Research in Veterinary Science 1991, 50, 185 189

Development of an enzyme-linked immunosorbent assay for the detection and measurement of the trypanocidal drug isometamidium chloride in cattle D. D. WHITELAW*, E. A. GAULT, P. H. HOLMES, I. A. SUTHERLAND, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, F. J. ROWELL, A. PHILLIPS, School of

Pharmaceutical and Chemical Sciences, Sunderland Polytechnic, Sunderland SR2 7EE, G. M. URQUHART, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH

An enzyme-linked immunosorbent assay (EL1SA) was developed to measure accurately levels of the trypanotidal drug isometamidium in the serum of treated cattle. The assay requires only 5 pl of test serum, is sensitive to a level of 0.5 pg ml -I and is highly specific. Cross reactivity does not occur with the two other widely used trypanoeidal drugs diminazene aceturate and homidium bromide. Serum drug levels are detectable for up to six months in cattle after a single dose of 1 mg kg -t intramuscularly, the maximum period under field conditions for which effective prophylaxis can be maintained against tsetse challenge. Application of the assay will aid the rationalisation of treatment campaigns and assist in assessing the occurrence of drug-resistant trypanosome populations.

THE use of prophylactic trypanocidal drugs, in particular isometamidium chloride (Samorin; RMB Animal Health), remains the most widely applied measure for the prevention and control of animal trypanosomiasis under field conditions (Boyt 1985). Despite this, guidelines for their use are of limited assistance in indicating either the frequency of treatments or the optimal drug dosage for a given situation, or in offering an alternative strategy to adopt in situations where a reduction in the efficacy of prophylaxis is encountered after treatment. This latter possibility, with regard to isometamidium, is now apparently occurring with increasing frequency in the field (Jones-Davies and Folkers 1966, Na'Isa 1967, Bourn and Scott 1978, Pinder and Authie 1984, Rottcher and Schillinger 1985) and may be the result of the development of drug resistance in the trypanosome population. Isometamidium (Berg 1960) has been in widespread use for 30 years and, at a dosage of 1 mg kg 1, prophylaxis of between four weeks and seven months

has been recorded (Pinder and Authie 1984, Whitelaw et al 1986, Sones et al 1988). While it has been assumed that numerous factors unrelated to drugs may influence the duration of prophylaxis (Whiteside 1962), of paramount importance must also be, first, the drug sensitivity of the trypanosome population and, secondly, the length of time for which trypanocidal levels of drug persist in the circulation of a treated animal. Methods by which the first may be evaluated rely on in vivo or in vitro screening of isolates against specific drugs, the results of which are often difficult to interpret. Analytical methods by which the second factor might be measured have hitherto not been available and, accordingly, it has been impossible to make a correlation between drug levels in a treated animal and the development of parasitaemia, particularly in situations where the emergence of drug-resistant parasites is suspected. For this reason, an enzyme-linked immunosorbent assay (ELISA) for the detection of isometamidium was developed. The criteria which an ELISA had to fulfil were: first, it had to have the necessary sensitivity of detection to measure the (assumed) low levels of drug present at the time when inoculated trypanosomes are able to establish and multiply, indicating the end of effective prophylaxis and, second, simplicity for easy adaptation to field use.

Materials and methods

Samorin Isometamidium chloride was obtained from RMB Animal Health, UK. It is stable in its powdered formulation, but rapidly loses activity in aqueous solution. Accordingly, all preparations were made up freshly for immediate use.

Preparation of conjugates *Present address: The British Council, 203 New South Head Road, PO Box 2027, Australia

Isometamidium was conjugated to two protein 185

Whitelaw, Gault, Holmes, Sutherland, Rowell, Phillips, Urquhart

186

carriers, either porcine thyroglobulin (PTG) o r ovalbumin (both Sigma). All incubations were carried out in light-proof containers. Fifty milligrams isometamidium was dissolved in 1 ml hydrochloric acid (1 + 2 dilution by volume concentrated hydrochloric acid in water) to which 1 ml sodium nitrite (8-8 mg m1-1) was added. After 10 minutes at 4°C, the isometamidium solution was added to protein (in 200 mM phosphate buffer, pH 7) to yield a final ratio of 0.2 (w/w) PTG:isometamidium. After a two hour reaction at 4°C, the conjugate was dialysed overnight against 5 per cent acetic acid and stored in small samples at 4°C. The integrity of the isometamidium moiety within the PTG-iSOmetamidium conjugate, in comparison to native compound in aqueous solution, was assessed by its fluorescence characteristic (excitation at 380 nm, emission at 610 nm: Phillips et al 1967). The procedure for preparing the ovalbumin-isometamidium conjugates was the same as for PTG above. A ratio of 0"0125 was selected as optimal (see below).

