CHAPTER IV

IMMUNOLOGICAL STUDIES
 59

Immunology is fundamentally a measure of the interaction between

parasite and host. In the course of many infectious diseases, antibodies
appear in the host which are found to react strongly with antigens from
species of plants, animals or microorganisms that are entirely unrelated to
the parasite responsible for the infection. The antibodies have, therefore,
the appearance of 'rogue' antibody, are considered to be non-specifically
derived and are thus termed 'heterophile' antibody. The heterophile
antigens can be defined as antigens occurring in the tissues of many
different species of animals, plants or microorganisms, that show extensive
cross-relationship (Parratt and Herbert, 1979). In kinetoplastid flagellates
such cross-relationships have also been reported. McGhee et^ al_. (1963)
used agglutination method and found 100% agglutinating cross-reaction
between Crithidia oncopelti and C. fascicuiata (Culex strain) and also
between C. lucilae and C. fascicuiata (Culex strain) but only 12-25% cross­
reaction between C. fascicuiata (Culex strain) and Crithidia sp. from
Euryophthalmus. Vitetta and Cuttman (1967) have shown that three
Crithidia fascicuiata strains and C. oncopelti are immunologically similar.
Souza et^ al_. (1974) have shown that sera from mice immunized uJith insect
trypanosomatids are able to cross-react with T. cruzi extracts.

In the present study, efforts have been made to evaluate the

immunological cross-reactivity among the three organisms namely B. indica
n.sp., H. indica n.sp. and L. donovani in the homologous and heterologous
systems using some serological tests viz. gel diffusion precipitation (GDP),
immunoelectrophoresis (1EP), indirect fluorescent antibody (IFA), enzyme-
linked-immunosorbant assay (ELISA) and electro immunotransfer blot (EITB).

4.1. MATERIALS AND METHODS

4.1.1. Preparation of antigens

For the preparation of antigen(s) each of the three cultures (72 to

96 hours old) was harvested by centrifugation at 5,000 g for 30 minutes at
4°C, then washed three times in phosphate-buffered-saline (PBS), PH 7.2.
The organisms were counted by Neubaur haemocytometer and their concen­
tration was adjusted to a known value. The organisms were then suspended
in double distilled water containing protease inhibitor phenylmethyl
 60

sulfonyl fluoride (PMSF) (Sigma) and leupeptin (Sigma) at a final concen­

tration of 2 mM and 2 Ul/ml respectively. The antigen suspensions were
freeze (-70°C) thawed (4°C) three times and then sonicated at -80 Hz
using Labsonic 2000 ultrasonicator (B. Braun, Melsungen AC, Malaysia) for
about 3 minutes (six bursts, each of a 30 seconds duration) in an ice
bath. Sonicated suspension was also examined microscopically to ensure
that there remained no intact organisms. The sonicated suspension was
centrifuged at 10,000 g for 30 minutes at 4°C in a Sorvall centrifuge using
SS34 rotor, and the clear supernatant was collected and stored at -20°C.
This clear supernatant was used as antigen. The pellet was also collected.
Protein contents of supernatant and pellet were estimated according to the
method of Lowry et_ aj_. (1951) using bovine serum albumin (BSA) (Sigma)
as a standard.

4.1.2. Production of immune serum in rabbit by injecting the antigen and

collection of antisera

Rabbit antiserum was prepared by injecting albino rabbits with

soluble antigen. The albino rabbits, weighing 1.5 to 2 kg were immunized
with B. indica n.sp., H. indica n.sp. and L. donovani soluble antigen(s).
A total of 3 mg antigenic proteins from the above parasites were
administerted to each rabbit in six doses. The first three immunizations
were given subcutaneously at 4 different sites with equal volume of
Freund's complete adjuvant (Gibco) at 7 days interval and the last three
doses were injected intravenously with antigen alone. Sera from these
animals were collected after 10 days of last immunization and were tested
for reactivity against soluble antigen by GDP test. Normal pre-immune sera
(NRS) were collected from all rabbits prior to immunization for their
subsequent use as healthy controls. All the sera were stored at -20°C and
heat inactivated at 56°C for 30 minutes prior to use.

To find out the nature of antigen, the soluble antigens were treated
with heat, trypsin and sodium metaperiodate.

4.1.3. Heat treatment

The test material was taken in physiological saline (0.154 N NaCI) in

a screw capped tube and heated in boiling water bath for 20 to 30
 61

minutes. The material was allowed to cool and tested for its antigenic
activity by GDP test.

