The typing of Trypanosoma evansi isolates using mobile genetic element (MGE) PCR.

The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference of <15%. Changes in the banding profiles were observed after serial passage of T. evansi type B in mice, while those of T. evansi type A were identical. It is apparent that significant random insertion mobile element positional variation occurs when T. evansi isolates are introduced into a new host, a factor that needs to be considered when MGE-PCR is used to determine genetic variation in T. evansi isolates that have different host origins.

Genetic variability of Trypanosoma evansi isolates detected by inter-simple sequence repeat anchored-PCR and microsatellite.

Studies on genetic variability in Trypanosoma evansi have been limited by a lack of high-resolution techniques. In this study, we have investigated the use of inter-simple sequence repeats (ISSR) and microsatellites in revealing polymorphism among T. evansi isolates. Twelve ISSR primers and five microsatellite loci were used to generate polymorphic bands and alleles, respectively, to investigate the genetic variability among T. evansi isolates from Africa and Asia. Seven of the twelve ISSR primers showed variability between isolates with a total of 71 fragments of which 49(69%) were polymorphic. Microsatellite analysis revealed a total of 60 alleles. On average the ISSR markers revealed a higher genetic diversity (23%) than microsatellites (21.1%). The two techniques showed a strong agreement of r=0.95 for Dice and r=0.91 for Jaccard indices in estimating the genetic distances between isolates. The distance UPGMA tree revealed two major clusters of T. evansi which correlate with the minicircle classification of subtype A and B. The cophenetic correlation coefficient between Dice and Jaccard based matrices were r=0.79 for microsatellites and r=0.73 for ISSR indicating a strong agreement between dendrograms. The results suggest that both ISSR and microsatellites markers are useful in detecting genetic variability within T. evansi.

A survey for Echinococcus spp. of carnivores in six wildlife conservation areas in Kenya.

To investigate the presence of Echinococcus spp. in wild mammals of Kenya, 832 faecal samples from wild carnivores (lions, leopards, spotted hyenas, wild dogs and silver-backed jackals) were collected in six different conservation areas of Kenya (Meru, Nairobi, Tsavo West and Tsavo East National Parks, Samburu and Maasai Mara National Reserves). Taeniid eggs were found in 120 samples (14.4%). In total, 1160 eggs were isolated and further analysed using RFLP-PCR of the nad1 gene and sequencing. 38 of these samples contained eggs of Echinococcus spp., which were identified as either Echinococcus felidis (n=27) or Echinococcus granulosus sensu stricto (n=12); one sample contained eggs from both taxa. E. felidis was found in faeces from lions (n=20) and hyenas (n=5) while E. granulosus in faeces from lions (n=8), leopards (n=1) and hyenas (n=3). The host species for two samples containing E. felidis could not be identified with certainty. As the majority of isolated eggs could not be analysed with the methods used (no amplification), we do not attempt to give estimates of faecal prevalences. Both taxa of Echinococcus were found in all conservation areas except Meru (only E. felidis) and Tsavo West (only E. granulosus). Host species identification for environmental faecal samples, based on field signs, was found to be unreliable. All samples with taeniid eggs were subjected to a confirmatory host species RLFP-PCR of the cytochrome B gene. 60% had been correctly identified in the field. Frequently, hyena faeces were mistaken for lion and vice versa, and none of the samples from jackals and wild dogs could be confirmed in the tested sub-sample. This is the first molecular study on the distribution of Echinococcus spp. in Kenyan wildlife. The presence of E. felidis is confirmed for lions and newly reported for spotted hyenas. Lions and hyenas are newly recognized hosts for E. granulosus s.s., while the role of leopards remains uncertain. These data provide the basis for further studies on the lifecycles and the possible link between wild and domestic cycles of cystic echinococcosis in eastern Africa.

Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA.

Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.