Acta Tropica 73 (1999) 49–58

Extension of the prophylactic effect ofisometamidium against trypanosome infections in

cattle using a biodegradable copolymer

S. Geerts a,*, B. Diarra b, M.C. Eisler c, J. Brandt a,Y. Lemmouchi d, P. Kageruka a, R. De Deken a, M. Ndao a,

O. Diall b, E. Schacht d, D. Berkvens a, N. Speybroeck a,P.H. Holmes c

a Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgiumb Laboratoire Central Veterinaire, Bamako, Mali

c Uni6ersity of Glasgow, Veterinary School, Bearsden Road, Glasgow G61 1QH, UKd Uni6ersity of Ghent, Laboratory of Polymers, Krijgslaan 261, 9000 Ghent, Belgium

Received 19 October 1998; received in revised form 13 January 1999; accepted 13 January 1999

Abstract

Two trials were carried out in order to compare the prophylactic effect of a subcuta-neously implanted sustained release device (SRD) containing a mixture of a biodegradablecopolymer, poly(caprolactone-co-L-lactide), and isometamidium (ISMM) with that obtainedafter intramuscular injection of the drug. In a first experiment under controlled conditions,two groups of cattle were treated with 0.5 mg/kg isometamidium either as a SRD orintramuscularly (i.m.), and exposed at monthly intervals to Glossina morsitans morsitansinfected with Trypanosoma congolense. The average protection period was at least 24 monthsin the SRD treated against 5.7 months in the i.m. treated group. Using an ISMMenzyme-linked immunosorbent assay, the drug could be detected until 140 days post-treat-ment in the latter group, whereas in the former group, traces of the drug were detectableuntil 330 days after treatment. Furthermore, a field trial was carried out at the MadinaDiassa ranch in Mali involving three groups of N’Dama cattle, each containing 23 or 24

* Corresponding author. Tel.: +32-3-247-6262; fax: +32-3-216-1431.E-mail address: sgeerts@itg.be (S. Geerts)

0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

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S. Geerts et al. / Acta Tropica 73 (1999) 49–5850

animals. Two groups were treated with 1 mg/kg ISMM either as a SRD or i.m. and a thirdgroup served as untreated control. Twelve months after treatment, the cumulative infectionrates were 56.5, 87.8 and 91.6% in the SRD implanted, the i.m. treated and the controlgroups, respectively. The ISMM concentrations were slightly lower than in the laboratorytrial, but the overall pattern of drug disappearance from the sera of the SRD treated cattlewas very similar in both trials. Statistical analysis showed that the incidence of trypanosomi-asis was significantly lower in the SRD treated than in the i.m. treated group. © 1999Elsevier Science B.V. All rights reserved.

Keywords: Trypanosomiasis; Isometamidium; Sustained release device; Cattle; Prophylaxis

1. Introduction

Significant extension of the prophylactic effect of isometamidium (ISMM)against challenge with pathogenic trypanosomes has been achieved using poly(D,L-lactide) sustained release devices (SRDs). Experiments under controlled conditionsshowed that it was possible to extend the protection period of ISMM by a factorof at least 3.2 (Geerts et al., 1997). Under field conditions, the same type of SRDgave a significantly longer protection than the intramuscularly (i.m.) injected drug.Eight months after the implantation of the SRD, only 27.7% of the animals becameinfected, against 58.5% of the i.m. treated cattle (Diarra et al., 1998).

The present experiments were set up to evaluate the prophylactic effect ofanother type of polymer. Instead of poly(D,L-lactide), a copolymer was used,consisting of caprolactone (80%) and L-lactide (20%). In vitro release studies usingthis polymer showed promising results (Lemmouchi and Schacht, 1997; Lemmouchiet al., 1998). This kind of copolymer can be produced at a much cheaper price thanthe homopolymer and is therefore potentially interesting for use by small farmers inAfrica. In this paper, the results of two trials are presented, one under controlledand one under field conditions, using a poly(caprolactone-co-L-lactide) SRD con-taining ISMM.

2. Materials and methods

2.1. Sustained release de6ice

The SRD consisted of 75% (w/w) copolymer and 25% (w/w) ISMM (Samorin®,Merial). The copolymer contained 80% caprolactone and 20% L-lactide (w/w):poly(caprolactone-co-L-lactide) (80/20). The mixture of copolymer and drug washeated at 140°C and extruded as rods of 3 mm diameter, between 10 and 30 mmlength. They were coated by dipping in a copolymer/chloroform solution (10% w/v)containing 0.5% (w/w) dexamethasone. The latter was added in order to reduce thetissue reaction at the implantation site.

