F1 cross-breed between susceptible BALB/c and resistant Swiss mice infected with Leishmania major exhibit an intermediate phenotype for lesion sizes and type 1 cytokines but show low level of total IgG antibodies.

Our current understanding of the host immune response during leishmaniases largely derives from studies performed in mice due to the intrusive techniques required to study infected human patients. Swiss mice are highly resistant to Leishmania infections in concordance with observed response in humans, while BALB/c mice indicate a high-susceptibility phenotype. Developing a cross-breed between BALB/c and Swiss mice may have important consequences on disease development, immune responses and parasite killing, as yet, response of the cross-breed to Leishmania infection is superficial. The aim of the present study was to determine disease course and immune responses in F1 cross-breed between BALB/c and Swiss albino mice infected with L. major. Three mice groups were infected intradermally with stationary-phase L. major parasites with parental strains (BALB/c and Swiss albino) as controls. Lesion development was monitored weekly for 8 weeks and monocyte chemotactic protein (MCP-1), macrophage inflammatory protein (MIP-1α), interferon-gamma (IFN-γ) and IgG antibody quantified by enzyme-linked immunosorbent assay. The data were analysed using one-way analysis of variance and Tukey-Kramer test. Results indicated F1 mice having intermediate lesion sizes, type 1 cytokine levels and footpad parasite loads as compared to the parental strains. However, the F1 mice had low levels of IgG antibodies and parasite burden in the spleen. (P < 0.05). This study concludes that the F1 cross-breed between resistant and susceptible mice may be used as a requisite model to study the role of genetics in leishmaniases and perhaps other intracellular parasites.

Vaccinations with live-attenuated Leishmania major promastigotes and challenge infection with L. major in BALB/c mice.

BACKGROUND: Currently there is no vaccine available in use against any form of leishmaniases worldwide. OBJECTIVE: To assess potential of a live-attenuated Leishmania major promastigates, for protection against a challenge infection with L. major in BALB/c mice. DESIGN: A laboratory based study. SETTING: Study was carried out at Centre for Biotechnology Research and Development, Kenya Medical Research Institute, Nairobi. RESULTS: The greatest protection against challenge with L. major was seen in mice immunised with live parasites (P < 0.001) compared to vaccinations with heat killed or soluble antigens. In general, immunised mice produced high level of antileishmanial antibodies and T cell stimulation to their respective antigens. CONCLUSIONS: Our live-attenuated parasites produced by serial sub-culture of L. major parasites 118 times showed the capacity to induce appropriate cell-mediated immune responses and protection against L. major infection in BALB/c mice. Data also suggests that these parasites do not revert to virulence when injected subcutaneously in mice.

The African Green Monkey model for cutaneous and visceral leishmaniasis.

Non-human primates are valuable models for biomedical research because of their similarities to human anatomy, immunology and physiology. Leishmaniasis, a disease caused by protozoan parasites, has a worldwide distribution and results in high morbidity and mortality. Availability of a non-human primate model of leishmaniasis would facilitate the study of different aspects of this disease and would accelerate the development of vaccines and new drugs. In this article, some interesting features of the vervet monkey (African Green monkey) model of human cutaneous and visceral leishmaniasis are discussed.

Phlebotomus guggisbergi (Diptera: Psychodidae), a vector of Leishmania tropica in Kenya.

Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.

Development of Leishmania major in Phlebotomus duboscqi and Sergentomyia schwetzi (Diptera: Psychodidae).

The extrinsic development of Leishmania major was observed in 2 man-biting sand flies, Phlebotomus duboscqi, a known vector, and Sergentomyia schwetzi, an assumed non-vector. Flies fed on a leishmanial lesion on the nose of a hamster were examined for infection at 0, 6, 12, 18, 24, 36, 48, and 60 hr and at approximately 24 hr intervals from day 3 to day 14 post-feeding. Infection rates, determined by light microscopy, were 47% (n = 258) in P. duboscqi and 5% (n = 162) in S. schwetzi. Transformation from amastigotes to "procyclic" promastigotes occurred in both species at 6-18 hr post-feeding. In P. duboscqi, the parasites multiplied rapidly and developed through as many as 10 forms, including at least 3 dividing-promastigote forms. Metacyclic promastigotes, the "infective" form, appeared at 6 days post-feeding, first in the region of the stomodeal valve, then in the pharynx, cibarium, and proboscis. In a single attempt 14 days post-feeding, a P. duboscqi transmitted L. major to a mouse by bite. In contrast, the parasites multiplied slowly in S. schwetzi, and did not develop beyond "procyclic" promastigotes. The parasites did not migrate anteriorly nor survive beyond 90 hr post-feeding, indicating that S. schwetzi is not a vector of L. major. Classical strategies for vector incrimination may be confounded by the isolation of non-infective early developmental forms of Leishmania from wild-caught non-vectors.

