WO1996037178A2

WO1996037178A2 - Novel method of enhancing efficiency of anti-arthropod agents against blood-feeding ectoparasites

Info

Publication number: WO1996037178A2
Application number: PCT/KE1995/000002
Authority: WO
Inventors: Suliman Essuman; Ahmed Hassanali
Original assignee: The International Centre Of Insect Physiology And Ecology (Icipe)
Priority date: 1995-05-17
Filing date: 1995-05-17
Publication date: 1996-11-28
Also published as: WO1996037178A3; AU7349596A

Abstract

The invention discloses a novel method of making systemic anti-parasitic agents more effective through prior treatment of host animals with appropriate antigens. It involves immunization of animals with antigenic preparations derived fron the gut of the target ectoparasite followed by administration of the animal with systemic anti-parasitic agent.

Description

NOVEL METHOD OF ENHANCING EFFICIENCY OF ANTI- ARTHROPOD AGENTS AGAINST BLOOD-FEEDING ECTOPARASITES

DESCRIPTION The invention disclosed herein relates to a novel method by which systemic anti-parasitic agents are made more effective when administered on animals previously immunized with antigenic preparations derived from the gut

of the target ectoparasites.

Ectoparasites and ectoparasite-borne diseases pose a major threat to livestock production throughout the world. Until recently, control of these parasites such as ticks, tsetse, blowflies etc. has been accomplished almost entirely by the use of synthetic pesticides ( acaricides or insecticides) with all their attendant problems: pollution of environment, contamination of milk and meat products and induction of resistance in the ectoparasites. Current research the world over is directed towards developing alternative approaches to ectoparasitic control that do not suffer from the disadvantages of synthetic pesticides. These approaches include the deployment of bait technology (eg for tsetse), exploitation of natural immune resistance of host animals (e.g for ticks), development of immunological methods for augmenting natural host resistance (eg for ticks) and the use of systemic anti-parasitic agents (eg for ticks and tsetse).

Although several systemic pesticides (eg famphur, ronnell, fenthion, coumaphos, imidan and crufomate) when administered in the daily feed of livestock have provided some measure of control of ectoparasites, such as ticks parasitising cattle or horses, the need for continual administration of the pesticides has made the method impractical and expensive. Sustained-release boluses or controlled-released injections that can prolong activity of single treatments have been tried. However, the high dosages of insecticides that

had to be used have deterred their practical use. A significant recent development in this regard involve the discovery that avermectins, a series of macrocyclic lactone fermentation products of actinomycete Streptomyces avermitilis, are highly active as animal systemic acaricides and insecticides. Avermectins have high selective toxicity against specific invertebrates (such as ticks, insects and nematodes) compared to mammals and have been devoloped for use in verterinary medicine (mainly as anthelmentic), agricultural and horticultural applications (insecticides) and even in human drugs (for control of the microfilaria Onchocerca volvulus. Their widespread use against ectoparasites such as ticks has been limited largely by high cost.

The present invention is based on the demonstration that sub-lethal doses of avermectins provide protection of mammalian hosts against ticks if the host animals are previously immunized with antigenic preparations derived from the guts of the ticks.

According to the invention, there is provided a method of protecting mammalian hosts such as cattle, horses, dogs, sheep, pigs, fowl and goats etc. from ectoparasites by sequentially immunizing them with immunologically active preparations the antigenic components of which are derived from the guts of target ectoparasites, followed (after appropriate periods) by injecting with systemic anti-parasitic agents. The gut-derived antigens may be made up of unfractionated crude mixtures of proteinaceous materials, partly enriched fractions, or purified proteins, the antibodies of which have the primary function of immunologically modifying the gut wall of the target ectoparasite. The systemic anti-parasitic agents may comprise of any of the hitherto tested systemlc acaricides or insecticides, including avermectins, or their derivatives, or compounds which when taken in by ectoparasites during feeding affect their normal growth, development or reproduction.

It is to be understood that the antigens used in immunizing host animals may be extracted from the guts of ectoparasites by direct dissolution in aqueous buffers or by detergent-induced solubilization of the guts. Moreover, the said antigens may be extracted from the whole organisms and comprise a fraction of such extracts. They also may be derived from enrichment of these extracts by physico-chemical procedures. Moreover, the antigens may be used in highly purified form through isolation from the extracts by the use of biospecific adsorbants comprising immobilised polycional or monoclonal antibodies raised against the active antigens. The guts of the ectoparasites represent suitable current sources of the said antigens. The preferred antigenic fraction is one obtained by extracting homogenized guts with a buffer containing a suitable detergent (such as Triton X-100 or Triton X-114) after the homogenate had been previously washed repeatedly with the buffer to remove blood meal and soluble materials. However, it is to be understood that recombinant DNA technology may be used to produce similar antigens which are able to simulate effects of the extracted material.

The modification of the gut wall may occur as a result of interaction with antibodies resulting from any antigenic sites of the active antigen. This 75 may comprise only amino acid sequences or carbohydrate chains or combination of the two.

It is to be understood that the invention includes treatment with all compositions that may contain the said antigens prepared for the purpose stated above. These may include formulations containing dilutions, solvents,

80 adjuvants, buffer systems, immunopotentiators, etc.

The systemic anti-parasitic agents may be administered subcutaneoulsy, intraveneously or intramuscularly. It may be administered in a single dose or periodically. As in the case of the antigen, it may be administered as a simple solution in a suitable solvent or as a formulation

85 containing any suitable additive (such as surface-active agents) which either maintains the active compound in solution or which controls its diffusion into the circulatory system.

