STUDIES ON THE IMMUNIZATION OF SAVANNA BROWN GOATS AGAINST HEARTWATER USING THE GROUND UP TICK SUPERNATANT (GUIS)
STUDIES ON THE IMMUNIZATION OF SAVANNA BROWN GOATS AGAINST HEARTWATER USING THE GROUND UP TICK SUPERNATANT (GUIS)
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Date
1999-08
Authors
IDRIS, ALAO LAWAL
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Abstract
The fatal side effect of heartwater vaccine derived from
Amblyomrna hebraeum infected Cowdria ruminantium has reduced its use
in Southern Africa despite its numerous advantages over the infected sheep
blood vaccine. The work reported here aims at eliminating the side effect
to enable its use within this environment. Poor technology and logistic
problems may not allow effective use of inactivated of molecular vaccine
locally. . Moreover, there is the emerging fear of antigenic diversity
among Cowdria isolates which could be worsened by the use of a sub unit
vaccine.
In preparing the crude vaccine (ground up tick supernatant).
Amblyomrna variegatum ticks were collected from resident domestic
ruminants in Zaria. Fully and partially engorged females of these ticks
were incubated at 27oC and 80% relative humidity, Giemsa stained
haemolymph smears were made, examined and found negative for
rickettsia bodies. Fully engorged ticks oviposited 7 to 10 days post
incubation while the partially engorged ones oviposited on the 9th day.
v i i i
The larvae progeny were fed using earbags, on a goal identified
as NNI infected with cryo-preserved Cowdria ruminantium (Jaji stock).
Engorged larvae were harvested from the earbags when the goat was
reacting to infection (indicated by a rise in temperature of 40°C - 41.3°C).
The lavae were expected to have been infected by feeding on the goat,
were subsequently incubated as described earlier and moulted to nymphae.
The nymphae were pre-fed on a susceptible goat which reacted 9 days
post infestation and the brain was positive for Cowdria colonies
confirming that the nymphae were infected and infective.
Crude vacciene was prepared by grinding them up in morta and
pestle with pnosphate buffered saline (PBS pH 7.4) added as diluent. Two
mls of the supernatant (vaccine) was inoculated into a parasite free
susceptible goat which had fever (temperature 4l°C) 10 days later
showing that the vaccine was potent.
Reaction-free minimum infective/dose (mid) was determined by
inoculating different levels of dilutions of the crude vaccine into 25
savanna brown goats that had been kept in arthropod-free pen. The
coneentration of each dilution was determined based on the calculated
number of nymphae per ml of the vaccine (N/ml). Two levels of
concentrated forms (2 N/ml; 3.6 N/ml) were used to determine the
threshold level. Other concentrations used were 0.025, 0.0125, 0.03125
N/DOSE. Inoculation of the concentrated forms (3.6 N/m; 7 N/ml) elicited
side reactions classified as violent. These included staggering, urination,
constant bleating, profuse salivation. The animal inoculated with 3.6 N/ml
recovered while 7 N/ml caused death of the animal. The side reactions
decreased with decreasing concentrations. Side reactions were completely
eliminated at 0.03125, 0.025 and 0.0125 N/dose. However, 0.0125 N/dose
did not give effective result as an animal which failed to react at this
concentration reacted at a higher concentration of 0.10 N/dosc. Incubation
period ranged from 0 to 14 days and were affected by the concentration of
the inoculum, being shorter at higher and longer at lower concentration.
Duration of febrile reactions were longer in younger than older animals.
There was no clinical reactions within the three weeks observation period
following homologous challenge using a uniform concentration of 0.10
N/dose.
Immunity and cross immunity studies were done using Cowdria
ruminantium isolates from Akure (Forest) and Shika (Savanna). Differential
white blood counts were done. Mice, rats and guinea pigs were inoculated.
Isolation of C. ruminantium from Akure (forest zone) was done by
collecting Amblyomma variegatum ticks from resident cattle in some farms
in Akure. The ticks were brought to Zaria and ground up using morta
and pestle with broken glass slides to facilitate grinding. Phosphate
buffered saline (P.B.S pH.7.4) was added as diluent at l.8ml/tick. The
mixture was allowed to settle on iee at room temperature and 2mls of the
supernatant was inoculated into a goat No. 5256, while the remaining
supernantanl was preserved as stabilate (CR4) alter dimethyl sulphoxide
(DMSO) was added at 1ml per 9 mls of the supernatant. The animal
showed a febrile reaction at day 13 post inoculation with maximum
febrile temperature of 40.5°C and stabilate CR7 was prepared and cryopreserved
in liquid nitrogen. The brain of the goat was heavily colonised
by Cowdria granules.
Isolate from Shika was similarly obtained from A. variegatum ticks
(collected from a sedentary herd in Shika) which were ground up as
described above and 2mls of the supernatant injected into a goat identified
as 5253 while the remaining supernantant was cryo-preserved as stabilate
(CR 6). The animal reacted 3 days post inoculation (pi) with a maximum
temperature of 40°C. The animal died and the brain was positive tor
Cowdria colonies which were scanty.
The forest isolate was used to immunize goats Nos. 5255. 271 and
NNII. Reactions to primary infection were followed by secondary
(homologous) challenges. There was no reaction to secondary infection.
None of the animals was treated. The savanna isolate was also used to
immunize animal Nos. 5253, 5254, 5257 which reacted between clays 2
to 4 clays post inoculation. Two of the animals (5252, 5257) that were
treated during pyrexia survived while those not treated died within two
weeks of infection. While none of the animals immunized with Shika
isolate reacted when cross challenged with forest isolate, one of the
animals, No. 0271, which was immunized against forest isolate died and
Cowdria colonies typical of the Shika isolate were observed in the brain.
Although no clinical reactions were observed after the heterologous
challenge.
The three isolates used in these studies were compared and found
to have differing characteristics. The forest isolate resembled closely
another savanna isolate (Jap stock) in terms of characteristic of the
colonies, length of incubatuion period and infectivity for the savanna
goats. Akcie and Jaji isolates showed extensive colonization of brain
capillaries while Shika isolate is scanty. However none of the isolates is
murinotrophic.
No definite pattern could be established as regards the percentage
circulating white blood cells in the infected animals. There is a wide
variation in % lymphocytes in Akure isolate infected animals.
Animals infected with Shika isolate showed a decrease in the
percentage of neutrophils and an increase in lymphocyte during pyrexia
although the mean pre- and post infection values did not vary much.
Monocytes remained within the normal range while a total absence of
eosinophils was observed during reactions.
The highlights of these studies are as follows
i) Eggs of fully engorged female Amblyomma variegatum are better
for preparing crude infected lick-derived Cowdria vaccine than
those from partially engorged ticks
ii) Concentrations of 0.03125 and 0.025 of A. variegatum nymphae
in 2 mls inoculum are both infective, immunizing and do not
produce side effects when inoculated intravenously
iii) An isolate from the forest zone (Akure) is immunogenic but
produces mild reaction in savanna brown goats
iv) Akure and Shika isolates are cross-protective but a goal that was
immunized against the Akure isolate reacted and died when crosschallenged
with the Shika isolate.
Description
A Dissertation
Submitted to
Ahmadu Bello University
In partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
August, 1999
Keywords
IMMUNIZATION,, SAVANNA,, BROWN GOATS,, AGAINST HEARTWATER,, TICK SUPERNATANT