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B A Allsopp

Publications and source records attributed to B A Allsopp.

At least 37 records · Page 2Linked to original sources
PubMed1999

A study of the systematics of Theileria spp. based upon small-subunit ribosomal RNA gene sequences.

The systematics of benign and moderately pathogenic Theileria isolates from cattle and deer originating from different geographic regions was undertaken by small-subunit ribosomal RNA (SSU rRNA) gene nucleotide-sequence analysis. A maximum-likelihood phylogenetic tree constructed from these sequences resulted in two major divisions, each with a common ancestor. One major division branches into four relatively divergent groups, including (1) bovine Theileria sp. Type D (USA and Korea), (2) T. mutans Intona and Theileria sp. MSD (Africa), (3) T. cervi (USA), and (4) well-characterized pathogenic Theileria spp. (Africa). The other major division branches into two groups: (1) T. buffeli Warwick and T. buffeli Marula and (2) a second branch of closely related isolates with SSU rRNA gene Types B, B1, C, E, and H. Putative geographically associated diversity was noted only in the Korean bovine Theileria spp. with SSU rRNA gene types C and H and in African T. mutans Intona and Theileria sp. MSD. The current results show that the United States bovine Theileria isolates are not T. mutans because they have T. buffeli Marula (Type A) and/or Type D (species undesignated) SSU rRNA gene sequences. The taxonomic separation of T. buffeli Warwick from African T. mutans is confirmed in this study.

Animals↗
PubMed1999

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo.

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5.8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93.5% of T. p. parva isolates examined were detected by probe TPP1 and 81.8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

Animals↗
PubMed1999

Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species.

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks.

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a gamecapture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33% were positive for T. parva by probing. Twelve (50%) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2.8%) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.

Animals↗
PubMed1998

Comparative evaluation of 16S, map1 and pCS20 probes for the detection of Cowdria and Ehrlichia species in ticks.

We have developed a panel of 16S ribosomal RNA gene probes for heartwater epidemiology; five of these detect different Cowdria genotypes (Ball3, Senegal, Omatjenne, Crystal Springs, and Mara 87/7); one detects all five of these genotypes; one detects any Group III Ehrlichia species other than Cowdria; one detects any Group II Ehrlichia species. We have used these probes on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different Cowdria isolates, field ticks were harvested from animals in heartwater-endemic and heartwater-free areas. All the samples were also examined by PCR amplification and probing for two other Cowdria genes (map1 and pCS20) which have been used for heartwater epidemiology. This paper describes the first direct comparison of all the currently available DNA probes for heartwater-associated organisms.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Isolation of Theileria taurotragi and Theileria mutans from cattle in Botswana.

Two Theileria species demonstrated in peripheral Giemsa-stained blood smears of sick cattle from various parts of Botswana were subsequently identified as Theileria mutans and T. taurotragi using DNA hybridization and the polymerase chain reaction. Initial screening for Theileria species was done using microscopy, the indirect fluorescent antibody technique and the micro Elisa test. The syndrome was characteristically that of high morbidity but low mortality and vague malaise. A low parasitaemia of pleomorphic intra-erythrocytic piroplasms and the absence of schizont stages in circulating lymphocytes and lymph node aspirates were evident. Dual infections were common and often complicated by intercurrent disease conditions.

Animals↗
PubMed1997

Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp. Sequenced difference within the V1 loop were sufficient for the derivation of four Cowdria genotype-specific oligonucleotide probes. Four further probes were designed; one for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp. and one for all Pseudomonadaceae. All the probes were specific except that for the Cowdria (Ball 3) genotype. The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmental contamination. The probes were used to screen DNA from goats in an area free of both Amblyomma ticks and clinical heartwater. A substantial proportion (42%) gave positive reactions for the apparently apathogenic Cowdria (Omatjenne), indicating that this genotype is relatively common.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1994

Evidence for two single copy units in Theileria parva ribosomal RNA genes.

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.

Animals↗
PubMed1994

Phylogeny and evolution of the piroplasms.

Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four independent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.

Algorithms↗
PubMed1993

Characterisation of a glutamine- and proline-rich protein (QP protein) from Theileria parva.

We have isolated a clone from a Theileria parva infected lymphocyte cDNA library which has the potential to encode a protein of 480 amino acids. This protein is particularly rich in glutamine and proline and has some short repeated amino acid motifs based on the sequences QPXP and QPXQ. We have called it the 'QP protein'. Southern blotting suggests that the QP protein gene is present as a single copy in the T. parva Muguga genome. Northern blotting revealed that the gene is transcribed in both schizonts and piroplasms. We have expressed part of the QP protein as a fusion with glutathione S-transferase in Escherichia coli and used this product to raise an anti-QP protein serum. Western blots of T. parva lysates using this serum showed a major polypeptide of approximately 100 kDa and two further polypeptides of approximately 67 and 72 kDa. Indirect immunofluorescence assays using the anti-QP protein serum on infected cells showed that the protein is associated with the schizont. The pattern of staining in the indirect immunofluorescence assays and the structure of the protein suggest that it is a component of the schizont membrane.

Amino Acid Sequence↗