Antiserum production Three sheep were immunised w i t h PTG-iSOmetamidium (0"2 ratio) conjugate using a schedule involving four antigen injections. These were, first, 2 mg conjugate combined with Freund's complete adjuvant, followed by three doses in incomplete Freund's adjuvant, 1 rag, 500/zg, 500 t~g, respectively, inoculated subcutaneously at four week intervals. The sheep were exsanguinated at euthanasia two weeks after the final inoculation. A high titre response against isometamidium was elicited in sheep by the immunisation procedure. This was confirmed to be IgG class by immunoelectrophoretic analysis.

Purification of sheep anti-isometamidium antibody A pool of serum was made from two sheep which

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screened positive for anti-isometamidium activity and 50 ml was precipitated with 50 per cent saturated ammonium sulphate for 16 hours at 4°C. The pellet was resuspended in phosphate buffered saline (PBS) and dialysed against PBS to remove excess salt. Immunoglobulins (Igs) were purified by anion exchange chromatography on diethylamino-ethyl (DEAE) cellulose. Igs were eluted in a PBs gradient (0.01 M to 0.3 M, pH 7.2), re-equilibrated in 0.01 M PBs by dialysis, the lgG fractions identified by immunoelectrophoresis and screened by the EL1SAfor anti-isometamidium activity. Anti-PTG activity in the antibody preparation was removed by preabsorption with PTG (500 #g ml- 1 in eBs/Tween).

Cattle Male Ayrshire and Friesian calves five to 10 months old at the beginning of each experiment were used. They were treated with isometamidium (I mg kg-l) by deep intramuscular injection either in the neck or the gluteal muscle of the hind leg and routinely bled by jugular venipuncture. Sera were stored at - 20°C until analysed. ELISA

method

The wells of 96-well microtitre plates (Alpha Laboratories) were coated by passive absorption from 100 ~tl samples of the ovalbumin-isometamidium conjugate (0.0125 ratio) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9"0). After 16 hours at 4°C, the plates were washed four times with distilled water, dried at 37°C and stored, sealed, at - 2 0 ° C in the dark. The plates were washed four times with r,Bs/Tween, pH 7.4, before use. Five microlitres of test serum sample or isometamidium standard were added to wells in triplicate. The standards were prepared as 10-fold dilutions of the drug from 5 tzg to 50 fg m l - i using normal bovine serum as the diluent. One hundred microlitres of the sheep anti-isometamidium antibody at a titre of 1:5000 was then added to each well. After overnight incubation at room temperature in the dark, the wells were washed five times with distilled water, and donkey anti-sheep IgG: alkaline phosphatase conjugate was added to each well (100 tzl of a 1:1000 dilution in PBS/Tween). After incubation for a further one hour in the dark, the plate was again washed five times with distilled water and 100 tzl (2 mg m l - l) enzyme substrate (Sigma 104 [paranitrophenyl phosphate] in 1 M diethanolamine buffer, pH 9-8) was added. The reaction was stopped after 30 minutes with 3 M sodium hydroxide and the plates read at 405 nm. Duplicate analyses on test samples and triplicates on standards were conducted. Sample concentrations were extrapolated from the isometamidium standard curve (Fig 1).

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Assay specificity Two animals were each treated with 3" 5 mg kg-J diminazene aceturate (Berenil; Hoechst) and 1 . 0 m g kg -I homidium bromide (Ethidium; FBC). Serum samples were collected at regular intervals for analysis, and tested on the ELISA together with standard concentrations of both drugs in normal serum, prepared in the same way as for isometamidium chloride.

