Dual culture method to determine the relationship of gut bacteria of sandfly (Phlebotomus argentipes) with promastigotes of Leishmania donovani.

A simple dual culture agar plating technique has been developed and evaluated for its efficiency in determining the relationship of gut bacteria of sandfly with Leishmania donovani promastigotes. There are about twenty morphologically distinct bacterial colonies have been isolated from the gut homogenate of Phlebotomus argentipes. In dual culture method, each bacterial isolate was inoculated in one half of the plate and the promastigotes of Leishmania was inculcated in the other half by streaking. After incubation, the type of association was determined based on the presence or absence of promastigotes colonies. The reliability of this method was compared with broth dilution method in 96 well plate.

Transfusion transmitted leishmaniasis: a case report and review of literature.

Leishmaniasis is caused by the infection of haemoparasite Leishmania . The disease is a major public health problem in at least 88 countries, including India. Various species of Leishmania are involved in causing this disease. In India, Leishmania donovani species causes visceral leishmaniasis or kala-azar. The parasite is mainly transmitted from infected to uninfected person through the bites of female sandfly. Rarely the parasite can transmit through placenta from mother to child, through sexual intercourse, as laboratory acquired and through blood transfusion. This paper reports a unique case of transfusion-transmitted fatal kala-azar in an Indian infant who acquired this infection within few days of his birth after receiving blood from his maternal uncle, who was asymptomatic at the time of blood donation but died due to severe kala-azar within three months. The baby started having fever and developed hepatosplenomegaly within one month of blood transfusion and in spite of repeated anti-leishmanial treatment with sodium antimony gluconate the child died at the age of 7 months. This paper details the clinico-pathological findings of this child and also reviews the literature on this aspect and its impact on transfusion medicine.

Preservation of Leishmania donovani promastigotes in blood agar slants.

Long-term cultivation of Leishmania promastigotes by weekly passage to fresh medium was reported to be disadvantageous because needs labor, risk of contamination, lowering in infectivity and virulence pattern. Cryopreservation and Lyophilization require expensive facilities which could be a burden and unaffordable to most laboratories of developing countries where the disease is endemic. These problems could be minimized by simple preservation of Leishmania donovani promastigotes in blood agar slants at 7-8 degrees C for 6-7 months. The preserved promastigotes were examined for viability up to one year at a regular interval of one month. Viable promastigotes were found and revived successfully from all the slants stored up to 7 months after that, the viability of promastigotes was found to be decreased in the slants of 8-9 month storage. No viable promastigotes were recovered from the slants stored up to 11-12 months. By this method, the promastigotes can easily be stored up to 7 months without loss of biological activity. The number of passage of promastigotes to fresh medium has been greatly reduced by this method from 30 times to 01 when compared with weekly passage in liquid medium. This simple and economical method can be recommended for short storage of Leishmania culture without loss of any activity.

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera.

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.

Successful replacement of fetal calf serum with human urine for in vitro culture of Leishmania donovani.

Leishmania donovani causes visceral leishmaniasis claiming several thousand lives every year in Indian sub-continent. The etiological agent is grown in cell free media supplemented with fetal calf serum (FCS). Urine from human beings and other mammalian species has also been reported to stimulate growth of Leishmania species No study has been carried out Leishmania donovani. Therefore, we studied the feasibility of culturing Leishmania donovani promastigotes in M199 medium supplemented with 10% human urine and compared the phenotypic and genotypic characters under supplementation with urine vis-a-vis FCS. The growth curve showed no significant difference in the promastigote counts in urine vs. FCS supplementation. The best growth was observed in cultures supplemented with the post-menopausal urine. No difference in antigenic bands and RFLP pattern was seen indicating that no alteration occurred in strain specific characters of the parasites cultured with human urine.

Is PGL-1 also present in Leishmania donovani promastigotes?

A soluble antigen complex (SAC) derived from the ruptured promastigotes of Leishmania donovani parasites (LD-SAC) was used for complement fixation test (CFT) in leprosy Cases of tuberculoid and borderline tuberculoid leprosy, post-kala azar dermal leishmaniasis (TT, BT, PKDL) and control sera gave negative CFT. Smear-positive cases of borderline (BB, BL) and lepromatous (LL) leprosy and drug-resisting cases of pulmonary tuberculosis gave positive CFT; smear-negative cases of LL leprosy sera also gave positive CFT. Sera of smear-negative inactive LL patients contained only PGL-1 and PDIM antigens for a long time after they become inactive. Therefore, the positive CFT in inactive LL makes us suspect whether PGL-1 is present in LD promastigotes.

New blood free biphasic medium with haemoglobin for cultivation of Leishmania donovani promastigotes.

A simple biphasic growth medium for cultivation of L. donovani promastigotes is described. A successful attempt has been made to replace the blood/serum from regular medium by bovine hemoglobin powder. Medium is easy to prepare within 4 hr. It is simple, reliable inexpensive and autoclavable. Ingredients used in the media preparation are readily available at low cost and components of the medium prepared in large quantities, could be stored at 4 degrees C for month without significantly altering their growth supporting potential. After an inoculation of 1 x 10(5) promastigotes/ml, it was possible to cultivate 1-3 x 10(2) promastigotes/ml from the above described media.

Antigenic differences between axenic amastigotes & promastigotes of Leishmania donovani.

