Lion (Panthera leo) and cheetah (Acinonyx jubatus) IFN-gamma sequences.

Cloning and sequencing of the full length lion and cheetah interferon-gamma (IFN-gamma) transcript will enable the expression of the recombinant cytokine, to be used for production of monoclonal antibodies and to set up lion and cheetah-specific IFN-gamma ELISAs. These are relevant in blood-based diagnosis of bovine tuberculosis, an important threat to lions in the Kruger National Park. Alignment of nucleotide and amino acid sequences of lion and cheetah and that of domestic cats showed homologies of 97-100%.

Molecular sequence evidence for the reclassification of some Babesia species.

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 beta-tubulin sequence data, suggesting that the organisms should be renamed and reclassified.

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number.

Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization.

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.

Molecular diagnosis of theileriosis and heartwater in bovines in Africa.

The advent of the polymerase chain reaction (PCR) coupled with the specificity of deoxyribocucleic acid (DNA)-DNA hybridization has led to the development of specific and sensitive molecular diagnostic tests to detect and characterize the organisms that cause theileriosis and heartwater. Theileriosis is a widespread disease of wild and domestic ruminants caused by apicomplexan parasites of the genus Theileria. Species-specific variations in small subunit ribosomal ribonucleic acid genes (SSUrRNA) have been used to develop probes that can distinguish between Theileria species such as T. parva, T. annulata, T. mutans, T. buffeli and T. taurotragi. Routine application of this test has led to the discovery of previously unknown species, such as Theileria sp. (buffalo) which is apparently apathogenic to both buffalo and cattle, and Theileria sp. (sable) which is pathogenic to sable and possibly also to roan antelope. In addition, characterization probes located in the internal transcribed spacer (ITS) can be used to distinguish between most isolates of the causative agents of East Coast fever (T. p. parva) and Corridor disease (T. p. lawrencei). Heartwater is an economically important disease of livestock and some wild ruminants, caused by the intracellular rickettsial parasite Ehrlichia (ex Cowdria) ruminantium. DNA probes used to detect and characterize E. ruminantium isolates include SSUrRNA (16S) probes, the pCS20 probe and map1 probes. A panel of eight 16S probes has been developed for the detection of E. ruminantium and related Ehrlichia species. There are probes for 5 different E. ruminantium genotypes, one which will detect all 5 of these genotypes, one to detect any Ehrlichia species other than E. ruminantium, and one for any Anaplasma species. The pCS20 probe is specific for E. ruminantium and is the most sensitive of the probes for E. ruminantium detection, but it is not able to distinguish among the different genotypes. The map1 gene has also been used for diagnosis, but the extensive polymorphism of this gene means that it is most useful for characterization of different genotypes of the parasite. Routine application of these tests has led to the discovery of new genotypes that are probably not E. ruminantium but are probably new species of Ehrlichia.