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) by bites of Phlebotomus duboscqi (Diptera: Psychodidae).

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) was accomplished by bites of Phlebotomus duboscqi sandflies. Three-day-old, laboratory-reared P. duboscqi were fed on leishmanial lesions on hamsters infected with L. major. The flies were re-fed on monkeys 10 d after infection. Five adult male vervet monkeys were used in concurrent transmission trials. Two of the monkeys received subcutaneous inoculations with stationary-phase promastigotes (2 x 10(6) promastigotes in 0.1 ml of medium) on the base of the tail. Putatively infected P. duboscqi were allowed to feed on the remaining 3 monkeys at sites on the base of the tail and on the right eyebrow. Challenges by sandfly bites resulted in multiple leishmanial lesions at all bite sites and, consequently, more lesion area than was produced by needle challenges. Post-feeding dissection of sandflies indicated that multiple lesions could be caused by bites of a single fly, and that probing alone, without imbibing blood, was sufficient for transmission. These first experimental transmissions of L. major to vervets by bites of P. duboscqi demonstrate that sandfly challenge is an efficient alternative to needle challenge, making available a unique Leishmania-sandfly-non-human primate model for use in vaccine development.

A simple method for maintaining, detecting and recovering virulent Leishmania donovani in hamsters.

The ability of hamsters (Mesocricetus auratus) to retain amastigotes of Leishmania donovani at cutaneous sites was examined. Following intradermal inoculation of L. donovani stationary phase culture promastigotes in fore and hind footpads, nasal area and belly skin, cultures of aspirates taken fortnightly from these sites showed that amastigotes can survive in the skin for up to 10 months without visceralizing. Hairless cutaneous sites were better at retaining L. donovani amastigotes than the hairy belly skin. L. donovani promastigotes cultivated from aspirates of sites of inoculation were highly virulent. The skin is suggested as one of the sites where viscerotropic L. donovani can remain cryptic for a long time before the infection either visceralizes or is aborted. Skins of hamsters when inoculated intradermally can serve as an easy site for maintaining, detecting and recovering virulent L. donovani without killing the hamster.

The chemotactic effect of Phlebotomus duboscqi (Diptera: Psychodidae) salivary gland lysates to murine monocytes.

The possibility that salivary gland lysates of Phlebotomus duboscqi are able to attract vertebrate monocytes was investigated. In vitro studies showed that salivary gland lysates of P. duboscqi, the vector of Leishmania major in Kenya, are chemotactic to mouse peritoneal monocytes. This attraction of monocytes by vector salivary gland lysates may form part of the mechanisms through which sandfly saliva ensures successful parasitization of macrophages in a susceptible host by Leishmania parasites.

The effect of immune sera from hamsters immunized with sandfly gut and whole body extract antigens on the fecundity and mortality of Phlebotomus duboscqi (Diptera: Psychodidae).

Phlebotomus duboscqi were fed on hamsters previously immunized with different concentrations of homogenized crude sandfly gut antigen and supernatant obtained from whole body extract. The humoral response in the rodents was quantified at different times post-immunization by the enzyme-linked immunosorbent assay. Sandflies were fed on either immunized or saline control hamsters and the effect of the blood meals on sandfly feeding, survival and fecundity was investigated. The humoral response in immunized hamsters as measured by the presence of P. duboscqi-specific IgG antibodies was significantly greater (P < 0.05) as compared to the controls. This difference was noted in sera collected on 15 and 25 days post-immunization. Sandflies fed on immunized hamsters had a significantly higher mortality (P < 0.05) and decreased egg production (P < 0.05) than those fed on unimmunized control hamsters.

