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M T E P Allsopp

Publications and source records attributed to M T E P Allsopp.

8 recordsLinked to original sources

Ehrlichia ruminantium variants which do not cause heartwater found in South Africa.

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.

Animals↗

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates.

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.

Animals↗

Phylogenetic relationships among Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

Africa South of the Sahara↗

Major outer membrane proteins of Ehrlichia ruminantium encoded by a multigene family.

Immune responses of infected animals and humans have been reported to be directed against variable outer membrane proteins of Ehrlichia species that are encoded by polymorphic multigene families. In Ehrlichia (= Cowdria) ruminantium, two immunodominant proteins have been identified, namely major antigenic protein 1 (MAP1) and open reading frame 2 (ORF2). The aim of the present study was to identify additional map1-like genes in the E. ruminantium genome. A 12 kb clone that hybridized with the map1 probe was amplified using long template PCR. The PCR product was partially digested, cloned, and sequenced. Four map1-like genes are located in tandem, namely map1-1 (orf2) and map1-2 upstream of map1 as well as map1+1 downstream of map1. A large ORF (2.4 kb) at the 3' end is homologous to secA genes of other organisms. The sequence data in this study support other findings that outer membrane proteins are located in tandem and are encoded by a polymorphic multigene family.

Amino Acid Sequence↗