4.1.4. Trypsin treatment

The test material was taken in 0.1 M Tris-HCI buffer (pH 7.6)
containing 0.005 M CaCI2 to give an antigen solution containing 0.05 to 0.1%
protein. Appropriate amount of freshly prepared trypsin solution (1 mg/ml)
(Sigma) was mixed to the solution to give a protein enzyme ratio of 10 1.
The mixture was incubated at 37°C with continuous stirring. After that
trypsin activity was inactivated by addition of Soyabean trypsin inhibitor
(Sigma) to an enzyme inhibitor ratio 1:1.1 (by weighing). The mixture was
dialysed for 24 hours against normal saline and tested for antigenic activity
as before.

4.1.5. Sodium metaperiodate treatment

The material to be tested was suspended to physiological saline
(0.154 N) and mixed with appropriate volume of freshly prepared sodium
metaperiodate (Sigma) solution to give a final periodate concentration of
0.05 M. The mixture was incubated for 15 hours at 4°C in the dark. The
material was dialysed for 72 hours against normal saline in dark and tested
for antigenic activity as before.

4.1.6. Adsorption experiment

Formaldehyde (2%) fixed and throughly washed (in PBS pH 7.2)
flagellates (IxlO7 cells) were suspended in 1 ml serum at room temperature
with constant agitation. The unadsorbed serum was collected by centrifuga­
tion at 5,000 g for 10 minutes. The clear serum was used in the dectection
of cross-reactivity on the surfaces of homologous and heterologous systems.

4.1.7. Determination of cross-reactivity

4.1.7.1. Ouchterlony gel diffusion precipitation (GDP) test
The test was done after Ouchterlony (1962).

4.1.7.1.1. Preparation of gel

Three ml of 1% agarose (Sigma) in PBS (pH 7.2) containing 0.02%
sodium azide was poured uniformly onto the microscope slides (2.3 cm x 7.5
cm) and kept at room temperature for solidification.
 62

4.1.7.1.2. Punching of gel

Wells were prepared as shown in Figures 53(a-c) by punching the
agarose layer with gel puncture (LKB producter AB, Sweden). The diameter
of each well was 5 mm, distances between serum wells were 3 mm and
between serum to antigen well were 2.5 mm.

4.1.7.1.3. Experimental procedure

The wells were filled with soluble antigens (200 Ug of proteins) and
the hyperimmune sera which were (40 Ul undiluted) raised against B.
indica n.sp. (HIRS-B), H. indica n.sp. (H1RS-H), L. donovani (H1RS-L)
antigens. The charged slides were incubated in moist chamber at room
temperature (25°C-30°C) for 24 hours and at 4°C upto 48 hours for
maximum precipitation. Results were noted after 24 hours of incubation.

4.1.7.1.4. Staining of the GDP slides

Gel diffusion precipitin slides were washed four times (each with 1
hour) in physiological saline (0.154 N NaCI), pressed for 30 minutes under
1 kg weight, dried in gel drier (Pharmacia LKB, Sweden) stained with
0.2% coomassie blue (KOCH-light) in 45% (v/v) methanol and 10% (v/v)
acetic acid and then destained in 25% (v/v) methanol, 5% (v/v) acetic
acid. The photographs of the destained slides were., taken.

4.1.7.2. Immunoelectrophoresis test

This test was done after Pan (1986).

4.1.7.2.1. Preparation of gel

For immunoelectrophoresis glass slides (8.4. cm x 4.7 cm) were
covered with seven ml of 1% agarose in Tris-barbital buffer (pH 8.6).

4.1.7.2.2. Punching of the gel

Three antigen wells of 3 mm diameter were punched and the distance
between each well was 9 mm apart, however, the antigen wells and the
serum troughs were 3 mm from edge to edge.
 63

4.1.7.2.3. Experimental procedure

Each antigen well was filled with 5 Ul of bromophenol blue in
sucrose and 15 pi of soluble antigen containing 200 UI of antigenic protein.
Electrophoresis was carried out at 4°C in Tris-barbital buffer {pH 8.6)
under constant current using horizontal 2117 multiphor II electrophoresis
unit {1.5 mAmp cm) until the bromophenol blue dye marker had traveled
to the anodal end of the plate (3.5 hours) (LKB - Producter AB Swden).
Cel was removed from the trough and 200 PI of undiluted antiserum was
added. The slides were then incubated in a humid chamber at room
temperature (25°C-30°C) for 24 hours and upto 48 hours at 4°C. Readings
were taken after 24 hours and 48 hours of incubation.