IFN-gamma and delayed-type hypersensitivity are associated with cutaneous leishmaniasis in vervet monkeys following secondary rechallenge with Leishmania major.

IFN-gamma levels and delayed-type hypersensitivity (DTH) responses were evaluated in vervet monkeys, following secondary infection with Leishmania major (L. major). The animals had previously been vaccinated with leishmanial antigen, exposed to a primary infection and allowed to self-cure. Supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with either L. major antigen or Concanavalin A (Con A), were examined for the presence of IFN-gamma in a double sandwich enzyme-linked immunosorbent assay (ELISA). Significant levels of IFN-gamma were detected during active disease and following self-cure in both antigen and Con A supernatants. Higher levels of IFN-gamma were, however, present during active disease as compared with after self-cure. Positive and strong DTH responses were elicited in all experimental animals, following intradermal injection of fixed promastigotes (5 x 10(7)/animal) before rechallenge, during active infection and following self-cure. Again, strongest DTH responses were obtained during active infection as compared with the other sampling points. There was a correlation between IFN-gamma levels and DTH responses. It was concluded that IFN-gamma secretion and positive DTH responses are associated with secondary L. major infection and represent specific immunological correlates of protection in this disease model.

Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63).

Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG). However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen. Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL. When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved. We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host. The molecule requires further improvement for vaccine and/or immunodiagnosis application.

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes.

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.

Leishmania major infections in Phlebotomus duboscqi fed on murine models immunized with L. major subcellular antigens and sandfly gut antigens.

The ability of antibodies in bloodmeals of mice and hamsters immunized with Leishmania major subcellular fractions and sandfly (Phlebotomus duboscqi) gut antigens to inhibit development of L. major in its vector P. duboscqi was examined. Antibodies from animals immunized with either L. major subcellular fractions alone or sandfly gut antigen alone were not very effective in inhibiting development of L. major in the sandfly. When P. duboscqi were fed on blood from animals immunized with both parasite flagella and sandfly gut antigen, development of L. major was significantly inhibited (P<0,05). Control sandflies fed on naive animals displayed a normal pattern of parasite development to the metacyclic stage. Electron microscopy studies showed that one of the mechanisms through which antisandfly gut antibody can cause inhibition of parasite development is by lysing sandfly gut epithelium. This study has demonstrated that it is possible to reduce transmission of leishmaniosis through immunization against both the parasite and its sandfly vector.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Experimental immunization against cutaneous leishmaniasis using Leishmania major subcellular fractions alone or in combination with Phlebotomus duboscqi gut antigens.

Leishmania major-derived flagella and nuclear fractions, and a combination of flagella and sand fly gut antigens were assessed for protection against L. major infection in BALB/c mice. Mice immunized with flagella antigen developed a severe infection while nuclear fraction-immunized animals were partially protected at the onset of infection from week 1 to 4 post challenge. A combination/cock tail of flagella and sand fly gut antigens protected animals at a later stage from week 10 to 14 post-infection. Surviving cocktail-immunized animals did not ulcerate, and parasites did not metastasize to the viscera. These results provide preliminary evidence of the potential of a cock tail antigen derived from Leishmania flagella and sand fly gut in the protection against cutaneous leishmaniasis caused by L. major.

Animal reservoirs of leishmaniasis in Marigat, Baringo District, Kenya.

A total of 1128 rodents belonging to seven genera were examined for leishmanial parasites over a period of sixteen months. Parasites were isolated from 36 (12.5%) Tatera robusta, 3 (0.5%) Arvicanthis niloticus, and 2 (0.8%) Mastomys natalensis. All isolates were characterised by isoenzyme analysis using nine enzymes. The enzymes examined were: malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), mannose phosphate isomerase (MPI), malic enzyme (ME) and 6-phosphogluconate dehydrogenase (6PGD). The enzyme profiles from these isolates were compared with those from Leishmania reference strains and also with isolates of Leishmania major from man and sandfly, P. duboscqci from the same area. All the isolates except one from a Mastomys were identified as L. major. The isolate from Mastomys was trypanosome-like and remains unidentified. The results in this study show that Tatera robusta is the main reservoir of cutaneous leishmaniasis in Baringo District. None of the animals trapped were found infected with Leishmania donovani suggesting that rodents do not play a role in the transmission of visceral leishmaniasis in this area.