PAPER SESSION IX: CHEMOTHERAPY AND PUBLIC HEALTH

CHAIRPERSON: Dr. F.W. Muli

PAPER 33

Characterization of Protective Antigens from Midguts of the Tick, Amblyomma variegatum

J.K. Kinyua¹ E.O. Osir², D.O. Ogoyi¹ an E.K. Nguu¹

¹Department of Biochemistry, University of Nairobi, P. O. BOX 30197, Nairobi, Kenya, E-mail: ketri@net2000ke.com

²International Centre for Insect Physiology and Ecology (ICIPE), P. O. BOX 30772, Nairobi, Kenya, E-mail: eosir@icipe.org

Midgut membrane proteins were isolated from the tick, Amblyomma variegatum, using a non-ionic detergent, Triton X-114, resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis using polyclonal antibodies against the DET and AQ fractions, 3 proteins (M_r ~95,000, 86,000 and 27,000) and two proteins (M_r ~67,000 and 54,000) were detected in the DET and AQ fractions, respectively. Two of the DET fraction proteins Mr~95,000 and M_r~27,000 were glycosylated since they bound to the lectin, Concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were shown to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of the eggs laid by ticks fed on rabbits immunised with DET (14.29%) and AQ (33.04%) fractions. Based on the egg hatchability, the reproductive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively.

PAPER 34

STATUS OF IMMUNISATION AGAINST EAST COAST FEVER USING THE INFECTION AND

TREATMENT METHOD IN KENYA

G.R. Muraguri & R.M. Rumberia

Kenya Agricultural Research Institute, National Veterinary Research Centre, Muguga, P.O. Box 32, Kikuyu, Kenya, E-mail: tbd-muguga@africaonline.co.ke

An assessment of the current status of immunisation against East Coast fever in Kenya was carried out. Between 1985 and 1991 immunisation trials covering 2,258 cattle were conducted on institutional farms mainly in the Coastal lowlands region to assess efficacy and safety of the candidate immunising parasite stocks. Logistics and field delivery pathways were assessed between1992 and 1995 when a total of 3,532 cattle were immunised in both smallholder and medium large scale farms across the country. Between 1995 and 1999, a total of 1,135 cattle were immunised in smallholder farms on cost recovery basis. The process of field delivery of the technology was commercialised in 1998. Despite the reported benefit / cost ratios of between 1.38 and 2.07, a marginal rate of return of up to 244 and a high demand by farmers, the level of adoption is still low. Constraints hindering its wide adoption include general fear by the target immunising agents, relative high costs attributed to monitoring and management of immunisation reactors and requirement for a cold chain in the delivery process. There is need for concerted efforts by all stakeholders to evaluate the future of this technology in Kenya.

PAPER 35

TICK-BORNE ENCEPHALITIS VIRUS INFECTION AT THE TICK-HOST INTERFACE

M. Labuda¹, M. Ličková², M. Kazimírová¹, E. Elečková² & M. Slovák¹,

¹Institute of Zoology, Slovak Academy of Science, 842 06 Bratislava, Slovakia, E-mail: uzaelabu@savba.sk

²Institute of Virology, Slovak Academy of Science., 842 56 Bratislava, Slovakia

Tick-borne encephalitis (TBE) virus is one of the most important flaviviruses causing severe disease in man and it is the most important human arboviral infection in Europe with increased importance in recent years. As based on numerous field studies in various European geographic areas the main vector of TBE virus in Europe is Ixodes ricinus tick. The role of vertebrate hosts in the transmission cycle is less precisely defined. An accepted dogma identified the importance of host species based on the levels of viraemia they produced. However, this view has been challenged by observation of very efficient transmission during co-feeding of infected and uninfected ticks on the same host irrespective of the levels of viraemia detected. It was indicated that the local skin site of tick feeding was an important focus of viral replication early after the TBE virus infected tick commenced to feed. Cellular infiltration of inflamed tick feeding sites, and the migration of cells from such sites, may provide a vehicle for „nonviraemic“ virus transmission. The transmission dynamics has been successfully monitored by short feeding nymphs of Haemaphysalis inermis ticks. Using these ticks the peak of transmission was identified to coincide with a peak of virus titre in the skin tick feeding site (as early as day two of infected tick feeding) rather than with viraemia peaks. In addition, viraemia did not induce generalised infection of the skin. The results also indicated the importance of local skin infection for the infection of other host target organs such as lymph nodes and spleen. The observed mechanism allowed virus transmission also on virus immune hosts, which was identified as an important contribution to the maintenance of TBE virus in natural foci. This novel concept of co-feeding transmission is probably not exceptional, valid for only TBE virus. In contrast, there was given sufficient evidence to consider it important in the ecology of other arboviruses and in more general sense, suggestive for other tick-borne pathogens as well.

PAPER 36

IMMUNISATION OF RABBITS USING MIDGUT MEMBRANE-BOUND PROTEIN DERIVED FROM RHIPICEPHALUS APPENDICULATUS

H.L. Kutima

Jomo Kenyatta University of Agriculture and Technology (JKUAT), Department of Zoology, P.O. BOX 62000, Nairobi, Kenya

The objective of this study was to immunise rabbits with Gut Membrane-Bound Proteins (GMBP) derived from partially engorged Rhipicephalus appendiculatus female ticks and to assess whether the elicited immunity was protective against both homologous and heterologous tick instars and to isolate and identify the protective antigens. Immunised rabbits acquired resistance to challenge infestation by all instars of the three tick species. Resistance was manifested by prolonged feeding, reduction in engorgement weights, egg mass weights, moulting and percentage hatchability and increased mortality. Immunisation of rabbits with GMBP antigens generated protection and cross-protection against challenge infestation with homologous and heterologous instars, respectively. Cross-protection was more pronounced in the homologous than heterologous systems. Enzyme Linked Immunosorbent Assay (ELISA) technique detected circulating antibodies in the immune sera to GMBP from homologous and heterologous systems one week after the primary dose. Ouchterlony double immunodiffusion reactions with anti-tick GMBP sera formed 2 to 4 precipitin lines with homologous GMBP antigens and 1 to 2 precipitin line(s) with each heterologous GMBP antigens. A line of complete identity was observed when immune sera to GMBP antigens reacted with GMBP from homologous and heterologous tick species, suggesting common antigenic epitopes. Western blot analysis on GMBP of R. appendiculatus, R. evertsi and A. variegatum with sera from immunised rabbits detected protein bands specific to the homologous GMBP antigens, and revealed considerable cross-reactions in the heterologous systems. These results suggested further the presence of common antigens. The presence of cross-reacting antigens conferred cross-protection.