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
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