Studies on the epidemiology and chemotherapy of trypanosoma evansi infections in camels
Abstract
The field studies reported in this thesis on
epidemiology and chemotherapy of Trypanosoma evansi in
camels were conducted for a period of 18 months on two
herds of camels located at Ngurunit and Olturot, in
Marsabit District, Northern Kenya. In this area, camels
provide subsistence for a nomadic population that own them.
During the field studies, data on the disease
incidence and patterns of the disease in different age
groups was collected. Serum samples were also collected
fortnightly and stored at -20oC, to be used later for
serological analysis.
The results of these studies show that
trypanosomiasis was the most important disease complex in
the area and epidemics occurred during and soon after the
rains.
Trypanosome infections were most severe in weaner and
adult camels. The weaners developed severe clinical
disease while in the adults, the effect of the disease was
mainly recognised in the pregnant dams which aborted.
Camel calves did not show infections until they were weaned
and were over one year of age.
Attempts to control the disease by individual animal
treatment with quinapyramine sulphate (Trypacide sulphate,
May and Baker Ltd, Dagenham, UK) failed while
chemoprophylaxis using quinapyramine prosalt (Trypacide
prosalt, May and Baker Ltd, Dagenham, UK) reduced
infections to manageable level.
Arising from the field studies, a number of questions
needed to be answered. The first question was: which
species of trypanosomes were responsible for the outbreak
of trypanosomiasis in the study area?
From morphometry, the trypanosomes isolated from the
camels were of the brucei-type. Because attempts to show
presence of tsetse in this area had failed, the
trypanosomes would have been termed as ~. evansi, in line
with Hoare's (1972) criteria of distinguishing T. evans1:-
from other Trypanozoon. The tsetse map of Kenya shows that
there are tsetse in the neighbourhood of the study area and
because camels cover long distances in search of pasture
and water, they could easily have traversed the tsetse
infested areas and therefore acquired ~. brucei brucei
infections.
To investigate further the identity of the stocks
collected the following characterization methods were used:
1) Tsetse transmissibility: Each of the trypanosome
stocks was raised in irradiated rats and at peak
parasitaemia, teneral Glossina morsitans mortisans
were allowed to feed on them. The tsetse were then
maintained by daily feeding on rabbits. On day 36,
the flies were dissected and checked for trypanosome
infections in the gut, proboscis and salivary
glands. None of the 48 isolates was infective to
~.m.morsitans. In comparison, mature infections
were found in the flies that had fed on rats infected
with a defined ~.Q.bruce! reference strain (KETRI
2502) which had also been obtained from a camel. The
rabbits used to maintain flies infected with the
reference ~.h.brucei developed chancres and
parasitaemia. Thus, by the criterion of tsetse
trnsmissibility, the 48 isolates were most probably
~. eyansi.
2 Isoenzyme typing: Samples of soluble enzymes were
prepared from each of the 48 stocks and analysed by
thin layer starch gel electrophoresis for the ALAT,
ASAT, PGM, lCD, ME and peptidases I and II. Except
for PGM, none of the other enzymes revealed
consistent differences between the 48 stocks and the
reference ~.h.brucei strain. However, stocks of I.
eyansi with a pattern similar to the one seen in ~.Q.
brucei have been described before by Gibson, Marshall
and Godfrey (1980). This approach was thus not
useful for determining whether these isolates were I.
evansi or I.Q. brucei.
3) Kinetoplast DNA (kDNA) minicircle analysis:
Kinetoplast DNA minicircles were analysed using
various restriction endonucleases. Digested samples
were then analysed in agarose and polyacrylamide
gels. The digested minicircles of the 48 stocks were
homogeneous. In contrast, the T.Q. brucei reference
strains used showed a complex of non-stoicheiometric
bands irrespective of the endonuclease used.
Analysis of kDNA minicircles was thus able to show
unequivocally that the 48 stocks were T. evansi.
