Microarray analysis of acaricide-inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus.

Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13 000 probes, having been derived from a previously described R. microplus gene index (BmiGI Version 2; Wang et al., 2007). Relative quantitative reverse transcriptase-PCR, real time PCR, and serial analysis of gene expression data was used to verify microarray data. Among the differentially expressed genes with informative annotation were legumain, glutathione S-transferase, and a putative salivary gland-associated protein.

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Development of improved attenuated and nucleic acid vaccines for heartwater.

Heartwater, an economically important tickborne disease of wild and domestic ruminants, is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium. The only commercially available immunization procedure is more than 50 years old and uses an infection and treatment regimen using a preparation of virulent organisms in cryopreserved sheep blood. Much research has been conducted into the development of attenuated, inactivated, and nucleic acid vaccines over the last half-century, with relatively little success until recently. We describe here the development of two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuation of virulent E. ruminantium was achieved by growing the organisms in a continuous canine macrophage-monocyte cell line. After more than 125 passages the cultures produced no disease when inoculated into mice or sheep, and the animals were completely protected against a subsequent lethal homologous needle challenge. The nucleic acid vaccine consists of a cocktail of four E. ruminantium genes, from a genetic locus involved in nutrient transport, cloned in a DNA vaccine vector. Sheep immunized with this cocktail were completely protected against a subsequent lethal needle challenge, either with the homologous isolate or with any one of five different virulent heterologous isolates. Protection against a field challenge in a heartwater endemic area, however, was relatively poor. Genetic characterization of the E. ruminantium genotypes in the challenge area did not identify any having major differences from those used in the heterologous needle challenge experiments, so lack of cross-immunity between the vaccine genotype and those in the field was unlikely to be the main reason for the lack of protection. We believe that a needle challenge is far less severe than a tick challenge, and that the immunity engendered by the DNA vaccine alone was not sufficient to protect against the natural route of infection. Boosting with live organisms after DNA vaccination results in much higher levels of protection against tick challenge than DNA vaccination alone, suggesting that improved methods of boosting could lead to more effective immunization.

Sequencing of a 15-kb Ehrlichia ruminantium clone and evaluation of the cpg1 open reading frame for protection against heartwater.

A 1.2 kb polymorphic fragment from the Gardel isolate of Ehrlichia (formerly Cowdria) ruminantium was used to isolate a 15kb clone from the E. ruminantium Welgevonden LambdaGEM-11 library. This clone, WL2EL1, was subcloned and sequenced. Eight open reading frames (ORFs) were identified. The ORF in WL2EL1 which contained the Welgevonden homologue of the 1.2 kb polymorphic fragment was designated Cowdria polymorphic gene 1 (cpg1). The cpg1 ORF was cloned into pCMViUB, a genetic vaccine vector. Mice and sheep were immunized with pCMViUB/cpg1 by intramuscular injection and gene gun inoculation. Although all of the immunized mice died, there was a trend for mice that received larger amounts of pCMViUB/cpg1 DNA to survive longer. Four out of five sheep immunized with the construct survived lethal challenge.

Genetic immunization with Ehrlichia ruminantium GroEL and GroES homologues.

Ehrlichia ruminantium GroEL and GroES genes were amplified from E. ruminantium Welgevonden genomic DNA and were cloned into genetic vaccine and Salmonella expression vectors. These constructs were used to inoculate Balb/c and C57BL/6J mice. Both GroEL and GroES induced low levels of protection in Balb/c and C57BL/6J mice immunized with the Salmonella expression vectors. None of the mice inoculated with the genetic vaccine survived. Immunological memory was also tested in these mice and a correlation between splenocyte proliferation and the survival rate was observed.

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.

Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens.

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp2 and msp3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Genome size and genetic map of Cowdria ruminantium.

Cowdria ruminantium is the cause of a serious tick-borne disease of domestic ruminants, known as heartwater or cowdriosis. The organism belongs to the tribe Ehrlichieae:, which contains obligate intracellular pathogens, causing several important animal and human diseases. Although a few C. ruminantium genes have been cloned and sequenced, very little is known about the size, gross structure and organization of the genome. This paper presents a complete physical map and a preliminary genetic map for C. ruminantium. Chromosomal C. ruminantium DNA was examined by PFGE and Southern hybridization. PFGE analysis revealed that C. ruminantium has a circular chromosome approximately 1576 kb in size. A physical map was derived by combining the results of PFGE analysis of DNA fragments resulting from digestion of the whole genome with KSP:I, RSR:II and SMA:I and Southern hybridization analysis with a series of gene probes and isolated macrorestriction fragments. A genetic map for C. ruminantium with a mean resolution of 290 kb was established, the first for a member of the Ehrlichieae: A total of nine genes or cloned C. ruminantium DNA fragments were mapped to specific KSP:I, RSR:II and SMA:I fragments, including the major antigenic protein gene, map-1.

