Basil A. Allsopp

Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences

The complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutants and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.

Detection of a carrier state in Theileria parva-infected cattle by the polymerase chain reaction

Two sets of oligonucleotide primers, one derived from a repetitive sequence and the other from the gene encoding a 67 kDa sporozoite antigen of Theileria parva, were used to amplify parasite DNA from the blood of T. parva-infected carrier cattle using the polymerase chain reaction (PCR). PCR amplification products were obtained from 15 carrier cattle infected with one of 4 different T. parva stocks. Successful amplifications were performed using DNA from 2 cattle infected with T. p. parva Pemba Mnarani, 10 cattle infected with T. p. parva Marikebuni, 2 cattle infected with T. p. bovis Boleni and 1 animal infected with T. p. lawrencei 7014. No amplification products were obtained from any of 7 cattle which had been infected with the T. p. parva Muguga stock. A synthetic oligonucleotide, which hybridized specifically to T. p. parva Marikebuni DNA among 6 T. parva stocks tested, was designed using sequence data from within the region of the T. parva genome amplified by the repetitive sequence primers. The oligonucleotide was used to probe PCR products and to increase the sensitivity and specificity of carrier animal detection. Southern blot analysis using a T. parva repetitive sequence probe demonstrated the existence of restriction fragment length polymorphisms between parasites isolated from T. p. parva Marikebuni-infected carrier cattle. The use of the PCR and other methods of carrier animal detection are discussed.

Molecular Sequence Evidence for the Reclassification of Some Babesia Species

Abstract:  Taxonomic characterization of organisms in the genera Theileria and Babesia was originally based on observations of morphology and certain general phenotypic characteristics, which enabled many parasites to be unequivocally assigned to a particular genus. However, application of molecular genetic techniques, such as the polymerase chain reaction (PCR) for gene amplification, and DNA sequencing, have revealed gross inconsistencies in the assignation of some parasite genetic variants, particularly those of the B. gibsoni and B. microti complexes, to the genus Babesia. These variants cannot be assigned, on the basis of sequence information and phylogenetic analysis, to either of the genera Theileria and Babesia. The gene for which most sequence information is available for phylogenetic analysis is the small subunit ribosomal RNA (srRNA) gene. This gene allows clear distinction of the genera Theileria and Babesia (sensu stricto) and reveals that many “Babesia” variants are phylogenetically distinct from both genera. This distinction is confirmed, for some of the variants, by β‐tubulin sequence data, suggesting that the organisms should be renamed and reclassified.

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

Abstract 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.

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.

Identification of novel Babesia and Theileria species in South African giraffe (Giraffa camelopardalis, Linnaeus, 1758) and roan antelope (Hippotragus equinus, Desmarest 1804).

Blood specimens were received from five cases in which young adult giraffe, from different geographic origins in South Africa, showed sudden onset of disease and subsequently died. Additional specimens from two translocated giraffe, as well as one specimen from a roan antelope, were also included in the study. Blood slides from some of these animals showed the presence of piroplasms. DNA was extracted; the V4 hypervariable region of the 18S rRNA gene amplified and analyzed using the Reverse Line Blot (RLB) hybridization assay. PCR products failed to hybridize with any of the Babesia or Theileria species-specific probes, and only hybridized with the Babesia/Theileria genus-specific probe suggesting the presence of a novel species or variant of a species. Full-length 18S rDNA was amplified, cloned and the recombinants were sequenced. 18S rRNA gene sequence similarity analysis revealed the presence of novel piroplasm species in both healthy giraffe and a roan antelope and clinically sick or dead giraffe. Phylogenetic analysis grouped five of these organisms in the Babesia sensu stricto clade and three in the Theileria sensu stricto clade. Although parasites were observed in blood smears, there is no direct evidence that piroplasmosis caused the death of five giraffe, although it certainly seems to be likely.

Ehrlichia ruminantium Major Antigenic Protein Gene (map1) Variants Are Not Geographically Constrained and Show No Evidence of Having Evolved under Positive Selection Pressure

ABSTRACT In a search for tools to distinguish antigenic variants ofEhrlichia ruminantium, we sequenced the major antigenic protein genes (map1 genes) of 21 different isolates and found that the sequence polymorphisms were too great to permit the design of probes which could be used as markers for immunogenicity. Phylogenetic comparison of the 21 deduced MAP1 sequences plus another 9 sequences which had been previously published did not reveal any geographic clustering among the isolates. Maximum likelihood analysis of codon and amino acid changes over the phylogeny provided no statistical evidence that the gene is under positive selection pressure, suggesting that it may not be important for the evasion of host immune responses.

Antigenic, phenotypic and molecular characterization confirms Babesia odocoilei isolated from three cervids.

Babesia isolates from an elk (Cervus elaphus canadensis) and a caribou (Rangifer tarandus caribou) with fatal infections were compared to Babesia odocoilei (Engeling isolate) from white-tailed deer (Odocoileus virginianus) by experimental infection, serologic, and small subunit ribosomal RNA (SSU rRNA) gene sequence analysis studies. Both the indirect fluorescent antibody test and immunoprecipitation assays demonstrated antigenic variation among the isolates. Experimental infection studies showed no clinical differences among the isolates. Nucleotide sequence analysis showed that the elk and caribou Babesia sp. isolates possessed SSU rRNA genes with identical sequences to that of B. odocoilei. A phylogenetic tree constructed from SSU rRNA gene sequences shows that B. odocoilei is most closely related to Babesia divergens, both of which branch together in the true babesia clade.

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.

Improved characterization of Theileria parva isolates using the polymerase chain reaction and oligonucleotide probes

Theileria parva DNA was purified from piroplasms isolated from cattle infected with 5 different East African isolates of the parasite, including the East Coast fever reference stock T. p. parva Muguga. Total cellular DNA was prepared from T. parva schizont-infected bovine lymphoblastoid cell cultures (11 isolates). Two probes, previously isolated from T. p. parva Muguga repetitive genomic DNA, were hybridized to restriction digests; closely similar restriction fragment length polymorphism (RFLP) patterns were produced, and no two isolates had the same RFLP pattern. The DNA sequences of probe PMB3, two further copies of the repeated sequence from T. p. parva Muguga, and homologous regions from T. p. parva Kiambu 4 and T. p. lawrencei 3081, were determined. Oligonucleotides were synthesized corresponding to two conserved sections flanking a region which varied between isolates. These oligonucleotides were used as primers in the polymerase chain reaction to amplify the variable region. Further oligonucleotides corresponding to sequences in this variable region were able to distinguish between isolates and no sample hybridized to both oligonucleotides. This is the first unequivocal plus/minus discrimination between groups of isolates to be achieved for T. parva.