| dc.description.abstract | Canine ancylostomiasis is a widespread nematode
disease. It has been shown that in endemic areas,
more than 50% .of all dogsa,re,infected and over 8%
of infected puppies die, while many more suffer
growth retardation. A live larval'vaccine has been
developed but it has given minimal protection to the
vaccinated animals. Therefore a search for purified
protective antigens was deemed necessary in view of
developing a protective vaccine. This work was thus
aimed at studying the antigenic fractions of the
adult A. caninum as well as the host immune response
to the whole worm extract and various fraction
products.
Adult A. caninum were collected from the intestinal
tract of sacrificed dogs, washed thoroughly in
phosphate buffered saline (PBS) to get rid of the
faecal material and were homogenised using sterile
frozen mortar and pestle. The homogenate was suspended
in PBS, clarified by centrifugation and standardized
to 2 mg of protein per millilitre of extract. The
above extract was combined with complete Freund's
adjuvant and used to produce hyper immune sera in
dogs which had no prior exposure to ancylostoma infection,
and also in rabbits.
to enable a study of the relationshir between
the faecal egg count and immune response a large
number of sera, together with corresponding faecal
samples were collected from free roaming dogs caught
within the City of Nairobi. Also in an effort to
find out whether antibodies to A. caninum were
excreted in the intestinal tract of infected dogs,
faecal material from infected dogs was suspended in
PBS. The suspension was clarified by centrifugation
and then filtered through a 0.45 micron millipore
membrane filter and reduced to 1/100 of original
volume. Also the mucosal lining of the infected dog's
intestinal tract was ground and suspended in PBS.
The mixture was treated similarly to the faeces, with
the final filtrate being 100 times more concentrated.
Both the faecal and mucosal extracts were tested
for the presence of antibodies with the whole worm
extract.
Eight mg of PBS worm extract was passed through
Sephadex G - 200 and 6.7 ml fractions collected.
Using the elution profile, the fractions were pooled
into six composite groups. The extract was also
fractionated in both analytical and preparative isoelectric
focusing. Analytical isoelectrofocusing
was done with 1.6 mg of extract applied on an
ampholine thin layer polyacrylamide gel with a pH
range of 3.5 to 9.5. The isoelectric points of the
various fraction bands were determined by overlaying
the plate over a predetermined pH curve. Preparative
isoelectric focusing was carried out with 10 mg of
extract in a 110 ml ampholine column stabilised with
sucrose o and glycerol at 9 c. One hundred and thirty
1 ml fractions were collected and the corresponding
pH values recorded. Seven composite fractions were
prepared from the 130 (1 ml) fractions.
The whole worm extract was also fractionated on
polyacrylamide precast gel with a density gradient
of 4 to 30 per cent. After staining with amido black
the protein bands were clearly visible and were
compared to the migration profile of a standard map
of known proteins, thus making it possible to determine
the molecular weights of the' various proteins.
The gel was sliced into 12 respective slabs from
which the proteins were €xtracted for serological
tests.
The fractions thus obtained from the various
analytical systems were tested for antigenic activity
with sera raised in rabbits, dogs, as well as sera
obtained from free roaming dogs. They were also
tested-with the faecal and mucosal extracts. Double
diffusion precipitation together with various electrophoretic
tests were applied to the extracts and the
sera. The fractions were also tested for their
ability to elicit active cutaneous anaphylaxis in .the
experimental dogs and rabbits. They were also tested
for their ability to induce indirect haemagglutination
with the various se~a.
In order to determine the location of the antigenic
component in the adult ~. caninum, hyper immune
sera, raised in rabbits and dogs were fractionated
to obtain IgG fractions. The latter was tagged with
fluorescein isothiocyanate and overlaid onto the
dissected A. caninum and examined in the fluorescent
mlcroscope.
The various fractionation methods applied to
A. caninum revealed a multiplicity of antigenic
components. Sephadex G - 200 showed that the major
component of the ~. caninum extract comprised of
small molecular weight substances of less than 4.0 x
104 Daltons which did not react with the hyper immune
sera raised in rabbits or dogs. However the substances
induced active cutaneous anaphylaxis in infected and
sensitised ~ogs and rabbits. A smaller fraction of
high molecular weight (2.0 x 105 Daltons) showed
immune precipitation with rabbit sera in various
electrophoretic test systems.
In the analytical isoelectrofocusing of the
whole worm extract, proteins focused at between pH
4.0 and 6.4 with a particular concentration at pH 5.6.
This material focusing at pH 5.6 was very reactive in
both double diffusion and agar gel electrophoresis
systems when tested with rabbit sera. When fraction
III of Sephadex G - 200 (the most reactive fraction
with rabbit sera), was isoelectrofocused on the
analytical gel it had an isoelectric point (pI) of
5.6 as well. When the whole worm extract was applied
on the preparative isoelectrofocusing column a heavy
protein concentration occurred at pH 5.3, which complemented
the analytical profile. In both systems it
was shown that the material with pI range of 5.3 to
5.6 had the highest antigenic activity in the electrophoretic
system. The material was shown to have an
approximate molecular weight of 1.7 x 105 Daltons as
determined by the polyacrylamide gel electrophoresis.
This material elicited low active cutaneous activity
in immunised dogs and rabbits.
While the rabbit antisera were very reactive with
the A. caninum whrile worm extract and various fractions,
the sera of both the infected and vaccinated dogs
reacted poorly in many of the immunologic precipitation
systems, using either whole or fractionation antigens.
This was so even after the sera were concentrated by
ammonium sulphate precipitation or lyophilisation
methods. It was also shown that the faecal and mucosal
extracts did not have appreciable reactivity with the
whole or fractionated antigens in either double
diffusion precipitation or in immune electrophoresis,
although they reacted in indirect haemagglutination
test leading one tb conclude that the faecal or
mucosal extracts did not have appreciable amounts of
precipitating antibodies detectable by the test
systems used here.
The canine sera were however shown to be similar
to rabbit sera in inducing passive cutaneous anaphylaxis
in rabbits. Both types of sera were also active
in the indirect haemagglutination test using the whole
worm extract and various fractionation products.
IgG from both dog and rabbit sera showed a weak
immunofluorescent reaction on the cuticular layer of
the dissected A. caninum. The reaction was stronger
with canine IgG. Fluorescence was also observed on
the oesophageal, gut and secretory gland cavities of
the adult worm using both canine and rabbit sera.
Thus it was concluded that the active antigens in the
worm originated from both structural components as
well as secretory materials of the worm. | en |