Results

Characterisation of the conjugates The fluorescence profiles of native and conjugated drug were the same (result not shown), indicating that, by this criterion, despite short-term exposure to weak acid during the diazotisation reaction for coupling, the isometamidium structure remained essentially unchanged after coupling to its protein carrier.

Optimisation of the assay conditions The IgG fraction obtained after purification from

187

the pooled sheep serum had an optimal working dilution of 1/5000 in the ELISA. Sensitivity of detection was increased by manipulation of the ratio of drug to ovalbumin in the conjugate used for coating the plates (solid phase). This was found to be optimal at a ratio of 0"0125. Under these conditions, sensitivity of detection was increased to 0" 5 pg m l - 1. Three to five hours was the minimum time required for the first incubation step, but overnight incubation (16 hours) was found to be optimal and for convenience this was subsequently adopted throughout.

Assay specificity Specific detection of isometamidium was obtained, and no significant cross reactivity with homidium bromide or diminazene aceturate was observed. Neither aqueous solutions of the drugs nor sera from animals treated with diminazene or homidium yielded a positive signal in the ELISA.

Isometamidium levels & treated animals After injection of cattle with isometamidium, levels in the sera of individual animals showed considerable variation but the trends were the same (Figs 2 to 4). An initial peak was observed four hours after injection and thereafter levels rapidly declined over the following 96 hours (Fig 2). In some cases they fell below the level of detection. Equilibration appeared to occur during the first week after injection, and thereafter a constant level of drug was maintained (Fig 3). Serum concentrations higher than the limit of detection (0.5 pg ml I) were still present five months after treatment (Fig 4).

Discussion Attempts have been made in the past to develop assay procedures by which levels of trypanocidal drugs might be measured in the serum of treated animals. The universal constraint which has applied

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188

Whitelaw, Gault, Holmes, Sutherland, Rowell, Phillips, Urquhart

to all of these has been the relative insensitivity of the spectrophotometric procedures on which drug measurement depended and the extraction methods which had to be used to separate the drugs from biological fluids before analysis. For example, diminazene aceturate was undetectable, by spectrophotometric analysis, in the bloodstream of dogs within 24 hours of therapeutic treatment with 7 mg kg -l (Raether et al 1972). Similar methods of quantitation for homidium bromide, after solvent extraction from blood, had a sensitivity of detection of 2 /~g m1-1 (Taylor 1960), but the trypanocidal activity of the drug persisted long after this level had been passed. Both Philips et al (1967) and Braide and Eghianruwa (1980) extracted isometamidium from serum and reported a detection limit of 1 Izg m l - l, but again this level did not reflect the longterm biological activity of the drug. Studies have been conducted using radioisotope-labelled drugs, but the orientation of these has been more towards residue analysis for the determination of withdrawal times for human consumption of animal products, than specific investigations into the residual biological efficacy of the compounds against the trypanosome (review, Kinabo and Bogan 1988a). More recently, high-performance liquid chromatography has been applied to isometamidium measurement, both as an analytical tool and to evaluate the pharmacokinetics of the drug in cattle (Perschke and Vollner 1985, Kinabo and Bogan 1988b). While these techniques have proved more sensitive (detection to 10 ng ml-1 serum), detection of isometamidium, in the absence of a radioligand, could not be made in cattle serum beyond seven days after administration. The advent of immunoassay systems has afforded the opportunity to overcome the shortcomings of other methods. Interference during direct measurement, by other components of the serum, is avoided and organic phase extraction, with its concomitant drawback of loss of material during recovery, is not required. Previous studies showed that a single intramuscular injection of cattle with 1 mg kg-l isometamidium conferred complete protection for at least five months, against single or repeated tsetse-transmitted challenge with the highly isometamidium-susceptible IL 1180 strain of Trypanosoma congolense (Whitelaw et al 1986). Studies with other stocks of Tcongolense and T vivax have indicated a spectrum of susceptibility to the drug (Peregrine et al 1987, Sones et al 1988). The stocks used in these studies possessed a higher degree of resistance to isometamidium, with ~L 1180 representing the extreme of sensitivity to the drug. It follows, therefore, that, with a dose of 1 mg kg 1, about five months is the maximum length of prophylaxis against infection which can confidently be expected after single dose treatment.

