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EFFICACY AND LOCAL TOLERANCE OF CYMELARSAN® IN HORSES INFECTED WITH TRYPANOSOMA EVANSI
EFFICACITE ET TOLERANCE LOCALE DU CYMELARSAN® CHEZ DES CHEVAUX INFECTES PAR TRYPANOSOMA EVANSI
Raymond E. Mdachi1, John M. Kagira1, Grace A. Murilla1 & Frans van Gool2
1Kenya Agricultural Research Institute-Trypanosomiasis Research Centre
P.O. Box 352 Kikuyu, Kenya.
2Merial SAS, 29Avenue Tony Garnier BP 7123, 69348 Lyon Cedex 07 Lyon, France
Résumé
Une étude a été menée pour évaluer les manifestations cliniques de la trypanosomose à Trypanosoma evansi (KETRI 2426) et mesurer le niveau de tolérance locale et d’efficacité du Cymelarsan® chez les chevaux. L’étude a été entreprise en deux phases. Lors de la première phase, 104 trypanosomes ont été inoculés à trois chevaux qui ont par la suite été traités au Cymelarsan® à raison de 0,25 mg/kg lorsque leurs taux d’hématocrite ont baissé de 20% par rapport à leurs valeurs initiales. Lors de la deuxième phase, le même nombre de trypanosomes que pour la première phase a été inoculé à six chevaux traités avec le même médicament à une dose égale à celle utilisée au début de la parasitémie.
Les chevaux ont présenté des signes cliniques typiques de trypanosomose qui comprenaient la fièvre, les œdèmes, le pelage hérissé, l’accélération du pouls et du rythme respiratoire qui entraîne l’essoufflement et la paralysie partielle des pattes de derrière à partir du 25ème jour après l’infection. Les hématocrites des trois chevaux ont considérablement baissé en l’espace de 30 jours (p<0,001) pour atteindre des valeurs situées autour de 20 % en-dessous des valeurs pré-infection. Le Cymelarsan®, administré par voie intramusculaire à la dose de 0,25 mg/kg, a été bien toléré par les chevaux qui n’ont présenté aucun signe majeur de toxicité, et le médicament a guéri tous les chevaux lorsqu’il leur a été administré au début de la parasitémie ou lorsque les chevaux n’étaient infectés que pendant un mois. Après le traitement, les trypanosomes ont disparu du sang en l’espace de 24 heures, et les signes cliniques, la température et l’hématocrite sont redevenus normaux. Le taux d’élimination des trypanosomes du sang circulant ne dépendait pas de la période à laquelle le médicament a été administré après l’infection. C’est là une preuve que le Cymelarsan®, administré à la dose recommandée, peut guérir des infections à la fois précoces et chroniques de T. evansi chez les chevaux.
Summary
A study was conducted to assess the clinical manifestations of trypanosomosis caused by Trypanosoma evansi (KETRI 2426) in horses and to evaluate the local tolerance and efficacy of Cymelarsan® in horses. The study was undertaken in two Phases. In phase 1 three horses were infected with 104 trypanosomes and treated with Cymelarsan® at 0.25 mg/kg when the Packed Cell volumes (PCV) had dropped by 20% from initial values. In phase 2 six horses were infected with a similar number of trypanosomes as in phase 1 and treated with the same drug at the same dose rate at onset of parasitaemia.
The horses exhibited typical clinical signs of trypanosomosis which included fever, oedema, raised hair coat, increased pulse rate and respiratory rate leading to gasping and partial paralysis of hind legs from day 25 post infection. Packed Cell Volumes (PCVs) of the three horses dropped significantly (p<0.001) to values that were 20% below pre-infection values in 30 days.
Cymelarsan® administered at 0.25 mg/kg intramuscularly was well tolerated in horses with no major toxicity signs noted and cured all horses when administered at on set of parasitaemia or when the horses had harbored infection for a month. Following treatment the trypanosomes disappeared from the blood within 24 hrs and the clinical signs, temperatures and PCVs normalized. The rate of clearance of trypanosomes from the blood circulation was not dependent on the period after infection that the drug was administered. This demonstrated that Cymelarsan® at the recommended dose is able to cure both early and chronic T. evansi infections in horses.
Introduction
Trypanosoma evansi is the most widely distributed of the pathogenic animal trypanosomiases, affecting domesticated livestock in Asia, Africa and Central and South America. The principle host varies geographically. In Africa, camels are the most important host (Dia et al., 1997), whilst in Central and South America, it is the horse (Monzon et al., 1995; Silva et al., 1995). In Asia, besides bactrian and dromedary camels and horses, the other hosts include buffaloes and pigs (Payne et al., 1991; Tuntasuvan et al., 2000).
Cymelarsan is an anti-trypanosomal drug, introduced on to the market in 1991 for the treatment of Trypanosoma evansi, in camels. Since its introduction, several studies have been carried out to determine its efficacy as well as tolerance in camels (Zelleke et al., 1989; Otsyula et al., 1992; Nyang’ao et al 1995). The systemic reactions of Cymelarsan in camels have been studied and documented and include, very sporadically, lachrymation, drooling saliva and muscle tremors. However, treatment of T. evansi in horses using the drug is not documented leading to poor understanding of its use in this animal species. Therefore, the aim of this study was to assess the clinical manifestation of T. evansi in horses and evaluate the local tolerance and efficacy of Cymelarsan against T. evansi infection in order to determine its usefulness in the control of the disease in this animal species.