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2.2. Laboratory experiment

A preliminary experiment under controlled conditions was carried out in anexperimental fly-proof stable of the Institute of Tropical Medicine (ITM, Antwerp).Two groups of three adult cows each were treated with ISMM at a dose of 0.5mg/kg body weight either as an i.m. injection (Samorin®, Merial, 2% w/v solution)or as a SRD. The latter was implanted subcutaneously in the shoulder region usinga commercially available implanter (Crestar®, Intervet). The prophylactic effect ofboth drug formulations was evaluated by monthly challenging the animals withTrypanosoma congolense clone IL 1180 (Nantulya et al., 1984) as described previ-ously (Geerts et al., 1997). From 1 month post-treatment onwards, the cows wereexposed to an average of eight Glossina morsitans morsitans (line MALL) (Elsen etal., 1993), infected with T. congolense clone IL 1180. Dissection of each batch offlies after use was carried out as described by Kazadi et al. (1994) and showedinfection percentages of at least 50%.

The infectivity of the challenge infection was confirmed by exposing one controlcalf (a different one each time) to a similar number of infected flies as the drugtreated animals. Each of these challenge control animals subsequently becameparasitaemic. Blood samples were taken weekly and examined by the dark groundbuffy coat technique (Murray et al., 1977). Monthly challenge infections were givenuntil 20 months post treatment (m.p.tr.). At 21 m.p.tr., the remnants of the SRD inone of the cows were surgically removed and two further challenge infections weregiven to the three remaining cows at 22 and 24 m.p.tr.

2.3. Field trial

The field experiment was carried out at the Madina Diassa ranch in Mali, wherea heavy tsetse challenge is present (Diall et al., 1986, 1992). The experimentalprotocol was similar to that described earlier for the evaluation of the poly(D,L-lac-tide)–ISMM SRD (Diarra et al., 1998). Briefly, two herds of male and femaleN’Dama cattle were used. Each of these herds was randomly divided into threegroups. One group (n=24) was treated with ISMM at a dosage of 1 mg/kg liveweight (Samorin®, Merial; 2% w/v aqueous solution) i.m., another one (n=23)received the ISMM SRD at the same dose as a subcutaneous implant in theshoulder region, and a third group (n=24) served as untreated control group.Parasitological examination was carried out using the dark ground buffy coattechnique 2 weeks prior to treatment and every month until 12 months post-treat-ment. Two weeks before the start of the experiment, all animals were treated with7 mg/kg body weight diminazene aceturate (Berenil®, Hoechst; 7% aqueous solu-tion) in order to remove any existing trypanosome infections. Any animal whichbecame parasitaemic during the course of the experiment was immediately treatedwith the same dose of diminazene aceturate. The animals of the ISMM SRD groupwhich became infected, however, were not treated the first m.p.tr. in order to seewhether there was spontaneous clearance of the parasites when levels of the drug incirculation built up to steady state concentration.

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2.4. Tsetse challenge

The apparent density (AD) of tsetse flies at the ranch was measured as describedearlier (Diarra et al., 1998). Briefly, 10 biconical traps (Challier and Laveissiere,1973) were used at two different sites (20 in total) during two consecutive days at2-monthly intervals. The AD was expressed as the mean number of flies per trapper day. At each fly collection, between 70 and 140 flies were dissected as describedby Pollock (1982) in order to identify the trypanosome species involved.

2.5. Isometamidium analysis

Serum samples were collected before treatment and at regular intervals aftertreatment in order to determine the ISMM concentration. ISMM analysis wascarried out using the enzyme-linked immunosorbent assay method (ELISA) asdescribed by Eisler et al. (1996).

2.6. Trypanolysis test

The presence of trypanolytic antibodies in the serum of the cattle involved in thelaboratory trial was examined according to the technique as described by VanMeirvenne et al. (1975) using T. congolense IL 1180.

2.7. Statistical analysis

The trypanosomiasis incidence in the different groups of cattle during the fieldtrial was compared using Cox proportional hazard regression (Collett, 1994).

3. Results

3.1. Laboratory trial

Table 1 clearly shows that the duration of the protection period in laboratorycattle under experimental tsetse challenge which received the ISMM SRD (\24months) was at least 4.2 times longer than in the cows which were injected i.m. withthe drug (5.7 months). The animal from which the remnants of the implants hadbeen removed became positive at the second challenge, 3 months after the removalof the SRD, whereas the two other animals were still parasitologically negative 4weeks after the last challenge, which was given 24 m.p.tr. Trypanolytic antibodiescould not be detected in any of the cattle as long as they were protected by thetrypanocidal drug.

