| dc.description.abstract | Parasitological techniques employed in the diagnosis of
patent trypanosome infection often fail to detect animals with
low or cryptic infection. Yet, serological tests lack precision
and accuracy in detecting patent infection due to the presence
of antibodies in circulation for a long time. So, there arises a
need for a better test for the diagnosis of patent trypanosome
infection in the field.
In an effort to improve on the diagnosis of infection with
T. (T) •b. evenei , murine t-1cAbs were developed against antigens of
the bloodstream forms of T.(T}.b.evansi, KETRI1342 and used as
antigen detecting probes for diagnosis of patent T.(T).b.evansi
infection in experimental goats and field camels. The murine
McAbs were produced by immunizing Balb/c mice with plasma
antigen I and membrane antigen II preparation from bloodstream
forms of T.(T).b. evansi KETRI1342. Later, spleen cells· from
the immunized Balb/c mice were harvested and fused with NS-I
myelomacells. A total of 69 hybridomas secreting McAbs to the
above antigens were raised. The hybridomas were cloned and where
applicable, recloned by limiting dilution. The classes and
subclasses of McAbs secreted by hybridoma reclones were
determined by the double immunodiffusion and
immunoelectrophoresis techniques and their stability to freezing
and thawing, and salt precipitation investigated.
Twenty two hybrid reclones were selected and inoculated
intraperitoneally into pristane-primed Balb/c mice so that
antibody rich ascites could be produced from each hybrid
reclone. Mouse IgM class McAbs were purified from the antibody
rich ascites by gel filtration on Sepharose 6 column. Mouse IgGrich
fractions were obtained by ammonium sulphate precipitation.
In an indirect ELISA and/or IFAT, the McAbs showed a wide
range of cross-reaction with antigens of other mammalian African
trypanosome spp, but no cross reaction was observed with
Theileria parva, Babesia bi gemi ne , Anaplasma mer g i ne l e ,
Plasmodium falciparum or Leishmania donovani lysate antigens.
Two McAbs TE M5/17.4.6. and TE M3/12.3.6, showed high
specificity for KETRI 1342 stock but no reaction with other
trypanosome species. However, their reaction with thirteen
T. (T).b.evansi Stocks of defined serodemes did not show that
they were T(T).b.evansi specific. McAb TEAI/23.4.6. was selected
for application in the sandwich antigen - ELISA (Ag-ELISA)
studies for the detection of circulating trypanosome antigens in
serum or plasma and cerebrospinal fluid of experimental goats
infected with KETRI 1342. This McAb is a mouse IgM antibody
which cross-reacts with all the 'species of mammalian African
trypanosomes. In this experiment, group one goats in which each
goat was inoculated intravenously by needle and syringe
challenge with 2x106 trypanosomes of KETRI 1342 stock, the
sandwich Ag-ELISA technique was able to detect circulating
trypanosome antigens in plasma or serum 24 hours after the
inoculation of the trypanosomes. In the group two goats, in
which the trypanosomes were inoculated by intramuscular route,
antigens could not be detected until day 6 after inoculation. In
both groups, there was no parasitological or serological
evidence of trypanosomes in cerebrospinal fluid. In these
experiments, a high positive correlation was observed between
plasma Ag-ELISA and serum Ag-ELISA values. A low positive
correlation was observed between serum Ag-ELISA values and
parasitaemia or rectal temperature. A negative correlation was
observed between serum Ag-ELISA values and pev or total
haemolytic complement. A positive correlation was observed
between serum Ag-ELISA and antibody-ELISA (Ab-ELISA) values.
After treatment with the trypanocidal drug, Berenil, antigen
levels dropped more sharply in group one than in group two
goats. By day 12 and day 41, antigen levels had fallen to preinfection
levels in group one and two, respectively. However, by
day 56 and 44, IgG antibody levels were still very high in group
one and group two respectively. On the other hand, IgM
antibodies had fallen almost to pre-infection levels by this
period. Field studies were carried out in four camel herds to
evaluate the usefulness of McAb TEA1/23.4.6.as an antigen
detecting probe in the diagnosis of patent trypanosome infection
in camels. In an Ag-ELISA sandwich assay designed as for the
goat experiment, a very high significant difference (P<.OOOl)
was observed between plasma or serum Ag-ELISA values of infected
and non-infected camels. In the third camel herd, it was
observed in 25% of camels, that by day 14 after treatment, there
was no evidence of detectable trypanosome antigenaemia, while in
only few 'camels ant.i.genaemi.apersisted beyond day 28. On the
other hand, by day 48, anti-trypanosome antibody levels were
still very high in about 95% of the previously infected camels.
Thus McAb TEA1/23.4.6. was a very successful probe in
detecting circulating T.(T).b evansi antigens in plasma or serum
of both experimental goats and field camels. Since antigens were
detected much earlier than the antibodies, it was concluded that
Ag-ELISA technique employing McAb TEA1/23.4.6. was a superior
technique to Ab-ELISA in detecting patent infection. There was
a very high agreement between TEA1/23.4.6. Ag-ELISA and the
parasitological test (HCT and MI) used. Since it is possible to
modify the assay conditions so that Ag-ELISA technique can be
used as a field test, McAb TEAl/23.4.6. could be employed in
such a test as a diagnostic reagent in trypanosomiasis control
programmes. | en |