The invention is illustrated but not limited by the following examples. EXAMPLE 1

90 Adult ticks were fed on rabbits. Five days after attacment the female ticks were removed and embeded alive on wax. The guts were removed and dropped into liquid nitrogen and stored at -70C until equired. The collection of guts was suspended in 0.1 M phosphate-buffered saline (PBS), PH 7.2, containing 1mM ethylenediaminetetraaceic acid and 1 mM

95 phenylmethanesulfonyl fluoride, washed several times in the same buffer and homogenized 5 times for 30 sec each, interspersed with periods of colling. Homogenization was carried out at the setting of 6 on a Polytron homogenizer (Kinematic GmbH, Littau-Luzern, Switzerland). The homogenate was centrifuged first at 10,000 g for 30 min. The supernatant fraction was 100 removed from the pellet and centrifuged again at 100,000 g for 1 hr and the supernatant fraction separated from the second pellet. The pellet from the first centifugation was washed two times with the PBS. The 2 pellets were pooled, suspended in PBS with 0.5% Triton X-100 (Pierce Chemical Co., Rockford, Illinois), and left for 30 min at room temperature. The suspension then was 105 mildly sonicated 3 times for 30 sec each at an amplitude of 4 urn (Soniprep

150, MSE Scientific Instruments, Sussex, U.K.) interspersed with periods of cooling. The sonicate was centrifuged (100,000 g, 1 hr), and the supernatant fraction containing solubilized membrane bound proteins (SMP-Gut) was stored at -70C until required. Alternatively when fractionation of SMP-Gut into 110 peripheral and integral proteins was required, Triton X-114 was used instead.

All operations were carried out either on ice or at 4C. Protein estimates were done by the bicinchonnic acid protein assay reagent (Pierce Chemical Co.) with bovein serum albumin (fraction V, Pierce Chemical Co.) as the protein standard.

115 EXAMPLE 2

Immunization and treatment of rabbits: Rabbits in groups of 1 - 6 were immunized with SMP-Gut. Another 7 groups (1b - 6b and 7) were left unimmunized. Intramuscular and subcutaneous routes were used for immunization. The immunization schedule consisted of one initial injection 120 followed by three boosters at 2-weekly intervals. For the initial injection 0.2mg of SMP-Gut was emulsified in equal volumes of Freund's complete Adjuvant. 0.15 mg SMP-Gut emulsified in equal volumes of Freund's incomplete Adjuvant was used for the first two booster injections. An animal received 0.15 mg sample, without adjuvant, for the third booster. Ten days after the last

125 booster the rabbits in all groups were challenged with 50 adult (25 males and

25 females) R. appendiculatus. Then from day one of challenge till day 7 the animals in the various groups (1,1b; 2,2b; 3,3b; 4,4b; 5,5b and 6,6b) except group 7, were given total ivermectin doses of 21.0//g, 31.5//g, 42.0 g, 52.5//g, 105.0//g and 210.0//g per rabbit admininstered in equal amounts for 7 days

130 respectively. Group seven served as the control. The ivermectin was dissolved in a solution made up of 60% propylene glycol and 40% glycerol formal from (Sigma Chemicals, U.K). Intravenous route was used for the administration of the compound. The effect on mortality, feeding performance and fecundity of the ticks were observed. 35 The mean effects of the doses 31.5 g/animal and 42.5/yg/animal compared with the controls have been summarized in the attached table. These concentrations were chosen because they are the levels where the role of immunity was clearly observed. In all cases involving immunization there has been very drastic increase in the effect of the compound on feeding 40 performance, mortality and fecundity. The relative progeny size of the mean of

31.5//g and 42.0 g doses in the presence of immune response was 1.8 compared with that of the controls, ivermectin and immunization alone, which were 20.2 and 42.0 respectively. These results show clearly that a substantial improvement of a sublethal dose 45 of ivermectin (31.5//g/rabbit) may be achieved through prior immunization of rabbit with SMP-Gut.

Claims

WHAT IS CLAIMED

1. A method of controlling ticks which involves immunizing tick host animals (goats, sheep, cattle, horses, dogs etc) with an immunogenic

150 preparation obtained by extracting tick guts with a buffer containing a detergent (Triton X-100, Triton X-114 etc), followed by administration of the host animals with an avermectin or a mixture of avermectins.

2. A method of controlling ticks which involves immunizing tick host animals (goats, sheep, cattle, horses, dogs etc) with an immunogenic

155 preparation obtained by extracting whole ticks with a buffer containing a detergent (Triton X-100, Triton X-114 etc), followed by administration of the host animals with an avermectin or a mixture of avermectins.

3. A method of controlling ticks as described in Claims 1 and 2 in which the antigenic fraction is obtained from homogenate previously washed

160 repeatedly with a buffer solution.

4. A method of controlling ticks as described in Claims 1, 2 and 3 in which the active antigen is present in an enriched fraction or purified form obtained by suitable physico-chemical, biochemical or biotechnological procedures.

165 5. A method of controlling other ectoparasites such as tsetse, blowflies etc as described in Claims 1 , 2, 3 and 4 in which the antigen is derived from the guts of the target ectoparasites or from the whole ectoparasites.

6. A method as described in Claims 1 - 5 in which avermectin is replaced by other systemic anti-parasitic agents which may either be toxic to the target

170 ectoparasite or deliteriously affect its growth, development or reproduction.