-MCF while 63% (95% C.I. 54–71%) were positive by indirect ELISA. The LCM demonstrated the indirect ELISA had poor sensitivity 63.3% (95% PCI 54.4–71.7%) and specificity 62.6% (95% PCI 39.2–84.9%) while the nested PCR performed better with sensitivity 96.1% (95% PCI 90.7–99.7%) and specificity 92.9% (95% PCI 76.1–99.8%). The sensitivity and specificity of clinical diagnosis were 99.1% (95% PCI 96.8–100.0%) and 71.5% (95% PCI 48.0–97.2%) respectively. Conclusions Clinical diagnosis was demonstrated to be an effective method to identify affected animals although animals may be incorrectly classified resulting in financial loss. The study revealed indirect ELISA as a poor test and nested PCR to be a more appropriate confirmatory test for diagnosing acute WA-MCF. However, the logistics of PCR make it unsuitable for field diagnosis of WA-MCF. The future of WA-MCF diagnosis should be aimed at development of penside techniques, which will allow for fast detection in the field. Electronic supplementary material The online version of this article (10.1186/s12917-019-1818-8) contains supplementary material, which is available to authorized users.

We present a draft sequence of the genome of Aedes aegypti , the primary vector for yellow fever and dengue fever, which at ∼1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae . Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of ∼4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster . Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of ∼2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent., This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (NIAID, NIH, DHHS) under contract numbers N01-AI30071, HHSN272200900007C, HHSN266200400001C and 5R01GM77117-5. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH. Additional grants and contracts supporting work described in this manuscript were from the NIH-NIAID (HHSN266200400039C and HHSN272200900039C) to F.H.C., and a subcontract under HHSN272200900039C to C.A.H. and J.M.M., the Australian Research Council Discovery Project (DP120100240) to S.C.B. and R.S., the Ministerio de Ciencia e Innovación of Spain (BFU2007–6292; BFU2010–15484) to J.R., BIO2009–07990 and BIO2012–37926 to J.V. NIH-1R01AI090062 to Y.P., L.S., and J.K., NIH 1R21AI096268 and NSF IOS-0949194 to R.M.R., the Xunta de Galicia of Spain (10PXIB918057PR) to J.M.C.T. and M.T., BFU2011–23896 and EU FP7 ANTIGONE (278976) to J.F., the USDA-NRI/CREES (2008-35302-18820) and Texas AgriLife Research Vector Biology grant to P.V.P. and European Research Council Starting Independent Researcher Grant (205202) to R.B., J.M.R was supported by the intramural program of the NIAID, R.M.W. by a Marie Curie International Outgoing Fellowship PIOF-GA-2011–303312, E.M.Z. by Swiss National Science Foundation awards 31003A-125350 and 31003A-143936, J.M.G. by an NIH-NCATS award TL1 TR000162 and NSF Graduate Research Fellowship (DGE 1333468), V.C. by a Boehringer Ingelheim Ph.D. Fellowship, F.G.V. by a Fundação para a Ciência e a Tecnologia, Portugal fellowship (SFRH/BD/22360/2005), C.J.P.G. and F.H. by The Lundbeck Foundation (Denmark), and J.J.G. by NIH awards HHSN272200900040C, R01AI017828 and R01AI043006. Support from the Broad Genomics Platform is gratefully acknowledged.

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide–MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle. Fil: Hansen, Andreas M.. Universidad de Copenhagen; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Svitek,Nicholas. International Livestock Research Institute; Kenia Fil: Harndahl, Mikkel. Universidad de Copenhagen; Dinamarca Fil: Golde, William T.. United States Department of Agriculture. Agriculture Research Service.; Estados Unidos Fil: Barlow, John. University of Vermont; Estados Unidos Fil: Vishvanath, Nene. International Livestock Research Institute; Kenia Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Technical University Of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.

Background The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing. Results The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing. Conclusion Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database.

Background Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome. Results Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses. Conclusion Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.

