An Experimental Trial of Vaccination Hamsters Against Leishmania donovani Using Attenuated Subcultures

This experiment is a trail to use an attenuated Leishmania donovani promatigote as a mean for protection the hamsters against visceral leishmniasis. By subculturing the promastigotes from 1-18 times in NNN media, it was found that subcultures 16, 17 ,18 were effective in protecting the hamsters against challenge dose of the parasites.


INTRODUCTION
Leishmania species are dimorphic, obligatory intracellular parasites.During their life, these organisms cycle between the midgut of the sandflies in which they proliferate as extracellular promastigotes and the phagolysosomes of mammalian macrophages where they differentiate and grow as intracellular amastigotes ( Dey et al., 2002).
The genus Leishmania are responsible for a spectrum of human diseases termed leishmaniasis.Visceral leishmaniasis (VL) or kala-azar caused by Leishmania donovani (LD) is the most fatal form of leishmaniasis afflicting millions of people worldwide (Bhaumik and Naskar, 2009).World Health Organization (WHO) considers leishmaniasis to be one of the most serious, epidemic-prone parasitic diseases of the poor and disadvantaged.An estimated 350 million people are at risk of Leishmania infection worldwide with about 1.5 million new cases of cutaneous leishmaniasis (CL) and 500000 cases of visceral leishmaniasis (VL) reported each year (Kumari et al., 2009).The numerous relapses of Leishmania are due to the increasing incidence of drug resistance associated with the toxic anti-leishmanial drug in use (Sundar, 2001).No vaccination is available against leishmaniasis and the fast spreading drug resistance in these parasitic organisms emphasizes the need for a safe, effective vaccine (Bhaumik and Naskar, 2009).
Effective vaccines for VL require their activity to elicit cell mediated immunity (CMI) capable of activating macrophages to a microbicidal states as the protective immunity in human and experimental leishmaniasis is predominantly of Th1 type (Bhaumik and Naskar, 2009;Awasthi et al., 2004).It is desirable to have a suitable animal model for human kala-azar adequate for the realistic evaluation of vaccine safety and efficiency.Golden hamster ( Mesocricetus auratus) is the best animal model for human VL in having the same clinic-pathological features, including a relentless increase in visceral parasitic burdens, progressive cachexia, hepatosplenomegaly, pancytopnea, hypergammaglobulinemia and ultimately death (Garg and Dube, 2006;Melbey et al., 1998;Al-Harmni, 2009).
Several vaccination strategies using live, killed and defined vaccines have been attempted with particular emphasis on their efficacy against CL instead of VL.The use of live vaccines is very promising since they most closely mimic the natural course of infection and therefore may elicit similarly effective immunity after cure.However its use has been restricted or abandoned entirely due to safety concern (Al-Harmni, 1988;Molan and Al-Harmni, 1989).
Many studies have established the efficacy of inoculating live attenuated (irradiated ) parasites as a vaccination procedure against Leishmania in murine models of infection.However, this approach is currently considered not acceptable in human due to the possible persistence of virulent parasites in the inoculums that can potentially cause disease in the situations of immunodiffiency (Al-Harmni, 1988;Molan and Al-Harmni, 1989;Rivier et al., 1999).Other potentially usable live vaccines include Leishmania administred at a low dose, drug attenuated Leishmania or recombinant Leishmania cytokines.However these experimental vaccines have yet to reach the clinical trial stage .Hence no effective vaccine currently exist against any form of human leishmaniasis (Bhaumik and Naskar, 2009;Kumari et al., 2009;Al-Harmni, 1988).

Experimental animals :
Eighty seven adult male golden hamsters ( Mesocricetus auratus) each weighting 45-50 gram were obtained from Syria and maintained under conventional condition.

Cultivation of parasites:
The stock culture of Leishmania donovani was maintained successfully in the laboratory through weekly serial subcultures.
Stock cultures were maintained on NNN medium at 26C° in Mc-Contry vials containing 5.0ml of slants solid phase, overlaid with 3.0ml of Lockś solution were inoculated with 0.1ml from stock culture during their stationary phase, subcultures were carried out every 5days.The third subcultures were used to infect experimental animals.
The activity and the virulence of the parasite was maintained by cyclic passage in vivo in golden hamsters, the latter were sacrificed their liver and spleen were removed aseptically, and inoculated in a fresh NNN medium and kept at 26C°.Examination of these cultures was performed once a week for a period of one month.