4.1.7.2.4. Staining of immunoelectrophoresis slides

The immunoelectrophoresis slides were stained and destained as also
done in CDP slides.

4.1.8. Determination of class specific antibodies (IgG) both in homolo­

gous and heterologous systems by indirect fluorescent antibody
(IFA) test
The IFA test was followed by Shaw and Voller (1964).

4.1.8.1. Antigen
Active cultures (72 hours old) showing exponential growth were
washed thrice with PBS (pH 7.2) and fixed at 4°C in cold methanol for 15
minutes. The fixed organisms were washed five times with PBS (pH 7.2)
and finally cell suspensions were adjusted to yield 50-60 organisms per
microscope field (400 x). Six drops of antigen suspension were smeared on
clean microscopic slides. The antigen was air dried, briefly washed in
PBS (pH 7.2) and then dried again.

4.1.8.2. Fluorescent conjugate

Antirabbit IgG (heavy and light chains) immunoglobulins tagged

optimally with fluorescein isothiocyanate (FITC, molar fluorochrom
Protein F/P ratio being 4:1) was obtained from Sigma. Preliminary studies
indicated that working dilution of 1:40 and 1:160 of this conjugated was
optimal for obtaining specific fluorescent staining in case of L. donovani,
 64

H. Sndica n.sp. and B. indica n.sp. respectively.

4.1.8.3. Performance of the test

About 20 to 30 Ml of two fold serial dilutions of test serum (pre­
immunized and immunized) was delivered to each drop of antigen and
incubated for 45 minutes in a humid chamber at 37°C. The slides were
once again throughly washed in PBS (pH 7.2) and then dried. About 20-30
Ml of the working dilution of fluorescent conjugate was added to each drop
of antigen spot and the slides were incubated as before. Finally the slides
were washed thrice in PBS (pH 7.2), dried and mounted in 90% glycerol
buffer (pH 7.2).

The slides were examined under a photomicroscope (Carl Zeiss, West

Germany) with ultra-violet assembly (air-cooled mercury vapor lamp, Osram
HBO - 200) with five excitor filters and light combinations of barrier
filters covering the entire ultraviolet range. The degree of fluorescence
was classified as reactive (4+, 3+ and 2+), weakly reactive (1 + ) and non­

reactive (-). Reactivity was indicated by the presence of bright and

intense apple green fluorescence.

4.1.9. Determination of class specific antibodies (IgG) both in homologous

and heterologous systems by enzyme linked immunosorbent assay
(ELISA)

The micro-ELISA test was similar to those described by Anthony et^

al_. (1980).

4.1.9.1. Sensitization of micro-ELISA plates

The test was performed in disposable, microtiter ELISA plates (A/S
Nunc, Denmark) containing 96 flat bottomed wells. Soluble antigens of all
the three parasites (20 Ml/ml - an amount which had been found in
preliminary titration to be an optimum antigen concentration) were diluted
with a carbonate-bicarbonate buffer (0.05 M, pH 9.6) and were added to
each well of the plate. The plates were incubated overnight at 4°C. Of
the 96 micro-ELISA plate wells, 94 received 100 Ml antigen solution.
 65

4.1.9.2. Treatment of sera

Next morning the antigen coated plates were removed from
refrigerator and washed thrice with washing solution which consists of
0. 85. saline containing 0.05% tween 20(Sigma). A 100 Pi volume of
appropriate diluted sera was added to each duplicate well and the contents
were incubated for 90 minutes at 37°C in a humid chamber.

4.1.9.3. Treatment with horse raddish peroxidase conjugated antirabbit

(IgG)
After incubation excess sera were washed as above. After final
wash, the plates were dried, and 100 Ul of optimally diluted (1:1000 in
PBS, pH 7.2 which was determined by preliminary studies) conjugate
(horse raddish peroxidase - HRP, conjugated antirabbit IgG, Fab part.
Sigma) was added to each well except the conjugate control wells which
received 100 Ul of PBS pH 7.2 and incubated as above.