The axenic amastigotes of an Indian strain of L. donovani have been generated from the promastigote form at 37 degrees C in RPMI-1640 medium, pH 6.0 +/- 0.5, supplemented with 10 per cent heat inactivated foetal calf serum and these are being maintained through serial subculturing under the same conditions. The present study was carried out to differentiate axenic amastigote from the promastigote stage on the basis of their antigenic constitution and also to look for any immunoreactive antigen(s) specific to axenic amastigotes. SDS-PAGE analysis revealed a few stage specific and some conserved antigenic fractions in both the stages. On immunoblotting with immune sera raised against the membrane fractions of the axenic amastigotes, the 200 kDa antigenic fraction of axenic amastigote was found to be highly reactive. When the immune sera raised against the membranes of both stages were checked by immunofluorescence no cross reactivity was observed at higher dilutions. These findings showed that there are some antigenic diversities as well as similarities between the two stages of L. donovani cultured in vitro. Also, the 200 kD fraction of axenic amastigote appeared to be an immunodominant antigen specific to that stage.

A simple medium without blood modified for successful isolation & cultivation of LD bodies.

A simple growth medium for primary isolation and subsequent cultivation of Leishmania donovani promastigotes without using whole blood is described. This medium is modified from Aljeboori's biphasic medium (used originally only for cultivation), containing only beef extract, peptone, sodium chloride, bactoagar and foetal calf serum (FCS). We have modified the medium by adding glucose and ascertaining the pH in the solid phase and by drastically reducing (91%) FCS in the liquid phase. The medium helps in isolation of L. donovani promastigotes from kala-azar patients, in addition to luxurious growth of parasites. The medium is simple, reliable, reproducible and convenient, with minimal interference in using the parasitic cells for immunological, molecular and isoenzyme studies.

A simple method for cryopreservation of Leishmania donovani promastigotes.

Cryopreservation of promastigotes of L. donovani in NNN and Tobie's media was attempted. The media containing the promastigotes were kept directly at -80 degrees C. The cryopreserved media were examined for living promastigotes after 9, 12, 15, 20, 23, 26, 29, 32, 42, 56, 65 and 126 days after the culture tubes were brought directly from -80 degrees C to room temperature and examined after 30 min. All tubes showed living promastigotes which were used for further growth with no apparent morphological changes in subsequent subcultures. Viability was optimum. This method of short term cryopreservation is simple, reliable and reproducible for cryopreservation of culture adapted parasites and also important because different strains can be stocked without using any other chemical, or special equipment and liquid nitrogen chamber, for further use in immunological or other purposes.

Development of Leishmania donovani in Phlebotomus argentipes & Ph. papatasi fed on kala-azar patients in Bihar.

A total of 258 laboratory bred Ph. argentipes was fed on untreated parasitologically confirmed kala-azar patients. Successful development of parasites was noted in 0.54 per cent Ph. argentipes fed during the day and 5.33 per cent fed during the night. However, none of the 245 laboratory bred Ph. papatasi fed on the same patients, was found positive for successful development of L. donovani in the foregut.

Sex-influenced population kinetics of Leishmania donovani in hamsters.

Susceptibility of animals to infections depends upon various factors including sex of the host which plays a pivotal role. The intake of L. donovani was investigated in male and female hamsters as also in gonadectomized and hormone (sex) treated animals. Male hamsters developed more parasites (55/100 cell nuclei) than their female counterparts (22/100 cell nuclei). The hamsters receiving testosterone (250 micrograms/animal for 7 days) exogenously (im) had enhanced parasitic count (1.1-fold in male and 1.5-fold in females with respect to their respective controls). Administration of estradiol (3 micrograms/animal for 3 days) suppressed the infection in males by 2.5-fold and in female by 1.94-fold. Castration lowered the parasite 'in take' while ovarectomy promoted infection. In these (gonadectomized) animals the administration of testosterone in males restored parasite load while the estradiol therapy in females suppressed the infection. The results suggest a definite modulatory role of sex hormone, in the susceptibility of hamsters to L. donovani infection.

Growth study of a nonpathogenic strain of Leishmania donovani with different nutrients.

The studies on growth pattern of a nonpathogenic Leishmania donovani, strain UR6, in different media showed that it can be regularly cultivated amd maintained in modified Ray's Medium (Agar) and three other liquid media, namely DME Medium, Medium 199 and RPMI-1640 which are manufactured in India. The well known N.N.N. Medium provided quantitatively poorer growth in comparison to these medium. Measurement of L. donovani cell concentration by optical density in a spectrophotometer has been worked out for expressing immunochemical observations on antigens in terms of cells per mililitre.

Impact of seasonal variation on Leishmania donovani infection in hamsters.

The impact of seasonal variation on the course of L. donovani infection in hamsters was investigated. Though the animals were maintained in controlled climatic conditions (25 degrees C +/- 2), parasites exhibited seasonal rhythm. During summer (April-July) when the atmospheric temperature ranged from 20.5 degrees C to 41.8 degrees C, the parasite load from an inoculum of 1 x 10(7) amastigotes/animal was found to be less than 1 to 9 per 100 cell nuclei (based on spleen biopsy) on day 25-35 post infection. An escalation in count was observed from August onwards, which reached the peak (approximately 30/100 cell nuclei) in February-March (temp. range 11.3 degrees C to 31.4 degrees C). The multiplication rate monitored 15 days after the initial assessment also showed a similar pattern. The secondary organs examined showed no parasites. The study revealed that despite the non-involvement of the vector in experimental infection in hamster, the parasites retained its periodic character as in man, corresponding to cyclicity of vector.