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R P Bishop

Publications and source records attributed to R P Bishop.

At least 19 recordsLinked to original sources
PubMed1995

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.

Animals↗
PubMed1995

Generation and characterization of cloned Theileria parva parasites.

A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.

Animals↗
PubMed1994

Cloning and characterisation of a repetitive DNA sequence from Theileria mutans: application as a species-specific probe.

Repetitive DNA sequences were isolated from a Theileria mutans genomic library by screening with T. mutans total DNA. DNA sequence analysis demonstrated that a section of one of the clones contained a complex series of overlapping perfect repeats ranging between 99 and 20 bp in size. The T. mutans repetitive sequence did not contain large open reading frames (ORFs), unlike T. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between the EcoRI-digested DNAs of two T. mutans stocks. The T. mutans repetitive probe gave a signal with 1 ng of purified T. mutans piroplasm DNA and detected T. mutans sequences in whole-blood DNA isolated from an experimentally infected animal when the piroplasm parasitaemia was equal to or above 0.4%. Oligonucleotide primers derived from the repetitive sequence allowed more sensitive detection of T. mutans DNA by polymerase chain reaction (PCR) amplification. Using the PCR, T. mutans DNA was amplified from an experimentally infected animal with a parasitaemia of < 0.1%.

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

Molecular characterization of Theileria parasites: application to the epidemiology of theileriosis in Zimbabwe.

Forty Theileria schizont-infected lymphocyte culture isolates from Zimbabwe were characterized using a panel of antischizont monoclonal antibodies (MAbs) and 4 Theileria parva DNA probes containing cloned extrachromosomal element, Tpr repetitive, ribosomal and telomeric sequences. The Theileria isolates were assigned as T. parva or T. taurotragi on the basis of reactivities with MAbs and restriction fragment length polymorphisms (RFLPs) detected using the extra-chromosomal element probe. Cattle-derived T. parva isolates were relatively homogeneous on the basis of reactivities with MAbs and RFLPs detected using Tpr repetitive and ribosomal DNA probes. In contrast to previous results from Kenya, most of the cattle-derived isolates from Zimbabwe exhibited very similar Tpr restriction fragment patterns, although the Tpr genotypes of buffalo-derived isolates were heterogeneous. This suggests that selection for a particular Tpr genotype may be occurring in cattle. Many isolates with similar Tpr genotypes were differentiated by RFLPs detected using the telomeric DNA probe. The T. parva Boleni immunizing stock was distinguished from all other isolates by telomeric RFLPs. The T. parva Boleni Tpr repetitive DNA probe cross-hybridized with T. taurotragi DNA and detected RFLPs between different T. taurotragi isolates.

Animals↗
PubMed1994

Characterization of Theileria parva which infects waterbuck (Kobus defassa).

Theileria-free waterbuck (Kobus defassa) born in captivity were successfully infected with Theileria parva sporozoites derived from ticks infected by feeding on African buffalo (Syncerus caffer). All waterbuck underwent mild infections with the development of sporadic schizont and piroplasm parasitosis when inoculated with sporozoite doses lethal to cattle. A carrier state of T. parva was demonstrated by feeding clean R. appendiculatus nymphs on two of these infected waterbuck. Tick batches from these waterbuck on 2 of 5 occasions transmitted lethal Theileria infections to cattle. In a separate experiment, waterbuck cells were infected and transformed in vitro by T. parva sporozoites derived from buffalo but not by cattle-derived T. parva (Muguga) sporozoites. Waterbuck cells infected in vitro with T. parva isolated from buffalo were inoculated into autologous waterbuck but no infections developed. Theileria parva isolates generated in this study from various sources were characterized using anti-T. parva schizont monoclonal antibodies (MAbs), and it was found that buffalo-derived and waterbuck-passaged isolates had different profiles. Species-specific synthetic oligonucleotide probes, restriction fragment length polymorphism (RFLP) analysis with cloned T. parva DNA probes, and DNA sequence analysis of the p67 sporozoite antigen gene confirmed that the waterbuck-passaged parasite was T. parva. The Tpr repetitive probe hybridization patterns from the waterbuck-passaged parasites were different from the other samples tested. The ribosomal genotype of the waterbuck-passaged T. parva was similar to that of cattle-derived T. parva Muguga. Analyses with both probes and MAbs suggested that a minor parasite population present within the T. parva 7014 buffalo-derived stock had been selected during waterbuck passage.(ABSTRACT TRUNCATED AT 250 WORDS)

Africa, Eastern↗
PubMed1993

Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies.