4.1.9.4. Detection of enzyme activity

After incubation with HRP conjugate the plates were washed thrice
with washing solution and dried. Enzyme activity was estimated using 0-
phenylene diamine dihydrochloride (OPD) (Sigma) as the substrate. To each
well, 100 ul of freshly prepared substrate solution was added and the
reaction was allowed to proceed for exactly 15 minutes in the dark. The
reaction was stopped by the addition of 25 Ul of 5N H2S0lt to each well.
The colour intensity that developed in each well was recorded as optimal
density (O.D.) at 492 nm wave length in a micro ELISA reader (Dynatech
Laboratories, Inc. USA) taking substrate control as blank.

Different duplicate controls described below were incubated in the

study.

1. Sensitization by antigen was ommitted i.e., antigen control.

2. Treatment with antiserum was omitted, i.e., antiserum control.

3. Treatment with horse-raddish peroxidase labelled antirabbit IgG was

omitted, i.e., conjugate control.

4. Treatments with antigen, antiserum and conjugate were omitted but

only substrate was added, i.e., substrate control.
 66

4.1.10. Determination of class specific antibodies (IgG) in both homolo­

gous and heterologous system by electro immunotransfer blot
(E1TB) technique

4.1.10.1. Electrophoretic blotting

The electrophoretic blotting was done according to the method of
Towbin et_ al_. (1979). One hundred micrograms ofthree antigens (proteins)
were first subject to electrophoresis in vertical gel electrophoresis unit
(Bio-rad Laboratories, USA). The gel was 5-20% SDS-polyacrylamide linear
gradient. The gel was run at 25 mAmp constant current for 6 hours at 15°C
temperature. The SDS-PAGE separated antigens were transferred to
nitrocellulose sheets as follows:

A sheet of nitrocellulose (0.45 Urn pore size in roll form, Millipore)

was briefly wetted with Trans-blot buffer (pH 8.6) and laid on a scouring
pad (Scotch-Brite) which was supported by a stiff plastic grid
(Disposable micropipette tray, Medical Laboratory Automation, Inc. New
York).
The gel to be blotted was put on the nitrocellulose sheet and care
was taken to remove all air bubbles.

A second pad and plastic grid were added and rubber were strung
around all layers. The gel was thus firmly and evenly pressed against the
nitrocellulose sheet.

The assembly was put into an electro-trans-blot chamber with the

nitrocellulose sheet facing the cathode. The electro-trans-blot chamber was
filled with electrode buffer (pH 8.6). The EITB was done at 4°C for 5
hours at a constant current of 500 mAmp with continuous stirring.

4.1.10.2. Immunological detection of proteins on nitrocellulose sheets

4.1.10.2.1. Treatment with sera

After transfer the nitrocellulose sheets were washed three times with
Tris-saline buffer (pH 7.4). The sheets were then incubated in 3% bovine
serum albumin (BSA) solution (blocking) for overnight at 4°C in PBS (pH
7.4). In the next morning, the sheets were washed twice with Tris-saline
buffer (TBS) containing tween-20 (Sigma) and twice with TBS only, and
incubated with 1:100 dilution of antisera to different parasites in TBS-3%
BSA solution at 37°C for 90 minutes with continuous agitation. The serum
 67

dilution was earlier determined to be optimal for obtaining clear cut

distinction between a known positive (immunized rabbit) and a known
negative (preimmune normal rabbit) serum.

4.1.10.2.2. Treatment with horse raddish peroxidase conjugated antirabbit

IgC
After incubation with sera, the sheets were washed as before. The
sheets were then exposed to peroxidase - labelled antirabbit IgG (Sigma)
for 90 minutes at 37°C with continuous agitation. The optimal dilution of
conjugate was 1 :1000 in TBS - 3% BSA solution which was determined by
the preliminary studies.

4.1.10.2.3. Detection of enzyme activity

After incubation with HRP conjugate the sheets were also washed as
before. Enzyme activity was estimated using substrate 3-3' diamino
benzidine tetrahydrochloride (Sigma). The washed sheets were then
developed in substrate solution. The reaction was stopped by washing with
deionized water as soon as brown bands appeared (5-10 minutes). The
blots were then dried. Drying considerably reduced the background
staining. The blots were stored in dry and dark place.

