| dc.description.abstract | The immunological control of African trypanosomiasis
is currently one of the problems occupying the minds of many
scientists. Earlier studies have shown that during infections
with African trypanosomes serum IgM levels are markedly
elevated. It has also been shown that activities like
agglutination variant serotype formation and protection in
different species of trypanosomes are influenced by IgM. But
nowhere in literature has it been shown that the IgM fraction
of immune rat sera can confer passive protection in
T. congolense infections in rats.
The objective of this study was therefore to try and
transfer passive protection against T congolense infection
in rats using the IgM fractions of immune rat sera.
Rats were inoculated intraperitoneally (i.p) with
2x105 thawed trpanosomes from the stabilates in order to
raise some immune sera. The sera were collected after the
first peak of parasitaemia that was 10 days later, before
there was much antigenic variation of the parasites. The
infected blood was left to coagulate for 4-6 hours at 4°C
and the sera were obtained by centrifugation for 10 minutes
at 4,500 r.p.m. (MSE, Superminor Centrifuge, U.K.) at room
temperature (22oC). The pooled sera were concentrated
using polyethylene gloycol carbowax 4000 and then filtered
using 0.22um filter (Millipore Corporation, Bedford,
Massachusetts) before being inactivated at 56°C for 30
minutes. Storage was done in 4ml. portions at -20°C until
needed.
Normal rat sera were prepared as the antisera. Blood
from clean rats was left to coagulate for 4-6 hours at 4°C
and the sera were obtained by centrifugation for 5 minutes
at 3,000 r.p.m. at room temperature. Concentration was done
using polythylene glycol. It was then filtered inactivated
and stored as for antiserum.
Rabbit anti whole rat serum was prepared by immunising
rabbits with normal rat sera which had been emulsified in
equal volumes with Freund's Complete Adjuvant (Difco, Detroit).
The rabbits were injected intramuscularly (i.m.) in each thigh
and at several sites along the back. Booster injections in
Freund's Incomplete AdjuvAnt prepared as for Freund's Complete
Adjuvant were given ~subcutaneously (s.c.) at several sites ,
along the back, 2 weeks later and then once every week for
next two weeks. Each rabbit received a total of about 20mg
of the antigen. Ten days after the,last injection the rabbits
were bled, sera inactivated at 56°C for 30 minutes and then
stored asceptically at -20°C.
Rabbit anti rat IgM sera were provided by Miles Laboratories
(Miles, Laboratories, slough, Bucks, U.K.).
Protein concentrations were determined on the basis of
absorbance at 280nm with OU2 Beckman Spectroprlotometer
(Beckman Instrument Company, Fullerton, California)
The antisera were tested for protective antibody
activity by mixing 106 trypanosomes from the stabilates in
phosphate buffered saline (PBS) pH 7.2 with 2ml of antisera
or control sera diluted or undiluted in the same buffer and
then incubated at 4°C for 30 minutes. Normal rat sera were
used as controls. Each of the above dose was inoculated
intraperitoneally (i.p.) into each rat. The number of deaths
were recorded in each group of tats and the results analyzed
by student's t-test.
Fractions of IgM were isolated from the pooled antisera
using ascending flow gel filtration chromatography on a
column (2.5x100cm) packed with Sephadex G200 equilibrated
against 0.05M Tris-HCL in 0.15M sodium chloride buffer pH
8.0 with 0.1% sodium azide as the preservative. Material under
the first peaK was poo Lad 'and concentrated using polythylene
glycol and dialysed against phosphate buffered saline pH 7.2
for 24 hours at 4°C before b~ing tested against rabbit antiwhole
rat serum and anti IgM in immunoelectrophoresis for
the presence of IgM.
Electrophoresis was performed in the shandon electrophoretic
chamber (Shandon-London) containing barbital buffer
pH 8.6. The slides had 1% Ionagar in the above buffer
applied to them.
The isolated IgM was used to immunise some rats which
were then challenged one hour later by intraperitoneal injection
(i.p.) of 106 trypanosomes from the original stabilates
diluted in phosphate buffered saline (PBS) pH. 7.2. Controls
received normal rat sera. The number of deaths in each group
were recorded and the results analyzed by student's t-test.
The observations made were that IgM levels are raised
during I. congolense infections in rats. The rise occurred
from around day 10 after infection. The IgM levels were still
high after the disappearance of trypansomes from the peripheral
blood after the initial peak of parasitaemia. The
immune rat sera had some protective antibody activity. The
average survival time of rats which were infected with trypanosomes
which had been incubated with immune rat sera was
17.3±4.4 (s.d.) days and the controls survived for an average
of 10.0±2.7 (s.d.) days. The difference in the survival time
of the two groups of rats was found to be statistically significant
(P<O.IT~). There was also some variation in the time
of death in rats which were infected with trypanosomes which
had been incubate 1 with -the immune rat sera as compared to
the controls.
Protective capacity was found to be associated with
IgM, and IgM fractions could be used to transfre passive
protection. Rats immunised with IgM fractions survived the
infection tor an average of 12.5±5.7 (s.d.) days while the
controls had an average survival time of 5.8±2.1. (s.c.)days.
The difference in survival time between the two groups of
rats was found to be statistically significant (P value
between 0.01 and 0.02). Similarly as for protective antibody
activity test for antiserum there was some variation
in the time of death of rats which were immunised with
IgM fractions as compared to the controls. | en |