The pharmacokinetics of ISMM in the sera of the cows are presented in Fig. 1.Peak levels in the i.m. treated group (16.4, 8.5 and 3.6 ng/ml; mean 9.5 ng/ml) wereobserved on day 1. Thereafter, the serum isometamidium concentrations followed abi-exponential decline, the second phase having a half-life of approximately 21.5

S. Geerts et al. / Acta Tropica 73 (1999) 49–58 53

Table 1Prophylactic effect of isometamidium implanted as a SRD or injected i.m. at 0.5 mg/kg in laboratorycattle exposed to G. m. morsitans experimentally infected with T. congolense clone IL 1180

Group 2Group 1

SRD LiquidDrug formulationS.c. I.m.Administration

3Number of treated cattle 3Protection period (months)

\20a, \24, \24Individual data 3, 4, 10\24 5.7Average

a The remnants of the implants of this animal were removed at 21 m.p.tr. It is not taken into accountto calculate the average protection period.

days. By approximately 90 days, concentrations reached the limit of quantificationof the assay (0.2 ng/ml), although traces (i.e. measured as \0.1 ng/ml) could bedetected until approximately 140 days following treatment.

The pharmacokinetics of the drug in the SRD treated group were markedlydifferent. Isometamidium was barely detectable on day 1, after which drug levels

Fig. 1. Mean isometamidium concentrations in the sera of cattle after implantation of a sustained releasedevice (SRD) or an intramuscular injection (i.m.). Results of the laboratory experiment. The horizontalline represents the limit of quantification of the isometamidium ELISA (0.2 ng/ml); concentrationsshown below this level are unreliable and should be interpreted as representing traces of the drug.

S. Geerts et al. / Acta Tropica 73 (1999) 49–5854

Fig. 2. Cumulative trypanosome infection rate in cattle, which received isometamidium as a sustainedrelease device (SRD) or as an intramuscular injection (i.m.), as compared with the control group.

rose to remain relatively consistent within the range 0.3–0.7 ng/ml for the next 10weeks. Peak levels, occurring on day 27 (0.60 ng/ml), on day 48 (0.64 ng/ml) andon day 6 (0.73 ng/ml) in each of the three animals (data not shown) were muchlower than those in the i.m. treated group. Thereafter, drug levels graduallydeclined, but in general remained above the limit of quantification (0.2 ng/ml) forapproximately 220 days following treatment. Between days 130 and 160, there wasa tendency for concentrations to fall below this level, although traces of drug (i.e.measured as \0.1 ng/ml) were still detectable throughout this period. Followingday 220, although concentrations were generally below the limit of quantification,traces of the drugs could be detected until approximately day 330 post-treatment.

3.2. Field trial

The cumulative trypanosome infection rate in the three groups of N’Dama cattleis shown in Fig. 2. Twelve months after treatment, there was a significant differencebetween the cumulative infection rates of the SRD (56.5%) and the i.m. (87.8%)treated groups (hazard ratio 0.33; P=0.003). Until 8 m.p.tr., the cumulativeinfection rate of the i.m. treated group still differed significantly from that of thecontrol group, but this was no longer the case at 12 m.p.tr.

The AD of G. m. submorsitans and of G. p. gambiensis during the course of theexperiment is shown in Fig. 3. The AD, expressed as mean number of flies per trapper day varied between 2 and 10.2 for G. tachinoides at the watering site of thecattle, whereas these flies were rare (ADB0.3) at pasture. The infection percentageof the flies varied during the course of the experiment between minimum values of

S. Geerts et al. / Acta Tropica 73 (1999) 49–58 55

4.4, 0 and 0, and maximum values of 31.3, 13.3 and 12.0 for G. m. submorsitans, G.p. gambiensis and G. tachinoides respectively. T. 6i6ax was present in 68.6% of thedissected flies, whereas T. congolense and T. brucei were observed in only 19.6 and2% of the flies, respectively.

The kinetics of ISMM in the sera of the drug treated cattle are presented in Fig.4. The sera were collected at monthly intervals. A rapid drop in the ISMMconcentration was observed in the i.m. treated cattle, whereas in the SRD im-planted animals, it remained more or less stable (between 0.2 and 0.3 ng/ml) until3 months post-treatment. At 121 days, the mean ISMM concentration droppedbelow the limit of quantification of the ISMM ELISA (0.2 ng/ml), although traces(i.e. measured as \0.1 ng/ml) could be detected in 13 of 23 cattle.

4. Discussion

The results obtained under experimental conditions with the poly(caprolactone-co-L-lactide)–ISMM SRD confirm those reported by Geerts et al. (1997) using apoly(D,L-lactide) type of polymer. The average protection of 20 months obtainedusing the latter polymer was extended to at least 24 months in the presentexperiment using the copolymer. It is of interest that the cow, from which theremnants of the SRD had been removed (cow number 13), became positive only atthe second challenge (3 months after the removal of the SRD), although thechallenge control cow developed a parasitaemia at both challenges. A possibleexplanation for this phenomenon could be that it takes some time before the ISMMat the level of the skin is depleted after the removal of the implants.