4.2. RESULTS

4.2.1. Effect of heat, trypsin and sodium metaperiodate treatment

The soluble form of three organisms were subjected to heat and

other biochemical treatments and the immunological activity of the treated
preparations were subsequently determined. Results showed that heat and
trypsin treatments completely destroyed its immunological reactivity against
Leishmania and Bodo antisera in gel diffusion precipitation test. However,
sodium metaperiodate treatment did not affect its immunological reactivity
against either of anti Leishmania and anti Bodo sera. So soluble antigen of
L.. donovani and B. indica n.sp. are polysacharide in nature.

Anti Herpetomonas sera showed immunological reactivity in gel

diffusion precipitation test after heat and trypsin treatment, whereas after
sodium metaperiodate treatment no immunological reactivity was found. So
soluble antigen of H. indica n.sp. is protein in nature.
 y
68

4.2.2. Gel diffusion precipitation test

In reaction between HIRS-L and its homologous antigen two prominent

precipitin bands were observed. An analysis of reactions between
Leishmania antigen with HIRS-B and HIRS-H showed a precipitin band which
was weaker than the homologous reaction. The band between Leishmania
antigen and HIRS-B was found much more weaker than the HIRS-H (Figure
53a). No band was observed with any of pre-immune sera tested.

The stronger reaction was once again observed between HIRS-B with
its homologous antigen. Of the two bands observed one was found more
prominent. The reactions between Bodo antigen with Leishmania and
Herpetomonas antisera also showed a precipitin band. The precipitin band
between Bodo antigen and Leishmania antibody was found stronger than the
Herpetomonas antibody (Figure 53b).

In reaction between HIRS-H and its homologous antigen 2 main groups

of precipitin band A and B were recognised. Group A lines always
developed closer to the sera wells with their concave sides facing to
these wells. Group B lines, on the other hand, were located closer to the
antigen wells, and their concave sides were turned towards the antigen
wells. Three distinct lines in group A and group B were observed. In
heterologous system common precipitin reactions were observed only with
the group B bands between Herpetomonas antigen to Leishmania and Bodo
antisera. Once again the reaction between Herpetomonas to Leishmania
antisera was found much more pronounced than Herpetomonas antigen to Bodo
antisera (Figure 53c).

4.2.3. Immunoelectrophoresis test

When HIRS-H was used to compare the homologous system, 13 bands
appear to extend along the gel from the anode towards the cathode region.
The strongest, probably compound band (No. 1, 2 and 3) migrated towards
the cathode part of the gel. Band 6,7,8,9,10,11,12 appeared to be the
second strongest; however, they were located close to the antigen well
(Figure 54a). The reactions between HIRS-H and Bodo antigen showed two
precipitin bands which, were labeled as A, B, towards the cathode part of
the gel. When HIRS-H was tested against Leishmania 5 weak bands were
seen. The most prominent difference in this reaction was the absence of
 c

Figure 53a Cel diffusion reactions between (1) Leishmania antigen (40 Pi
containing 200 Pg of protein) and (2) HIRS-L (40 pi undiluted),
(3) HIRS-B (40 yl undiluted), (4) HIRS-H (40 y I undiluted).
(5) NRS (40 yl undiluted).
b Gel diffusion reactions between (1) Bodo antigen (40 PI contain-
200 Pg of protein) and (2) HIRS-B (40 Pi undiluted) (3) HIRS-H
(40 pi undiluted), (4) HIRS-L (40 Pi undiluted), (5) NRS (40
Pi undiluted).
c Cel diffusion reactions between (1) Herpetomonas antigen (40 Pi
containing 200 Pg of protein) and (2) HIRS-H (40 Pi undiluted),
(3) HIRS-L (40 PI undiluted), (4) HIRS-B (40 PI undiluted),
(5) NRS (40 PI undiluted)
 69

band No. 1,2,3 and 4. Therefore, the bands appeared using Leishmania
antigen were labeled as a.b.c etc.

The reactions between HIRS-L with its homologous system showed 10

bands all along the gel. Bands No. 1,2 etc. formed in this reaction were
labeled as a,b,c etc; implying their homology with those noted in the
HIRS-H and Leishmania antigen reaction. The strongest, probably compound
bands (no. c,d,e) appeared towards the cathode part of the gel. The
second strongest bands (no. g,i) were located below the antigen well.
When HIRS-L was tested against Herpetomonas and Bodo antigen, the
reaction was generally weaker. The most outstanding characteristic of the
reaction between HIRS-L and Herpetomonas antigen was the absence of
precipitin band 3 and 8, and the bands were weaker. The precipitin
patterns were more or less similar to that noted in reaction between HIRS-
H and Herpetomonas antigen. The reaction between HIRS-L and Bodo antigen
was similar to that noted in HIRS-H and Bodo antigen reaction (Figure
54b).