Experiment 1:
Seventy two, 8-10 weeks old male golden hamsters were allocated in to 18 equal groups (4 per group).All the animals were infected intraperitoneally with 100×10 6 promastigotes of L. donovani in stationary phase from subcultures (1-18) respectively.Forty days later all animals dissected under aseptic condition to avoid contamination, the liver and spleen were cultivated in NNN medium and incubated at 26 C°, an impression Giemsa stained smears were also prepared from the spleen and liver of each sacrificed animal.Table1.

Experiment 2:
Fifteen male hamsters were divided into three equal groups.The animals of the first group injected intraperitoneally with100×10 6 promastigotes of L. donovani subculture 16.Those of the other groups were injected with the same number of promastigotes from subcultures 17 and 18. Month later, all fifteen hamsters, together with 5 previously uninfected hamsters were challenged with 20×10 6 .Forty days after challenge, all animals were dissected under aseptic condition, weights of spleen, liver and spleen length were recorded, the liver and spleen were cultivated in NNN medium, an impression Giemsaś stained smears were prepared from the spleen and liver of each animals.Table 2.

Experiment 1:
It can be seen from Table 1 that the intraperitoneally inoculated hamsters with 100×0 6 Leishmania donovani promastigotes from subcultures 1-15 were positive, this confirmed by the presence of promastigotes in the cultures inoculated with the liver and spleen of these animals in NNN media.In addition to the presence of amastigotes in impression smears prepared from the liver and the spleen.While the animals inoculated with subcultures 16,17 and 18 showed negative results, this result confirmed by the disappearance of promastigotes in the cultures inoculated with the liver and spleen of these animals in NNN media.In addition to the disappresence of amastigotes in impression smears prepared from the liver and the spleen of these animals.

Experiment 2:
Table 2 showed that there was statistically significant variation( Studentś t test p<0.05) of mean liver and spleen weight between the challenged control group inoculated with 20×10 6 promastigotes and the animals inoculated intraperitoneally with 100 × 10 6 Leishmania donovani promastigotes from subcultures 16,17 and 18.These observations were supplemented with the disappearance of promastigotes in the cultures inoculated with the liver and spleen of these animals in NNN media.In addition to this disappresence of amastigotes in impression smears prepared from the liver and the spleen.