Fig. 3. Apparent density (flies/trap/day) of G. m. submorsitans at pasture and G. p. gambiensis at thewatering site of the cattle at the Madina Diassa ranch.

S. Geerts et al. / Acta Tropica 73 (1999) 49–5856

Fig. 4. Average isometamidium concentrations in the sera of cattle after implantation of a sustainedrelease device (SRD) or an intramuscular injection (i.m.) during the field trial in Mali. The horizontalline represents the limit of quantification of the isometamidium ELISA (0.2 ng/ml); concentrationsshown below this level are unreliable and should be interpreted as representing traces of the drug. SRD,�; i.m., �.

The results of the field trial are very similar to those obtained previously at thesame location using poly(D,L-lactide) SRD (Diarra et al., 1998). Eight monthspost-treatment, the latter authors observed cumulative infection rates of 27.7, 58.5and 77.4% in the SRD implanted, the i.m. injected and the control groups,respectively. In the present experiment, these figures were 26.1, 41.6 and 70.8%,respectively, at 8 m.p.tr. and rose to 56.5, 87.8 and 91.6% at 12 m.p.tr.

The breakthrough infections during the first weeks after the implantation of theSRD, which were reported by Diarra et al. (1998), were also observed in the presentexperiment and in a higher number of animals than in the former trial (four out of23 against only three out of 44 at 1 m.p.tr. in the field trial using the poly(D,L-lac-tide) SRD). These infections were left untreated and disappeared spontaneously by2 m.p.tr. Whether this was due to the effect of ISMM or to the intrinsic capacityof the trypanotolerant N’Dama cattle to eliminate the infections remains a matterof speculation.

The pharmacokinetics of ISMM in the sera of the SRD implanted animals in thelaboratory trial followed a pattern similar to that described earlier in cattle using apoly(D,L-lactide) SRD (Geerts et al., 1997).

Concentrations of the drug could be measured in most serum samples takenduring the first 220 days following treatment (laboratory trial), although there wasa tendency for concentrations to fall below the limit of quantification of theisometamidium ELISA (0.2 ng/ml) between days 130 and 160. However, during thisperiod, and from 220 to 330 days following treatment, traces of the drug weredetected in almost all samples. This was based on the observation that, although

S. Geerts et al. / Acta Tropica 73 (1999) 49–58 57

the measured concentration was below the limit of quantification, the opticaldensity responses corresponded on calibration curves to concentrations above 0.1ng/ml.

Under field conditions, although the overall pattern of drug disappearance fromsera of SRD treated cattle was similar to that in the laboratory experiment, ingeneral, slightly lower concentrations were observed. For example, at 30, 61 and 92days after SRD treatment of cattle in the field, the mean concentrations were 0.33,0.28 and 0.24 ng/ml, respectively, whereas at 27, 62 and 90 days after SRDtreatment of cattle in the laboratory, mean concentrations were 0.54, 0.40 and 0.27ng/ml, respectively. These differences may have been related to the different breedsand body weights of cattle, since Friesians (weight 640–690 kg) were used in thelaboratory, whereas the cattle in the field were N’Dama (average weight 144 kg atthe beginning and 195 kg at the end of the trial).

5. Conclusions

There are several advantages in using the copolymer poly(caprolactone-co-L-lac-tide) over the homopolymer poly(D,L-lactide) in a sustained release device forisometamidium: (1) the protection period is slightly longer under controlled condi-tions; (2) under field conditions, it is at least as good; (3) it is easier to manipulate(the rods are less fragile and more flexible); and (4) it can be produced at a cheaperprice. The copolymer, however, is less biodegradable than the homopolymer. Thebreakthrough infections during the first few weeks after the implantation of theSRD remain a problem, although they are self-limiting in N’Dama cattle. Itremains to be seen whether this will also be the case in zebu animals. However, assuggested by Diarra et al. (1998), simultaneous injection of diminazene at the timeof the implantation might resolve this problem.

Acknowledgements

The assistance of the technical personnel of the Veterinary Departement of theInstitute of Tropical Medicine, Antwerp, of the Laboratoire Central Veterinaire,Bamako, and of the Department of Veterinary Physiology, University of Glasgowis gratefully acknowledged. The authors would like to thank particularly C. Fodefor his help during the field trial.This research project was financially supported bythe EU-STD3 (contract No. TS3-CT93-240) and INCO-DC (contract no. IC18-CT95-0006) programmes.

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