Background As the developmental costs of genomic tools decline, genomic approaches to non-model systems are becoming more feasible. Many of these systems may lack advanced genetic tools but are extremely valuable models in other biological fields. Here we report the development of expressed sequence tags (EST's) in an orthopteroid insect, a model for the study of neurobiology, speciation, and evolution. Results We report the sequencing of 14,502 EST's from clones derived from a nerve cord cDNA library, and the subsequent construction of a Gene Index from these sequences, from the Hawaiian trigonidiine cricket Laupala kohalensis. The Gene Index contains 8607 unique sequences comprised of 2575 tentative consensus (TC) sequences and 6032 singletons. For each of the unique sequences, an attempt was made to assign a provisional annotation and to categorize its function using a Gene Ontology-based classification through a sequence-based comparison to known proteins. In addition, a set of unique 70 base pair oligomers that can be used for DNA microarrays was developed. All Gene Index information is posted at the DFCI Gene Indices web page Conclusion Orthopterans are models used to understand the neurophysiological basis of complex motor patterns such as flight and stridulation. The sequences presented in the cricket Gene Index will provide neurophysiologists with many genetic tools that have been largely absent in this field. The cricket Gene Index is one of only two gene indices to be developed in an evolutionary model system. Species within the genus Laupala have speciated recently, rapidly, and extensively. Therefore, the genes identified in the cricket Gene Index can be used to study the genomics of speciation. Furthermore, this gene index represents a significant EST resources for basal insects. As such, this resource is a valuable comparative tool for the understanding of invertebrate molecular evolution. The sequences presented here will provide much needed genomic resources for three distinct but overlapping fields of inquiry: neurobiology, speciation, and molecular evolution.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasiteTheileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8+cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted byT. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8+CTL from immune cattle. CD8+T cell responses to these antigens were boosted inT. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

East Coast fever (ECF) in cattle is caused by the tick-borne protozoan parasite Theileria parva. The major sporozoite surface antigen of T parva (p67) is an important candidate for inclusion in a subunit vaccine. Recently, we reported the expression and production of different parts of p67 as fusions to either GFP or to the baculovirus GP64 envelope glycoprotein in insect cells, which resulted in stable proteins recognized by a monoclonal specific for native p67. The immunogenicity of these fusion proteins was examined in out-bred mice and cattle. In mice, the full length p67 molecule without its signal peptide and transmembrane region, but fused to GFP (GFP:p67 DeltaSS) was the best immunogen followed by the C-terminus of p67 fused to GP64 (GP64:p67C). These two immunogens also provoked a high level of sero-conversion in cattle when formulated in a water-in-oil or saponin-derived adjuvant with only 100 mug of protein and a single booster. The vaccine-elicited antibodies efficiently inhibited the infectivity of T parva sporozoites in in vitro neutralization assays. This study demonstrated that these new baculovirus-derived p67 vaccines were highly immunogenic, and that in combination with a suitable adjuvant, they have a clear potential to induce protective immunity in cattle. (C) 2004 Elsevier B.V. All rights reserved.

Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.

Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attempt to express this parasite protein in a native form and thereby increase the protective capacity of the antigen. However, Spodoptera frugiperda SF21AE cells infected with recombinant virus expressed p67 as a 100-kDa molecule. The host cells exhibited a limited capacity to glycosylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule of about 70 kDa. Since the bulk of insect cell-derived p67 was not expressed in an appropriate form, we tested the immunogenicity of these partially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals developed high levels of anti-p67 antibodies that neutralized sporozoite infectivity in vitro, but antigen-specific T-cell proliferative responses were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the molecule of choice for the development of vaccines against East Coast fever.

Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.

Bacterial strains carrying poorly suppressed amber mutations in the RNA polymerase beta subunit gene (rpoB) exhibit regulatory compensation. This compensation allows these strains to produce an adequate content of RNA polymerase to support a near normal rate of growth despite the poorly suppressed amber mutation. The primary compensatory mechanism permitting the elevated expression functions by permitting a much more efficient (up to threefold) loading of ribosomes at the beta cistron translation initiation site on the mRNA. This result supports the concept that the production of beta and beta' RNA polymerase subunits are autogenously regulated at the level of mRNA translation; this translational mechanism is clearly distinct from the transcriptional mechanism regulating beta and beta' expression described previously (P. P. Dennis, Proc. Natl. Acad. Sci. U.S.A. 74:5416-5420, 1977).