A total of 21 Theileria parva stocks from 6 countries were characterized using T. parva repetitive and ribosomal DNA probes, a Plasmodium berghei telomeric oligonucleotide and a panel of anti-schizont monoclonal antibodies (MAbs). Hybridization of the repetitive DNA probe to Southern blots of EcoRI-digested T. parva DNA revealed 20 different restriction fragment patterns among DNA samples isolated from infections initiated using 16 parasite stocks. The panel of anti-schizont MAbs defined 8 different profiles among schizont-infected lymphoblastoid cell-cultures infected with the same 16 T. parva stocks. Many stocks, which were differentiated by the repetitive DNA probe, could not be distinguished using the anti-schizont MAbs. A cloned T. parva small subunit ribosomal RNA (SSUrRNA) gene probe separated 17 T. parva stocks into 2 groups, exhibiting either 1 or 2 restriction fragments, when hybridized to EcoRI-digested T. parva DNA. When hybridized to PvuII-digested DNA from 8 T. parva stocks, the ribosomal probe identified 4 groups with similar restriction fragment patterns. A synthetic oligonucleotide derived from a P. berghei telomeric sequence hybridized to 7 or 8 size-polymorphic restriction fragments in the EcoRI-digested DNA of most T. parva stocks. The telomeric and ribosomal probes defined the same 4 groups among 8 T. parva stocks as assessed by similarities in restriction fragment patterns. Based on the comparison of repetitive DNA sequences from the T. parva Uganda and Muguga stocks, a synthetic oligonucleotide was developed which distinguished the DNA of the T. parva Uganda stock from that of 4 other T. parva stocks on a positive/negative basis.

Animals↗
PubMed1991

An unusual repetitive gene family in Theileria parva which is stage-specifically transcribed.

Tpr1 is a repetitive DNA element from Theileria parva which has previously been shown to be of value in strain characterisation. Further characterisation, described here, has shown that Tpr1 is present in long tandem arrays. The sequence of 8.1 kb from one end of an array has been determined. The sequence showed that Tpr1 is a 1.44-kb element which contains an ORF extending from its 5' end to the 3' end. The sequenced region contains 4 large ORFs; 2 of these consisted only of Tpr1 whilst the third consisted of Tpr1 and a 0.55-kb element (Tpr2) located 5' of Tpr1. The largest ORF consisted of Tpr1 plus Tpr2 as well as an additional 420-bp element (Tpr3) 5' of Tpr2, thus a continuous ORF arranged 5'-Tpr3-Tpr2-Tpr1-3' was formed. This ORF potentially encodes a 795 amino acid polypeptide commencing at an ATG close to the 5' end. In contrast the first in frame ATGs in the other 3 ORFs are at least 417 bp from the 5' end. Southern analysis confirmed that the sequenced region was typical of the rest of the Tpr array(s). Transcripts containing both Tpr3 and Tpr1 were detected in the piroplasm but not the schizont stages of the life cycle.

Amino Acid Sequence↗
PubMed1990

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family.

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.

Amino Acid Sequence↗
PubMed1990

Extensive homologies between Leishmania donovani chromosomes of markedly different size.

The smallest chromosome (230 kb) of the HU3 stock of Leishmania donovani was purified from an orthogonal field alternation (OFAGE) gel, digested with PstI and cloned into the plasmid pUC13. When used to probe Southern blots of OFAGE gels, the cloned sequences recognised one or more large chromosomes in all L. donovani stocks and a small chromosome in HU3 and two additional L. donovani stocks (Khartoum and DD8). These probes recognised a single band on Southern blots of restricted genomic DNA regardless of their homologies to only large or large and small chromosomes. Analysis of lambda EMBL3 genomic clones selected with the same probes suggested that at least 30 kb of DNA was common to large and small chromosomes. Most of the cloned sequences were mapped to the same 50-kb SfiI fragment present in both chromosomes. These data indicate that two or more L. donovani chromosomes of markedly different size on OFAGE gels are extensively homologous.

Animals↗
PubMed1990

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis.

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

Animals↗
PubMed1990

Immunization with Theileria parva parasites from buffaloes results in generation of cytotoxic T cells which recognize antigens common among cells infected with stocks of T. parva parva, T. parva bovis, and T. parva lawrencei.