The principal objective of the development of the ELISA for isometamidium detection was to be able to measure the levels of drug which remained in the serum of a treated animal beyond the time at which the drug was no longer effective. Extrapolating the experimental studies mentioned above, a technique was required which could detect drug levels for over five months after treatment. The ELISA fulfils this requirement, providing a sensitivity of detection in the serum of treated cattle at levels previously not possible. Only 5 lzl of serum is required and the assay can detect isometamidium to a level of 0-5 pg m1-1. The assay is specific for isometamidium. No cross reactivity occurs with either of the closely related compounds from which the drug is derived (diminazene and homidium). This suggests that, within the isometamidium-carrier protein conjugate, there is a degree of stoichiometric modification of the drug molecule through its mode of binding to the carrier. This results in the presentation of an epitope which is expressed by neither diminazene nor homidium and it is against this epitope that the immune response of the sheep is directed for the generation of the antibody used in the ELISA.

The ELlSA functions on the basis of competition. Isometamidium bound to the solid phase competes with the drug in the test sample for binding sites on the anti-isometamidium antibody. It follows that, at high sample concentration of the drug, the anti-isometamidium antibody complexes predominantly with the drug in the liquid phase and to a much lesser extent to that on the solid phase, hence low optical densities (ODs) result when the assay is developed with chromogen. Conversely, a high OD is obtained at low sample concentration of isometamidium. It was of interest that drug profiles observed in individual animals differed. High levels of drug have been found at the injection site for over six weeks (Braide and Eghianruwa 1980, Kinabo and Bogan 1988b), attributable to the drug's strong binding to tissue macromolecules which in turn results in inflammation and coagulative necrosis at the injection site (Braide and Isoun 1980). The absorption pattern may therefore vary considerably between individuals depending on the degree of the inflammatory reactions and the absorptive capacity of the tissues injected, for example, intra- or intermuscular sites, etc/(MacDiarmid 1983). Over the first four weeks after treatment, a smooth decay in serum/., levels was not observed and, in particular, ther~ was an initial oscillation in levels, during the first seven days, before an equilibration occurred. The latter may be of considerable practical importance. It is suspected that, despite meticulous care during the deep intramuscular administration of isometamidium, the rapid initial decline to a virtually undetectable level reflects a rapid efflux of a pro-

Isometamidium measurement by ELISA

portion of the isometamidium inoculum which failed to establish successfully within the prophylactic depot. The persistent levels which were observed after the excretion of this proportion of the inoculum reflect the 'true' effective dose derived from the depot. In practical terms, therefore, an animal under field conditions may receive a significantly lower effective prophylactic dose than assumed. If retention and mobilisation of the depot is a function of the inflammatory responses arising from the inoculum, which in turn is dependent on the volume retained, effective prophylaxis may vary from animal to animal. This would manifest itself as differences in susceptibility to infection between individuals within a herd under uniform management. This reduced protection might be interpreted as evidence of the development of drug resistance, rather than being the result of suboptimal drug administration. As well as providing new opportunities in pharmacokinetic studies of isometamidium, application of the isometamidium ELISA will be of great value in the rationalisation of treatment campaigns. The test can provide a direct measurement of the amount of drug present in a treated animal and indicate any requirement for supportive treatment should such levels be deemed insufficient. It will also give evidence of the existence of drug-resistant strains in situations where parasitaemias are detected in animals whose serum isometamidium remains at a level normally considered to be trypanocidal. Acknowledgements The authors wish to thank Dr Huw Smith of the Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, for valuable discussions and advice. The financial support of the ODA(UK)and the EEC Directorate General for Science, Research and Development, under research schemes R4415 and Js 2-0031-UK, respectively, is gratefully acknowledged. References BERG, S. S. (1960) Structure of isometamidium (M and B 4180A), 7-m-amidinophenyldiazoamino-2-amino-10-ethyl-9-phenylphenanthridium chloride hydrochloride, the red isomer present in metamidium. Nature 188, 1106-1107 BOURN, D. & SCOTT, J. M. (1978) The successful use of work oxen in agricultural development of tsetse infested land in Ethiopia. Tropical Animal Health and Production 10, 191 203 BOYT, W. P. (1985) A field guide for diagnosis, treatment and prevention of African animal trypanosomiases. FAO publication M / R 1662/E/5.85/1/1000, Rome BRAIDE, V. & EGHIANRUWA, K. I. (1980) Isometamidium residues in goat tissues after parenteral administration. Research in Veterinary Science 29, 111 - 113

