CONCLUSION

74

The kinetoplastid flagellates offer rich and accessible material for

the analysis of diversification within different genera belonging to an
order. With a number of species available in culture, some in defined
media, they become an excellent material for analysis of nutritional
relationship of host and parasites. Ideally, a single medium should be
used for all species; so that results would be comparable, however, we
have no such known medium and the goal remains to be attained. Since
there is no single common medium that serves for all, any medium that
supports vigorous growth may be used for morphological and growth
studies. For B. indica n.sp. modified glycerol beef extract medium, for
L. donovani and II. indica n.sp. modified Tobie's medium was found
suitable for routine cultivation.

Beginning with the work of Noguchi (1926), there have been some
attempts to describe and differentiate the trypanosomatid flagellates on the
basis of nutrition, morphological and biochemical characteristics. Till now
there is no evidence to characterize bodonine flagellates on the basis of
nutritional, biochemical or serological criteria. For studying morphological
response to variations in culture condition and also for biochemical and
immunological studies, the organisms were cloned.

The temperature 25°C was found optimal for the growth of all the
organisms. The pH 6.8 was optimal for B. indica n.sp. whereas pH 7.2
was found optimal for H. indica n.sp. and L. donovani. From growth
studies it can be concluded that for the growth of B. indica n.sp.
peptone, agar and glycerol are most important factors. Glucose or other
components present in the overlay of modified Tobie's medium are
necessary for the growth of L. donovani and H. indica n.sp. The result of
the present study (i.e. growth on agar plate) indicates that freshly
prepared beef heart infusion with soft agar is superior than the other
one. The ability of the organism to divide and formation of colonies may
depend on a number of factors including quality of the blood used (Hill et^
al., 1983), the stage of the particular cell cycle (Bhattacharya and
Ghosh, 1985). Although further studies will provide more information about
the medium which will increase the sensitivity of the method and allow
one to identify the organisms even in lower number of initial count.
 75

Morphological description of any organisms is necessary for the

designation of a new species. Light microscopy reveals the shape of
kinetoplast and also the location of kinetoplast which is the basis for
distinction between the promastigote, paramastigote and opisthomastigote
forms. Opisthomastigote form is the characteristic of the genus
Herpetomonas. Only promastigote forms are found in L. donovani and in 13.
indica n.sp.

The advancement of electron microscopy has already provided

valuable information particularly on structure of the flagellum and
kinetoplast. Transmission electron microscopy reveals compact kinetoplast
nucleoid in H. indica n.sp. and L. donovani whereas in B. indica n.sp. it
is reticular. According to Vickerman and Preston (1976) the compact
kinetoplast may be evolutionary advancement. Transmission electron
microscopy of other structure reveals little differences and so
taxonomically uninformative.

Characterization by isoenzyme analysis would be valuable in

classifying all these organisms. By isoenzyme studies the flagellates of
unknown origin may be identified. The accurate identification of flagellates
in wild insects will also aid in deciding whether they are the vector
phase of a vertebrate pathogen. The ACP, ALP, C6PDH, GPl, ICDH, and ME
show different banding patterns in B. indica n.sp., H. indica n.sp. and
L. donovani. So these six enzymes may be used for taxonomic purpose of
these organisms.

There have been few attempts to use serological methods for

identification and classification of kinetoplastid flagellates. But the
serological methods are relative and are subject to cross-reaction. Both
variable and common groups of antigens may be used in classification. The
immunological tests presently have been found the most useful for
characterizing kinetoplastid flagellates. Immunoelectrophoresis and
electroimmunotransfer blot analysis are of special value as it allow a
comprehensive characterization of the set of antigens displayed by an
organism and afford these tests for monitoring the purification of antigens
and the production of specific antibodies.
 76

So the Intrinsic character may be preferred than extrinsic ones, as

the latter involve factors other than those specifically related to the
organisms and which are not able to fully standardized. Of intrinsic
characters, macromolecular characteristics, biochemical ones such as
isoenzymes and immunological ones such as determination of the variables
antigen type appear to be most promising.