When HIRS-B was employed with Bodo antigen, 14 bands were

developed (Figure 54c). Here five bands were more prominent and
strongest. The bands were labelled as A, B, C and so on, implying their
homology with those noted in HIRS-L and HIRS-H with Bodo antigen. The
strongest and compound band appeared throughout the gel from anode region
to cathode region. When HIRS-B were tested against Herpetomonas and
Leishmania antigen, the reactions were generally weaker. The reaction
between HIRS-B and Herpetomonas showed 4 bands, one towards cathode end
and one above the antigen well. The other two bands were between these
two bands. The reactions between HIRS-B and Leishmania antigen also
showed 5 bands.

4.2.4. Indirect fluorescent antibody test

In IFA test different degree of positive reactions were observed both

in homologous and heterologous system which were ploted in table 7. In H.
indica n.sp. and L. donovani immunofluorescence was observed throughout
the body and flagellum when treated with homologous or heterologous sera.
In contrary, only homologous antibody showed bright fluorescence on body
and flagellum in B. indica n.sp. The heterologous antibodies i.e., L.
 i/i
!M

ir 13 i

&> -mt- > A “5

c

Figure 54a. Reactions between" (1) HIRS-H (175 pi undiluted) and (2)
Leishmania, (3) Herpetomonas and (4) Bodo antigens (16 pi
containing 120 pg of protein).

b. Reactions between (1) HIRS-B (175 pi undiluted) and (2)

Leishmania, (3) Herpetomonas and (4) Bodo antigens (16 pi
containing 120 pg of protein).
c. Reactions between (1) H1RS-L (175 pi undiluted) and (2)
Leishmania, (3) Herpetomonas, and (4) Bodo antigens (16 pi
containing 120 pg of protein).
Table 7. R esults o f in d ire c t flu o re s c e n t a n tib o d y te s t in hom ologous and heterologous s y s te m s
1

A ntigen A n tib o d y Serum d ilu tio n

CO
o
o
50 100 200 400 1600 3200 6400

4
4
4
4
4
4
4
4
4

4
4
4
-hi

L . donovani L . donovani
1
1

4-
+
4
4
4
4
4-1
4-1
L . donovani Herpetom onas
1

4-
4-
4-
+
4-
4-1
4-1
L . donovani Bodo

+
+
4-
4-
+
4-

+
4-
+
4-
4-
4-1

Herpetom onas Herpetom onas

1
1
1
1

+
4-

4-
4-
+
4-1
Herpetom onas L . donovani

1
1
1
1
!

4-
+
44
Herpetom onas Bodo
4•

4-
4-
4-
4-
4

+
+
4-
+
4-
4-

Bodo Bodo
1
1
1
1

4-
4-

+
+
4-1

Bodo L . donovani
1
1
i
!

4-

4
+\

4-1

Bodo Herpetom onas

++ - b r i 11 ia n t s ta in in g o f a ll organism s + = a ll organ isms v is ib le

± = o n ly some organism s v is ib le - = no organism s v is ib le
 70

donovani or H. indica n.sp. antisera produced diffused surface fluorescence

and no flagellar fluorescence with B. indica n.sp. antigen (Figure 55,
a-d).

4.2.5. Enzyme-1 inked-immunosorbant assay

The result of ELISA was plotted in Figure 56. To detect the cut off
point for IgC antibodies in ELISA test the positive and negative sera were
analysed at different dilutions viz., 1:20, 1:40, 1:80 and 1:160 with the
constant amount of antigens (20 jjg/ml). The 1:20 dilution was found optimal
in the sense that it completely separated out the negative serum from the
positive ones. At this dilution all positive (homologous) sera

showed mean O.D. > 0.40 values however the negative (control) sera
showed mean O.D. < 0.10 values. However, in heterologous system, L.
donovani antigen revealed greater O.D. value (mean O.D. = 0.28) with H.
indica n.sp. antibody than with B. indica n.sp. antibody (mean O.D. =
0.22). Similarly, when H. indica n.sp. antigen was reacted with L.
donovani and B. indica n.sp. antibodies, former showed greater O.D.
values (mean O.D.= 0.34) than the latter (mean O.D. = 0.18). However,
the reaction of Bodo antigen with Herpetomonas and Leishmania antibodies
showed O.D. values 0.16 and 0.30 respectively.