DISCUSSION
It is to be noted that subcultures (1-15) were infective and the inoculated hamsters have no immunity against a challenge dose ,while the subcultures of 16, 17, 18 were not infective and have the ability to build up an immune response among the inoculated animals.The hamster has long been utilized as an experimental host for Leishmmania donovani.The extreme susceptibility of this species is exploited for the detection and propagation of the parasite (Farrell, 1976) Loss of infectivity and /or virulence of promastigotes of Leishmania spp.After prolonged in vitro cultivation is well documented.The promastigotes reduced their pathogenicity when remained for a long period of time and the promastigotes in stationary phase were more pathogenic than the promastigotes in logarithmic phase (Abdul-Rahman and El-Hashimi, 1980).
Several vaccine strains of L. tropica have been shown to lose their infectivity for human after 1-5 years in culture, while other strains have maintained their infectivity but show a decrease virulence.Similarly, strains of L. donovani have lost their infectivity for hamsters after prolonged cultivation while others have retained infectivity but show a decreased virulence (Nolan and Herman, 1985).The decrease in infectivity seen in prolonged culture may be a reflection of the faster growth of a noninfectious subpopulation that might predominate after prolonged cultivation in vitro as has been reported for a strain of L .tropica (Handman et al.,1983).In a study in which hamsters were infected i.c. with 10 7 promastigotes of prolonged cultured population of L.donovani 1S, no parasites could be cultured from the spleen at 16 days postinfection (Giannini et al., 1981).(Nolan and Herman, 1985) concluded that, after prolonged culture, the parasite is apparently avirulent, causing none of the classical disease symptoms in hamsters (hepato-splenomegay and death of the hamster).
Leishmaniasis are unique among parasitic diseases because a single vaccine can potentially be successful at both treating and preventing the disease (Bhaumik and Naskar, 2009).Vaccination strategies are based on our current understanding of the characteristics of an effective anti-Leishmania immune response as they have been determined from human and murine studies (Vanloubbeck and Jones, 2004).Successful immunization protocol should be able to activate the requisite signaling between macrophages and T cells.IFN γfrom Th 1 or CD 8 + T cells as part of the acquired immune response, and from IL-12 activated NK cells as part of the innate response, mediates macrophage activation, nitric oxide production and parasite killing (Bhaumik and Naskar, 2009).
Resistance to leishmaniasis has been associated with a predominant IFN-gamma production from the antigen specific CD 4 + T lymphocyte population (Th1).These cells are effective in promoting macrophage activation at the site of the lesion, and the intracellular Leishmania are killed in a nitric oxide dependent manner.In addition, activation of the CD 8 + T cells have also been shown to be important in playing a protective role after L. major infection, and for effective vaccination in experimental murine leishmaniasis (Vanloubbeck and Jones, 2004).(Bhaumik et al., 2003) mentioned that during the course of in vitro propagation, Leishmania donovani promastigotes lose their infectivity.The lose of infectivity was accompanied by modifications of surface glycoconjugates with concomitant expression of terminal galactose residues.The B1-4 galactosyltransferase enzyme of the Leishmania donovani promastigotes found to be developmentally regulated and expressed only in the attenuated parasites, the total protein extracts of the attenuated parasites (LP-CSA late passage complete soluble antigens) could stimulate macrophage respiratory burst activity and could also reduce macrophage parasite burden in vitro experiments (Bhaumik et al., 2003).(Bhaumik and Naskar, 2009) reported that the terminal B(1-4)-galactose -linked glycan induce a novel TLR signaling pathway in macrophage leading to up-regulated IFNγ production with TLR and NF-kB p56 involvement.TLR-4 has been shown to contribute to the control of parasite growth in both the innate and acquired immune response to Leishmania infection, the overall impact of this signaling pathway would be the initiation of TH1-type immune response.(Nylen et al., 2001) stated that most effective protection against human leishmaniasis has been achieved following vaccination with live promastigotes.Live parasites had the potential to dramatically induce IFNγ secreting cells often in the absence of proliferation.Dead promastigote tended to have the opposite effects, stimulating proliferation but few cells to IFNγ production.These findings suggest that IFNγ inducing factors are located in the membrane of the parasite and that in the live organisms these factors are modified or accumulated.(Rivier et al., 1999) mentioned that the capacity of gp63 ( major surface protein of Leishmania promastigotes) to protect mice against L. major infection was compared to vaccination using live parasites.In all experiments but one, protection offered by the purified antigen ( without or with adjuvant ) did not reach the level obtained using radioattenuated or virulent promatigotes .
Previous attempts at immunization against L. donovani in the hamster have yielded primary negative results (Stauber et al., 1958;Al-Qubaisi, 1980) or partial immunity (Farrell, 1976).In these studies, an intracardial or subcutaneous route of inoculation of live amastigotes or promastigotes was used as a method of attempted immunization.It is probable that the direct intravascular or subcutaneous administration of parasites does not induce or induce weak acquired immunity, especially if cellmediated immunity rather than humoral immunity plays a role in protection against visceral leishmaniasis, in addition a severe challenge may overcome a weak immunity which would be sufficient to enable the test animals to withstand a milder challenging dose.(Nylen and Gautam, 2010) concluded that vaccination with attenuated paraites may be the solution for preventing leishmanial disease.Several methods have been used to develop live attenuated Leishmania parasites including long term in vitro cultures, selection for temperature sensitivity, chemical mutagenesis and irradiation.(Kedzierski, 2010) stated that the subunit vaccines tested so far did not lead to development of long term immunity, and the whole cell killed vaccine have been performed disappointingly in the field trail.Thus, the live attenuated vaccine provide an appealing alternative.By mimicking the natural infection, live attenuated parasites can deliver a complete spectrum of antigens to the antigen presenting cells, in principle leading to a better immune response that results in a better protective outcome than observed following immunization with a subunit vaccine.

Table 1 :
The subcultures effect on the liver and spleen of hamsters infected Intraperitoneally with 100 × 10 6 Leishmania donovani promastigotes and killed (40) days post infection.

Table 2 :
The effect of subcultures on the average spleen, liver weights and spleen length of hamsters infected intraperitoneally with 100 × 10 6 Leishmania donovani promastigotes, challenge with 20×10 6 promastigotes and killed (40) days post infection.