4) Chromosome-sized DNA analysis: Each of the
trypanosome stocks was embedded in an agarose slab
and chromosomes separated using contour-clamped
homogeneous electric field gel electrophoresis. From
the karyotype patterns, the intermediate chromosomes
and the minichromosomes were bigger in the reference
~. evansi and the 48 stocks than those of the
reference ~.R.brucei. The trypanosome stocks
ana lysed could be grouped into 9 molecular
karyotypes. Only one molecular karyotype was found
in the herd that was kept under chemoprophylaxis.
This herd had a long history of drug use and
recurring parasitaemias were often found soon after
treatment. When tested for drug sensitivity, the
trypanosomes were shown to be four times less
sensitive to quinapyramine sulphate than the
sensitive stock. It is possible that, the
trypanosomes in this herd could have been derived
from one drug resistant type.
with regard to the herd kept under individual
treatment, nine molecular karyotypes were seen. The
majority of the infections that occurred during the
second epidemic could be traced to similar karyotypes
seen at the beginning of the study. Thus, it appears
that karyotyping is a sensitive method for revealing
differences between T. evansi isolates and might be
useful in revealing multiple re-isolation of the same
trypanosome.
The next question that needed to be answered was
whether an antigen detection system would have been a
better method of detecting infections than
parasitological diagnosis. To answer this question,
over 3000 serum samples, collected fortnightly for a
period of 18 months, were analysed for the presence
of antigens and results compared with the
parasitological data. The results can be grouped into
four categories:
1) Group one comprised cases in which the presence of
trypanosomal antigens could be correlated with
parasitological diagnosis. This was observed in 52
out of 61 (85%) instances in which trypanosomes were
detected. On treatment, in most of the cases (80%),
antigens disappeared from circulation within a period
of 30 days further confirming the correlation noted
above and also indicating the potential for use of
this test to assess efficacy of treatment. In 20% of
the instances, antigens remained detectable for a
longer period of time, and in five cases even over
500 days. The reasons for 'persistence of the
antigens in the few instances where they did persist
could be due to failure of the trypanocides to effect
a complete cure either because the trypanosomes were
resistant to the drugs used or because the parasites
were located in tissues inaccessible to the drugs.
2) Group two comprised those cases in which sera from
parasitologically proven infections did not have
antigens. This was observed in 9 camels, 7 of which
were from a herd that was being examined for the
presence of trypanosomes weekly. Two possible
explanations were advanced. One was that the antigens
might have been mopped up by antibody to form immune
complexes and therefore the epitopes recognised by
the trapping antibody masked. Secondly, the
trypanosomes could have been detected too early
before sufficient parasite destruction had occurred
to give detectable levels of antigen in circulation.
Attempts to detect immune complexes failed and the
second possibility was thought more likely.
3) Group three comprised camels that were at no time
parasitaemic despite the presence of antigens. In
the herd where control of trypanosomiasis was by
prophylaxis, such antigens were noted to disappear
from circulation after trypanocide therapy,
indicating that, the presence of antigen represented
true cases of trypanosomal infections, which could
not be detected by the parasitological methods used.
That the antigens detected were indeed due to the
presence of trypanosomal infection was confirmed by
the presence of anti-trypanosomal antibodies in the
sera of antigen positive camels.
4) Group four comprised camel calves, in which no
trypanosome infections were detected during the early
period of their lives. Most of the calves also did
not have antigen during this period. The calves
appeared to have some form of protection from
trypanosome infections. Anti-trypanosome antibodies
were not found during this early period. This was
suprising for calves born in a trypanosomiasis
endemic area. What then was the source of
protection? Are there non-specific factors akin to
those that contribute to calfhood immunity against
babesiosis? These questions remain to be answered.
In six out of 40 calves, occasional antigenaemia was
detected but no corresponding antibodies were found
indicating absence of a patent infection. This observation
is intriguing in the light of the fact that cross-reaction
has not been observed between the monoclonal antibody used
in the antigen detection and other haemoparasites
(Nantulya, Musoke, Rurangirwa, Saigar, and Minja 1987).
Does this antigen represent disrupted trypanosomes that
were unable to establish infection. Clearly, further work
is needed to try and eludicate the nature of calfhood
immunity.
Citation
Doctor of Philosophy Degree in Veterinary Clinical StudiesPublisher
University of Nairobi Department of Clinical Studies