4.2.6. Electroimmunotransfer blot

The identification of the common antigenic polypeptides recognised by

the homologous and heterologous antibodies are shown in Figure 57. The
common immuno-reactive bands against the three antigens with homologous
and heterologous antibodies were observed at 60 kD. However, with
heterologous antibodies a variety of common and uncommon bands were also
observed. When H. indica n.sp. antibody was used as a probe against the
two heterologous antigens some common bands were observed with L.
donovani antigen (viz. 67 kD, 50 kD, 33 kD) and also with the B. indica
n.sp. antigen (34 kD). Similarly when L. donovani antibody was used in
blotting, three common immuno-reactive bands at 60 kD, 43 kD, and 23 kD
were observed with all the antigens, however, some more common bands
were with Herpetomonas antigen (68 kD, 52 kD 48 kD, 20 kD, 19 kD) than
with the Bodo antigen (29 kD). In the third experiment when Bodo
Figure 55. Indirect fluorescence cross-reactions a. Rabbit anti Herpeto-
monas or anti Bodo sera against Leishmania. b. Rabbit anti
Leishmania or anti Bodo sera against Herpetomonas, c. Rabbit
anti Bodo serum against Bodo, d. Rabbit anti Leishmania or
anti Herpetomonas sera against Bodo x 1 ,000.
 OD Qt
492 nm
Anti Bodo serum 0 5

£20 Anti Le'shmama serum 0 4

0 3
ffi Anti Herpetomonas serum
0 2
III Normal rabbit serum
0 1
0 0

Figure 56 Histogram showing the comparison of ELISA titres in HIRS-B,

H1RS-L, HIRS-H and NRS i.e., anti Bodo, anti Leishmania, anti
Herpetomonas and normal rabbit serum respectively. ABC are
■§££?£/ Leishmania and Herpetomonas antigen respectively.
a,b, c, and d represent serum dilution at 1:20, 1:40, 1:80 and
1.160 respectively. The reaction was done as described in
materials and methods. The data from 3 experiments are pooled
and presented as mean and standard deviations.
 D
T: 200 kd
4{
V>

Pj 97.4 kd
!i 4

i'H 68 kd
^ I

H 43 kd
\

i*s 25-7 kd

; 18 4 kd
14.4 kd

Figure 57. Immunoblot analysis of proteins (antigens) in Herpetomonas

indica n.sp., Leishmania donovani and Bodo indica n.sp. The
proteins were resolved by SDS-PACE, transferred to nitro­
cellulose and reacted with antisera (antiserum dilution 1:100).
Lane a, b and c contain antigen ,B. indica n.sp., H. indica
n.sp. and L. donovani respectively. A. Antigens recognized by
HIRS-H, i.e., anti HerpetOmonaS' serum. B. Antigens recognized
by HIRS-L, i.e., anti Leishmania serum. C. Antigens recognized
by HIRS-B, i.e., anti Bodo serum. Arrow indicates the common
immunogenic band in H. indica n.sp., L. donovani and B.
indica n.sp.
 71

antiserum was used the common immunogenic bands with three antigens were
observed at 60 kD, 52 kD, 28 kD and 23 kD. Three more common bands
were furthermore noticed between Leishmania antigen and Bodo antibody (84
kD, 67 kD, 48 kD). However, with Herpetomonas antigen to Bodo antibody,
only one common band (67 kD) was demonstrated.

4.3. DISCUSSION

Various serological tests have been discussed by different groups of

workers on this type of studies for their usefulness. Results of GDP test
with L. donovani antigen and antibody showed two precipitin bands which
are well correlated with the findings of Rassam and Muddhaffar (1980).
Presence of precipitin bands with L. donovani antigen to B. indica n.sp.
and H. indica n.sp. antisera definitely suggest the common antigenic
components(s) in all the parasites. Cross-reactivity of Leishmania and
Bodo antibody with only B group of Herpetomonas antigen (and not with A
band) is in well conformity with the observation of Goldman and Honigberg
(1968). These findings further suggest that Bodo and Leishmania have
common epitopes for only against one group antibody of Herpetomonas.