Background Although vector control strategies, such as insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS) have been effective in Kenya the transmission of malaria continues to afflict western Kenya. This residual transmission is driven in part by Anopheles arabiensis, known for its opportunistic blood feeding behaviour and propensity to feed outdoors. The objective of this research was to evaluate the efficacy of the drug eprinomectin at reducing malaria vector density when applied to cattle (Bos indicus), the primary source of blood for An. arabiensis, under field conditions. Methods A pilot study was carried out in the Samia District of western Kenya from September to October of 2014. Treatment and control areas were randomly designated and comprised of 50 homes per study area. Before cattle treatments, baseline mosquito counts were performed after pyrethrum spray. Cows in the treatment area were administered topical applications of eprinomectin at 0.5 mg/kg once a week for two consecutive weeks. Mosquito collections were performed once each week for two weeks following the eprinomectin treatments. Mosquitoes were first identified morphologically and with molecular confirmation, then screened for sporozoite presence and host blood using PCR-based methods. Results The indoor resting density of An. arabiensis was significantly reduced by 38 % in the treatment area compared to the control area at one-week post-treatment (Control mean females per hut = 1.33 95 % CI [1.08, 1.64]; Treatment = 0.79 [0.56, 1.07]). An increase in the indoor resting density of Anopheles gambiae s.s. and Anopheles funestus s.s. was observed in the treatment area in the absence of An. arabiensis. At two weeks post-treatment, the total number of mosquitoes for any species per hut was not significantly different between the treatment and control areas. No change was observed in An. arabiensis host preference as a result of treatment. Conclusions Systemic drugs may be an important tool by which to supplement existing vector control interventions by significantly impacting outdoor malaria transmission driven by An. arabiensis through the treatment of cattle.

Comparative genome analysis of two endosymbiotic polydnaviruses from Glyptapanteles parasitic wasps reveals new insights into the evolutionary arms race between host and parasite., Background Polydnaviruses, double-stranded DNA viruses with segmented genomes, have evolved as obligate endosymbionts of parasitoid wasps. Virus particles are replication deficient and produced by female wasps from proviral sequences integrated into the wasp genome. These particles are co-injected with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and, thereby, survival of proviral DNA. Here we characterize and compare the encapsidated viral genome sequences of bracoviruses in the family Polydnaviridae associated with Glyptapanteles gypsy moth parasitoids, along with near complete proviral sequences from which both viral genomes are derived. Results The encapsidated Glyptapanteles indiensis and Glyptapanteles flavicoxis bracoviral genomes, each composed of 29 different size segments, total approximately 517 and 594 kbp, respectively. They are generated from a minimum of seven distinct loci in the wasp genome. Annotation of these sequences revealed numerous novel features for polydnaviruses, including insect-like sugar transporter genes and transposable elements. Evolutionary analyses suggest that positive selection is widespread among bracoviral genes. Conclusions The structure and organization of G. indiensis and G. flavicoxis bracovirus proviral segments as multiple loci containing one to many viral segments, flanked and separated by wasp gene-encoding DNA, is confirmed. Rapid evolution of bracovirus genes supports the hypothesis of bracovirus genes in an 'arms race' between bracovirus and caterpillar. Phylogenetic analyses of the bracoviral genes encoding sugar transporters provides the first robust evidence of a wasp origin for some polydnavirus genes. We hypothesize transposable elements, such as those described here, could facilitate transfer of genes between proviral segments and host DNA.

Background The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important parasite of cattle and can transmit several pathogenic microorganisms to its cattle host during the feeding process. Understanding the biology and genomics of R. microplus is critical to developing novel methods for controlling these ticks. Results We present a global comparative genomic analysis of a gene index of R. microplus comprised of 13,643 unique transcripts assembled from 42,512 expressed sequence tags (ESTs), a significant fraction of the complement of R. microplus genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of R. microplus, including larvae exposed to heat, cold, host odor, and acaricide. Functional annotation using RPS-Blast analysis identified conserved protein domains in the conceptually translated gene index and assigned GO terms to those database transcripts which had informative BlastX hits. Blast Score Ratio and SimiTri analysis compared the conceptual transcriptome of the R. microplus database to other eukaryotic proteomes and EST databases, including those from 3 ticks. The most abundant protein domains in BmiGI were also analyzed by SimiTri methodology. Conclusion These results indicate that a large fraction of BmiGI entries have no homologs in other sequenced genomes. Analysis with the PartiGene annotation pipeline showed 64% of the members of BmiGI could not be assigned GO annotation, thus minimal information is available about a significant fraction of the tick genome. This highlights the important insights in tick biology which are likely to result from a tick genome sequencing project. Global comparative analysis identified some tick genes with unexpected phylogenetic relationships which detailed analysis attributed to gene losses in some members of the animal kingdom. Some tick genes were identified which had close orthologues to mammalian genes. Members of this group would likely be poor choices as targets for development of novel tick control technology.

Background Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. Methods A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. Results The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. Conclusion Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.