Macrorestriction fragment profiles reveal genetic variation of Cowdria ruminantium isolates.

Macrorestriction profile analysis by pulsed-field gel electrophoresis (PFGE) was used to distinguish between seven isolates of Cowdria ruminantium from geographically different areas. Characteristic profiles were generated for each isolate by using the restriction endonucleases KspI, SalI, and SmaI with chromosomal sizes ranging between 1,546 and 1,692 kb. Statistical analysis of the macrorestriction profiles indicated that all the isolates were distinct from each other; these data contribute to a better understanding of the epidemiology of this pathogen and may be exploited for the identification of genotype-specific DNA probes.

Cowdria ruminantium DNA is unstable in a SuperCos1 library.

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.

A partially protective clone from Cowdria ruminantium identified by using a Salmonella vaccine delivery system.

A strategy has been developed to screen the Cowdria genome, using a Salmonella vaccine delivery system, to identify genes that code for protection-stimulating proteins. We have cloned mini-libraries of Cowdria into this Salmonella system, used the recombinant bacteria to immunize outbred mice, and then challenged them after two weeks with a lethal dose of Cowdria. When one of these mini-libraries was tested in a group of 5 mice, one mouse lived much longer than the others. The experiment was repeated with each of the clones from the mini-library being tested individually in 10 mice, and one mouse survived the challenge. This clone has been tested repeatedly in larger groups of mice and is proven to protect 14% of outbred mice against a lethal Cowdria challenge.

Purification of Cowdria ruminantium organisms for use in genome analysis by pulsed-field gel electrophoresis.

Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick-borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed-field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra-chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.

Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes.

The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.

Development of the OPgun for bombardment of animal tissues.

A simple and inexpensive particle-bombardment device, the OPgun, was constructed for the delivery of DNA into animal tissues. This device is based on the particle-inflow gun first described for plant-cell transfection. The delivery of tungsten particles into the epidermis of the mouse ear, without the use of vacuum and without causing damage to the tissue, was demonstrated. The system was also shown to be capable of inducing antibodies to a foreign gene in mice.

Two genes for de novo purine nucleotide synthesis on human chromosome 4 are closely linked and divergently transcribed.

A cDNA encoding human glutamine phosphoribosylpyrophosphate amidotransferase for step one in de novo purine nucleotide synthesis was cloned, sequenced, and expressed in Chinese hamster ovary cells to yield functional enzyme. Enzyme function was dependent upon removal of an 11-amino-acid propeptide. A mutant enzyme having three propeptide amino acid replacements was not processed and was not active. The human genes GPAT, encoding the amidotransferase, and AIRC, encoding a bifunctional enzyme for steps six and seven in the pathway, were cloned and characterized. GPAT and AIRC are closely linked and divergently transcribed from an intergenic region of approximately 625 base pairs. Expression of a luciferase reporter from the GPAT promoter was approximately 3-4-fold higher than from the AIRC promoter. The GPAT gene was mapped to the q12 region of chromosome 4.

Coexpression of two closely linked avian genes for purine nucleotide synthesis from a bidirectional promoter.

Two avian genes encoding essential steps in the purine nucleotide biosynthetic pathway are transcribed divergently from a bidirectional promoter element. The bidirectional promoter, embedded in a CpG island, directs coexpression of GPAT and AIRC genes from distinct transcriptional start sites 229 bp apart. The bidirectional promoter can be divided in half, with each half retaining partial activity towards the cognate gene. GPAT and AIRC genes encode the enzymes that catalyze step 1 and steps 6 plus 7, respectively, in the de novo purine biosynthetic pathway. This is the first report of genes coding for structurally unrelated enzymes of the same pathway that are tightly linked and transcribed divergently from a bidirectional promoter. This arrangement has the potential to provide for regulated coexpression comparable to that in a prokaryotic operon.

Rat preprocarboxypeptidase H. Cloning, characterization, and sequence of the cDNA and regulation of the mRNA by corticotropin-releasing factor.

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of approximately 2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by approximately 30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.

Cloning, characterization, and sequence of a porcine cDNA encoding a secreted neuronal and endocrine protein.

This report describes the cloning, sequence, and characterization of a cDNA which encodes a protein synthesized in the brain and endocrine tissue, including pituitary, adrenal medulla, and ovary. The deduced 207-amino-acid sequence of the 23-kD protein contains a hydrophobic signal peptide suggesting that it is secreted. Northern blot analysis utilizing the cDNA clone identifies a single RNA of approximately 1400 nucleotides in porcine brain, adrenal medulla, pituitary, and ovary, as well as in human endocrine tumors. Very high levels of RNA were observed in one human pancreatic tumor. Southern blot analysis suggests that sequences homologous to the porcine cDNA are present in human, cow, rat, and salmon DNA, indicating that the gene(s) have been highly conserved during evolution.