Gel diffusion precipitation test has been extensively used by various

groups of workers for determination of, common non-varient antigens of T.
brucei (Beat et^aL, 1984), leishmanial excreted factor products (Schnur et_
al., 1972) and antigenically active glycoproteins of Leishmania (Decker -
Jackson and Honigberg, 1978). This method has also been used to classify
certain strains of Leishmania (Abdalla, 1977; Abdalla, 1980; Raffi et al.,
1980). This technique is considered for identification of certain soluble
antigens those precipitate with their homologous or heterologous antisera
(Dwyer, 1972). A stronger precipitin reaction between Bodo antigen and
Leishmania antibody than Herpetomonas antibody in the present study
probably suggest the presence of more closely related antigenic similarity
between Bodo and Leishmania than Bodo and Herpetomonas antigen.

In immunoelectrophoresis, the major differences were the absence of

band in reactions involving heterologous systems. The precipitin band
appears to be shared by all antigens. Judging from the intensities and
presence or absence of bands, there seem to be quantitative and perhaps
 72

qualitative differences among the three parasites e.g., when Herpetomonas

antigen and antibody were allowed to react, 13 precipitin arcs were
observed, however, when the same antibody was reacted with Bodo and
Leishmania antigen only two and five precipitin arcs respectively were
observed. A similar observationwas also noticed when Leishmania antigen
was reacted with Herpetomonas antibody. This observation suggests the
presence of more closely related antigen(s) in Leishmania and Herpetomonas
parasite than in the Herpetomonas with Bodo. Using this technique, both
qualitative and quantitative differences in amastigotes and promastigotes of
Leishmania has -been described by Pan (1986).

ELISA has been used for serodiagnosis of leishmaniasis by Hommel et^

al., (1978); Anthony ert al_., (1980) & Raffi et_ aL, (1980). In the present
study 1:20 dilution of positive and negative sera was found optimal in the
sense that it completely separates out the positive serum from the
negative. At 1:20 dilution positive sera showed >0.40 mean O.D. value
however, at the same dilution pre-immune normal sera (control) gave <0.10
mean O.D. value. In heterologous system Herpetomonas antigen and Leish­
mania antigen showed greater O.D. values with Leishmania or Herpetomonas
antibody respectively than in the Herpetomonas with Bodo antigen-antibody
reaction. These results once again support that Leishmania and Herpeto­
monas have moreclosely related antigenic epitopes than with the Bodo
antigen.

It is found that soluble antigen is more immunogenic than the

particulate fraction. Furthermore, the soluble antigen maximizes the total
number of antigens examined and for these reasons the above three tests
i.e., CDP, IEP, ELISA were done with soluble antigen.

Immunofluoresence assay has been discussed for serodiagnosis of

leishmaniasis by Duxbury and Sadun, (1964); Shaw and Voller, (1964);
Bray and Lainson, (1965); Walton et_ aj_., (1972); Hedge et^ al_., (1978) and
Lopez-Brea et^ al_., (1979). Results of IFA test once again suggest the
existence of common antigens among the three organisms. Preadsorption
experiment showed drastically reduced fluorescence with homologous and
heterologous system. This fact suggests the presence of common antigenic
epitopes on the surfaces of three parasites.
 73

In EITB studies common antigenic epitope in three parasites with

homologous or heterologous antibodies were observed in the region of 60
kD. However, with the homologous and heterologous system a variety of
immuno-reactive polypeptides were demonstrated. The immuno-reactive
bands of Leishmania and Herpetomonas at 68 kD corresponds well with the
published information of Chang and Fong (1983) and Pan (1986). Presence
of more common immuno-reactive polypeptides with Herpetomonas antigen to
Leishmania antibody or Leishmania antigen to Herpetomonas antibody
suggests the closer relationship in antigenic make up of these two
parasites. However, Bodo antigen showed more common polypeptides in EITB
test with Leishmania antibody than with Herpetomonas antibody, which
implies the presence of common epitopes between Bodo and Leishmania
antigens than Bodo to Herpetomonas. The higher molecular weight
polypeptides were noticed with the Herpetomonas (~10 to 180 kD) antigen
than Leishmania (~2Q to 95.6 kD) and lastly with Bodo, as the latter
parasite showed the highest protein band at 75 kD.
These results hint at the idea that trypanosomatid (monoflageilum)
originated from biflagellate (Bodonina) ancestor as demonstrated by Brooker
(1971) and also vertebrate host form originated from insect host form
(Hoare, 1948; and Baker, 1963).