An investigation of experimental Leishmania aethiopica infections with reference to host and parasite diversity
Leishmania aethiopica causes two distinct forms of cutaneous leishmaniasis in the highlands of Ethiopia and in northern Kenya: the generally self-healing localised cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL), which does not self-heal. Unlike other Leishmania parasites, the pathological and infectious processes associated with exposure to this parasite are not well studied in experimental systems, mainly due to the lack of a suitable in vivo model. This study was undertaken to develop an experimental model to examine the distribution and fate of parasites and pathological processes that take place after infection with L. aethiopica. It was also the aim of this work to develop a diagnostic system to directly identify parasites in potentially infected tissue with or without the presence of clinical signs of disease. Several laboratory animals including 12 strains of mice from a spectrum of genetic backgrounds, two strains of hamsters and other experimental animals such as rabbits, guinea pigs, gerbils and cotton rats were tested. All animals were inoculated with late stationary phase culture promastigotes of L. aethiopica and examined over a period of 18 months. Evaluation of the outcome of infection was monitored by observing the production of lesions and isolating cultivable parasites from sites of inoculation. The fate and distribution of parasites was investigated using culture, histology and PCR to identify L. aethiopica-specific DNA. The results revealed differences in susceptibility to L. aethiopica infection within the strains of mice used. Cultivable parasites were isolated from the footpads of BALB/c, BALB/c nude and CBA nude mice up to 16 days post infection whereas SCID mice retained live parasites for a longer period of 20 days. CBA and C57/BL mice cleared cultivable parasites from sites of inoculation within 8 and 12 days respectively. Overall, BALB/c mice retained detectable parasite DNA for the longest period (550 days) post-infection in the footpads, draining lymph nodes and the liver. Among mice with intact genetic backgrounds, only BALB/c mice showed cellular in vivo responses at the site of parasite inoculation that peaked at day 13. Hamsters were found to be the only animals that produced clinical lesions (46%). In addition, live parasites were isolated from the site of inoculation (55%) and from the draining lymph nodes (50%) after 390 days post-infection. L aethiopica kinetoplast DNA was detected up to 470 days post infection in 82% of noses and 91% of draining lymph nodes. Hamsters exhibited the ability to retain cultivable parasites late in the course of infection regardless of the presence or absence of lesions. These results indicate that hamsters are potentially suitable models to study the fate and distribution of L. aethiopica. The detection of parasites using the amplified DNA signatures was found to be superior to conventional methods such as histology and culture in that it was sensitive, specific and simple. The PCR method that was developed during this work involved extraction of nucleic acids from cultured parasites, tissues and smears, amplifying L. aethiopica-specific DNA using a universal primer for a conserved Leishmania kinetoplast sequence together with an L. aethiopicaspecific oligonucleotide. The amplified fragments were 864bp in length and were initially characterised by restriction digest analysis using Hae HI restriction enzyme digestion as a result of which several patterns were recognised. To confirm this diversity in k-DNA sequences, amplified fragments were cloned using pCRII® TA cloning kit and the recombinant clones were digested to confirm the patterns observed initially and were sequenced. The sequences that were generated were analysed using the BLAST alignment facility of the National Centre for Biotechnology Information (NCBI). All fragments were Leishmania kinetoplast in origin but were found to possess varying percentages of similarity ranging from 50% to 100% among themselves. Analysis of these results suggested that L. aethiopica isolates that originated from individual patients possess genetic differences in their k-DNA sequences. In summary, the results generated from this work indicate that the hamster is a suitable model for studying cutaneous leishmaniasis caused by L. aethiopica and that both hamsters and mice can be used to study parasite distribution in experimental leishmaniasis. It was also revealed that there are genetic differences within the kinetoplast of L. aethiopica isolates. These genetic differences within L. aethiopica DNA could be exploited to better understand the diversity within the causative agent of Ethiopian Cutaneous Leishmaniasis.