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BRAIDE, V. & ISOUN, T. T. (1980) Histopathology of tissue reaction to intramuscular injection of Samorin. Nigerian Veterinary Journal 9, 2 3 - 2 5 JONES-DAVIES, W. J. & FOLKERS, C. (1966) Report of International Scientific Council for Trypanosomiasis Research and Control, 11th Meeting, Nairobi, Kenya, 1966. OAU/STRC Publication 100. Publications Bureau, PMB 2359, Lagos, Nigeria. pp 3 5 - 4 0 KINABO, L. D. B. & BOGAN, J. A. (1988a) The pharmacology of isometamidium. Journal of Veterinary Pharmacology and Therapeutics 1 1 , 2 3 3 - 2 4 5 KINABO, L. D. B. & BOGAN, J. A. (1988b) Pharmacokinetic and histopathological investigations of isometamidium in cattle. Research in Veterinary Science 44, 267-269 MACDIARMID, S. C. (1983) The absorption of drugs from subcutaneous and intramuscular injection sites. Veterinary Bulletin 53, 9 - 2 3 NA'ISA, B. K. (1967) Follow up of a survey on the prevalence of homidium resistant strains of trypanosomes in cattle in northern Nigeria and drug cross-resistance tests on the strains with Samorin and Berenil. Bulletin of Epizootic Diseases of Africa 15, 231-241 PEREGRINE, A. S., MOLOO, S. K. & WH1TELAW, D. D. (1987) Therapeutic and prophylactic activity of isometamidium chloride in Boran cattle against Trypanosoma vivax transmitted by Glossina morsitans morsitans. Research in Veterinary Science 43, 268-270 PERSCHKE, H. & VOLLNER, L. (1985) Determination of the trypanocidal drugs homidium, isometamidium and quinapyramine in bovine serum or plasma using H P L C . Acta Tropica 42, 209-216 PHILIPS, F. S., STERNBERG, S., CRONIN, A., SODERGREN, S. A. & V1DAL, P. M. (1967) Physiologic disposition and intracellular localisation of isometamidium. Cancer Research 27, 333-349 PINDER, M. & AUTHIE, E. (1984) The appearance of isometamidium resistant Trypanosoma congolense in West Africa. Acta Tropica 4 1 , 2 4 7 - 2 5 2 RAETHER, W., HAIDU, P., SEIDENATH, H. & DAMM, D. (1972) Pharmacokinetische und chemoprophylaktische Untersuchungen mit Berenil an Wistar-Ratten (Trypanosoma rhodesiense). ZeitschriJ?ffir Tropenmedizin und Parasitologie 23, 418-427 ROTTCHER, D. & SCHILLINGER, D. (1985) Multiple drugresistance in Trypanosoma vivax in the Tana River District of Kenya. Veterinary Record 117,557-558 SONES, K. R., NJOGU, A. R. & HOLMES, P. H. (1988) Assessment of sensitivity of Trypanosoma congolense to isometamidium chloride: a comparison of tests using cattle and mice. Acta Tropica 45, 153-164 TAYLOR, A. E. R. (1960) The absorption of prothidium by Trypanosoma rhodesiense. British Journal of Pharmacology 15, 230-234 W H I T E L A W , D. D., BELL, I. R., HOLMES, P. H., MOLOO, S. K., HIRUMI, H., U R Q U H A R T , G. M. & MURRAY, M. (1986) Isometamidium chloride prophylaxis against Trypanosoma congolense challenge and the development of immune responses in Boran cattle. Veterinary Record 1 1 8 , 7 2 2 - 7 2 6 WHITESIDE, E. F. (1962) Interactions between drugs, trypanosomes and cattle in the field. In Drugs, Parasites and Hosts. Eds L. G. Goodwin and R. M. Nimmo-Smith. London, Churchill. pp 116-151

Received March 12, 1990 Accepted September 21, 1990