Immunity to infection by the protozoan parasite Theileria parva in cattle is partially attributable to cytotoxic T cells, which kill lymphocytes infected with the schizont stage of the parasite. Here we evaluated five stocks of buffalo-derived T. parva lawrencei parasites and two stocks of cattle-derived T. parva parva parasites for their ability to induce in vivo cytotoxic T cells which can kill lymphocytes infected with a wide variety of strains of T. parva parasites. A group of seven full-sibling cattle, produced by embryo transfer and matched for at least one major histocompatibility complex class I haplotype, were immunized by infection and treatment with the parasite stocks. Target cells used in in vitro cytotoxicity assays were infected with five buffalo-derived parasite stocks and five cattle-derived parasite stocks, including T. parva parva and T. parva bovis. Immunization with any of the seven parasite stocks resulted in the generation of cytotoxic T cells which recognized parasite antigens on most if not all of the target cell lines tested, although the T. parva bovis stock was the least effective at doing so. Further in-depth analyses performed with peripheral blood mononuclear cells from one of the cattle immunized with T. parva lawrencei parasites showed that the pattern of killing of the panel of target cells was altered when either cells infected with different parasite stocks or clones of infected cells were used as stimulator cells in vitro, suggesting the presence of more than one population of parasite-specific cytotoxic effector cells in the peripheral blood mononuclear cells. However, clones of these cytotoxic effector cells recognized common or cross-reactive antigen epitopes expressed by the entire panel of infected target cells. These T-cell clones will be useful for identifying common T-cell antigen epitopes of T. parva and the parasite genes encoding them.

Animals↗
PubMed1987

Chromosome size polymorphisms of Leishmania donovani.

A minimum of 22 chromosomes were found in all Leishmania donovani stocks examined by orthogonal field alternation gel electrophoresis (OFAGE). Chromosome sizes ranged from approximately 270 to 4000 kb. Certain chromosomes were polymorphic in size between stocks and chromosomes present in some stocks had no apparent equivalent in others. Specific polymorphisms were useful in distinguishing the subspecies L. d. donovani, L. d. infantum and L. d. chagasi and African L. donovani stocks but there were karotypic differences within these taxa. Radiolabelled DNA derived from whole chromosomes was hybridised to OFAGE Southern blots. Chromosome 1 of L. d. donovani was homologous to two larger chromosomes in all stocks. Chromosome 2 of certain L. d. chagasi and L. d. infantum stocks was homologous to both chromosomes 2 and 3 of L. d. donovani: this suggested that translocation between chromosomes may have contributed to the size polymorphisms. The smallest chromosome seen (270 kb) was unique to the African stock HU3. It was not homologous to small chromosomes in L. d. donovani, L. d. infantum or L. d. chagasi. The small chromosome did hybridise to two small chromosomes in another African stock, Khartoum, and to a large chromosome present in all stocks. The beta-tubulin gene was mapped to chromosomes 21/22, 13 and 7 with strongest hybridisation to 21/22. alpha-Tubulin was mapped to chromosomes 9. The alpha- and beta-tubulin arrangement was highly conserved.

Animals↗
PubMed1986

New findings in live, attenuated hepatitis A vaccine development.

Strain CR326F of hepatitis A virus, derived from a fecal specimen of Costa Rican patient 033-03, was passed 15 times in fetal rhesus monkey kidney (FRhK6) cell cultures plus eight times in human diploid lung (MRC5) cell cultures to yield variant F and 16 times in MRC5 cell cultures to yield variant F'. Both variants were purified by limit dilution passages. Virulence for marmosets was assessed at six different passage levels, including variants F and F'. There was a gradual loss of virulence with in vitro passage. Variant F retained slight virulence for marmosets; variant F' showed no evidence of virulence. Both variants induced hepatitis A antibody in most marmosets that received them, and the animals were immune to infection when challenged. Variants F and F' were also assessed in chimpanzees. As in marmosets, F retained slight virulence but F' did not. Experimental vaccines made from variants F and F' were then inoculated parenterally into adult human volunteers. A portion of recipients of variant F showed brief, low-order enzyme elevations; none was seen in recipients of F', although their occurrence could not be totally ruled out. As in the animal models, F' appeared more attenuated than F. Most persons developed hepatitis A antibody, indicating the feasibility of developing a live, attenuated hepatitis A vaccine for human beings.

Animals↗
PubMed1977

The automated classification of mitotic phase for human chromosome spreads.

A system has been developed that automatically recognizes the mitotic phase of human chromosome spreads for karyotyping. Suitable spreads are classified into one of five subphases of mitosis. Classification is performed on the basis of summed chromosome length and most probable chromosome width. Classification requires 100-500 msec. A television camera scans the spread through microscope optics; computer and special purpose electronics process the video signals to generate run length histograms. The histograms are used to determine mitotic phase. Unbanded spreads, 133, were classified with a 4.5% error rate. One hundred banded spreads were classified with a 15% error rate.

Autoanalysis↗