Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.

Study of factors affecting growth of leishmania in cultures

The rate of growth of Leishmania major and L. infantum in El-ON's culture media supplemented with human, dog, rat and avian blood was studied in vitro. Rabbit blood was used as a control. The effect of culture with these types of blood on the infectivity of both Leishmania strains to albino mice was also studied. The results showed that a good yield of both L. major and L. infantum parasites can be obtained in culture by using avian blood as substitute for rabbit blood in El-ON's medium. In addition, rat blood gave good results with L. infantum. The morphological forms of L. major and L. infantum on all types of blood supplemented media [elongated promastigotes, spindle promastigotes, promastigotes and amastigoes] were present all through the culture period with variable percentages. The infectivity of experimental animals was not affected by culture of both Leishmania strains on rabbit, human, rat, dogs as well as avian blood supplemented media

Vector-host parasite inter-relation in leishmaniasis-III-impact of blood meal from natural vertebrate hosts on the survival and the development of L. infantum and L. major in phlebotomus langeroni [Diptera: Psychodidae]

Phlebotomus langeroni collected from a leishmaniasis endemic focus at El-Agamy, Alexandria, Egypt, were found to fed on blood from man, dogs [Canis familiaris] and rats [Rattus rattus]. The effect of the kind of blood meal on the development and the life cycle of L. infantum and L. major in laboratory reared P. langeroni was investigated. A membrane feeding technique was used to infect sand flies. Gut smears of infected females were examined immediately after feeding and daily for 16 days. Nectomonads and short promastigote forms of L. infantum or L. major were detected in females fed on human, dog and rat blood at all intervals. Paramastigotes [infective stage] were present only in females fed on dog blood containing L. infantum or L. major and in those fed on rat blood containing L. major

Phosphorylation of proteins in virulent promastigotes from Leishmania major

Protein kinases are present in the plasma membrane of the human parasite Leishmania. A marked increase in enzyme activity has been detected as cultures entered into the stationary phase of growth. Since avirulent parasites can be separated from virulent forms by the peanut agglutinin (PNA), we have examined the change in the protein kinase activity of L. major during growth in vitro and the difference in phosphorylation with virulent promastigotes (PNA-) of L. major. Marked similarities were found between the phosphorylation patterns of the logarithmic and stationary phase promastigotes of L. major. On the other hand, when the phosphorylation pattern of those proteins, shared by both the metacyclic (PNA-) promastigotes and the stationary phase cells, was examined, a marked increase in both the total number of phosphoproteins and the extent of their phosphorylation was observed in PNA-. Both the increase in protein kinase activity in the stationary phase parasites and the marked changes in phosphorylation in the highly infective promastigotes, may provide a clue as to the adaptative mechanism which enable promastigotes to survive within the vertebrate host