We have assessed the phylogenetic status of the Leishmania genome project Friedlin reference strain by MLEE and multiprimer RAPD including a set of 9 stocks representative of the main Leishmania species and of the whole genetic diversity of the Leishmania genus. To our knowledge, the detailed genetic characterization of the Friedlin strain has never been published before. As previously recorded (Tibayrenc et al. 1993), MLEE and RAPD data gave congruent phylogenetic results. The Friedlin reference strain was definitely attributed to Leishmania (Leishmania) major Yakimoff et Schokhor, 1914. Five specific RAPD patterns made it possible to distinguish between the Friedlin strain and the 2 other L. (L.) major stocks included in the study. Various specific MLEE and RAPD characters permitted to distinguish between the Leishmania species included in the study. All these characters are usable to detect accidental laboratory mix-ups involving the Friedlin reference strain. In confirmation with previous studies involving a more limited set of genetic markers, the general genetic diversity of the Leishmania genus proved to be considerable. It must be made clear that only one strain cannot be considered as representative of the whole genetic variability of the genus Leishmania. In the future, it is therefore advisable to complement the results obtained in the framework of the Leishmania genome project with data from other strains that should be selected on a criterion of important genetic differences with the Friedlin strain.
There are many strategies to control leishmaniasis, but majority of them are inadequate. Killed Leishmania vaccine (KLV) has been applied for its immunogenicity in human and mouse model. Bacillus Calmette-Guerin (BCG) as adjuvant is an immunemodulator inducing humoral and cellular immune responses during zoonotic cutaneous leishmaniasis (ZCL). Both KLV and BCG have been applied for their immune responses in hosts for controlling leishmaniasis. In this study, KLV and BCG were applied to inhibit replication and visceralization of Leishmania major in BALB/c mice. Mice were injected with KLV and BCG, followed by infection with promastigotes of L. major. Six weeks after infection, a small nodule appeared, which was followed by development of a large lesion and visceralization. Effects of KLV and BCG, physiopathological changes, lesion size, delay of lesion formation, proliferation of amastigotes inside macrophages and detection of amastigotes in target organs were studied. Results showed that the KLV had anti-leishmanial activity by reducing lesion size on late infection. In KLV and BCG group, the average number of amastigotes in macrophages was lower than in other groups. Significant reductions in number of amastigotes in both spleen and lymph node were observed, indicating lower visceralization of Leishmania parasites in these target organs. No significant changes were presented in body weights, survival rates and degrees of splenomegaly in test group. It can be concluded that application of KLV and BCG had acceptable efficacy in reduction of skin lesions size and proliferation of parasites, even though a few side-effects were observed. It is indicated that KLV/BSG may have ability to modulate host immune responses against Leishmania parasites and to reduce pathophysiology of the disease during infection.
Zoonotic cutaneous leishmaniasis (ZCL) is an expanding disease and a public health issue in Iran. In the present study, rate of natural infection of rodent populations with Leishmania was investigated in six endemic foci including 28 villages in Golestan, Esfahan, Yazd, Fars, Khuzestan and Ilam provinces. A total of 593 rodents were captured and identified as Rhombomys opimus (n = 325), Meriones libycus (n = 171), Meriones persicus (n = 27), Tatera indica (n = 37), Nesokia indica (n = 12), Rattus rattus (n = 13) and Mus musculus (n = 8). Microscopic examinations of Giemsa-stained smears showed that 108 out of 593 (18.2%) rodents were infected with Leishmania spp., whereas infection of 186 out of 593 (31.4%) rodents with Leishmania was then confirmed by ITS1-PCR. The highest rate of infection was found in R. opimus (prevalence of 35%) and M. libycus (31%). Based on Restriction Fragment Length Polymorphism (RFLP), 145 (78%) of 186 samples detected as Leishmania DNA were identified as L. major, 8 (4%) samples as L. turanica and 33 (18%) as mixed infection (L. major and L. turanica). Samples from infected rodents were inoculated subcutaneously at tail base of BALB/c mice. In 35 of them, nodules and ulcers containing amastigotes appeared at the inoculation site. The samples prepared from infected rodents were cultured in NNN medium and only two samples were positive. Rhombomys opimus, M. libycus, M. persicus, T. indica and N. indica were confirmed as reservoir hosts of ZCL in the studied regions. Leishmania major infection was usually accompanied L. turanica in naturally infected gerbils (R. opimus and M. libycus) in Golestan, Esfahan and Fars provinces.
The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L. major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host.