Materials and methods
Nine trypanosome naïve ponys weighing approximately 170-300 kg bodyweight were purchased from a tsetse free area in Nairobi district. The animals were allowed to acclimatize for three weeks. During this period the horses were trained and accustomed to the handlers for ease of sampling and were treated for helminthes and other diseases. The horses were maintained on a ration of good quality hay supplemented with proteins and mineral salts and water ad libitum.
The study was carried out in two phases. In Phase One, three randomly selected horses were infected with the T. evansi KETRI 2426 intravenously through the jugular vein. The horses were treated with Cymelarsan prepared as a 0.5 % solution of active principle in de-ionised and distilled water under sterile conditions at 0.25 mg/kg bw, by deep intramuscular (im) injection into the neck muscles, when the PCVs had dropped by 20% from pre-infection value. In Phase Two, six horses were infected with the same trypanosome stabilate used in Phase One and treated with Cymelarsan at the same dose rate as in Phase One, on detection of trypanosomes in the blood by buffy coat examination of all the animals in the group.
v A cryo-preserved stabilate of T. evansi KETRI 2426 multiplied in irradiated mice was used to infect horses intravenously via the jugular vein with 1 x 104 trypanosomes administered in a volume of 4ml PBS. The horses were monitored for parasitaemia and PCV daily before treatment and three times a week thereafter for sixty days, by collection of blood from the jugular directly into heparinised vacutainer tubes and centrifugation.. Packed cell volume was determined as a ratio of red cells to plasma volume using a PCV reader (Hawksley). The presence of trypanosomes was determined by examination of the buffy coat according to Murray et al. (1977). Body temperatures were also monitored three times a week and body weights were determined weekly by measuring the girth and the length of the horses and estimating the weight using the formula W = G2 x L/11000, where, G is the girth and L is the Length from the shoulder to the ramp. Data generated from the study was analysed using Analysis Of Variance (ANOVA) to determine differences in PCV, bodyweight, temperature, within and between groups. Results
Phase One: Clinical study:
The pre-patent period of the T. evansi infection in the three horses was 5 days. The first parasitaemic peak occurred between day 4 and 9 post infection. The parasitaemia increased in waves from an average of 103 trypanosomes per ml by day 5-post infection to a maximum peak of 107 trypanosomes per ml by day 25-post infection. Following treatment on day 30-31 after infection the parasites cleared from the peripheral blood after 24 hrs and remained negative to the end of the trial.
During infection the horses exhibited typical clinical signs of trypanosomosis which included edema, raised hair coat, increased pulse rate and respiratory rate leading to gasping and partial paralysis of hind legs from day 25 post infection an indication of central nervous system involvement. These signs however disappeared completely and rapidly after treatment. There was an initial significant (p< 0.001) elevation of rectal temperatures to peak values of 37.6-38.0 ºC between 4 and 9 days post infection coinciding with first parasitaemic peak, which fluctuated at an average of 36.7 ºC thereafter. Post-infection body temperatures (Mean ± SD: 36.6±0.4 – 37.0 ± 0.5 ºC) were significantly (p<001) higher than pre infection temperatures (Mean ± SD: 36. 4± 0.3 – 36.5 ± 0.3 ºC). However following treatment body temperatures of the horses decreased significantly (p<0.01) to values close to pre infection values.
Following infection, the packed cell volume values of the three horses decreased significantly to 20% below pre-infection values in 30-31 days post infection. After treatment the packed volumes of all the horses increased significantly (p<0.001). However by the end of the experimental period the PCVs had not gone back to pre infection values.
During infection there was a slight increase in bodyweight to a maximum mean of 293.5 ± 28.0 kg on day 23 after infection and a drop to a mean of 280.0 ±10.0 kg on the day of treatment. After treatment the bodyweight increased to a mean of 309.0 ± 6.2 kg at the end of the experimental period, 60 days.
Phase Two: Efficacy study:
The pre-patent period of the T. evansi infection in the six horses was 4 days. All the horses became positive on the same day. Following treatment all the horses became aparasitaemic after 24 hrs. The horses remained trypanosome free to the end of the follow up period of 60 days.
No significant clinical manifestations that may be attributed to infection were observed. However, some of the animals appeared dull and less active and had decreased appetite the night before detection of trypanosomes in the blood.
Pre-infection bodyweights varied between 168 kg to 305 kg with a mean of 230.6 ± 49.4 kg. Following infection the bodyweights decreased to mean of 224.7 ± 52.3 kg on day 3 post infection then increased to a mean of 230.0 ± 47.5 kg by the end of the experimental period. The post treatment body weights were not significant different (p>0.05) from pre-infection body weights.
Pre-infection PCVs varied from 38% to 47% with a mean of 42.7 ± 3.0 %. Following infection the PCVs decreased to mean of 39.5 ± 2.4% by day 3 post infection but increased after treatment to a mean of 40.8 ± 3.1 % by the end of the experimental period of 60 days.
Discussion
The clinical manifestation of T. evansi infections in the horses was typical of trypanosomosis. As expected the parasitaemia pattern was in form of parasitaemia waves. The pre- patent period of 5 days in horses was longer than that observed in mice (3 days) infected with 104 trypanosomes. In addition all mice died by day 26 post infection (mean survival time: 11 ± 8.0 days). The parasitaemia in mice increased rapidly to maximum of 109 trypanosomes per ml by day 11-post infection, in contrast to a maximum of 107 per ml on day 25-post infection in horses. This difference between the two animal species observed cannot over emphasize the danger of extrapolation of data from one animal species to the other.
The current study also revealed that the rate of clearance by Cymelarsan of trypanosomes from peripheral blood was similar irrespective of the length of period of infection. Horses treated on detection of infection or 26 days after detection were all cleared of parasitaemia in the blood within 24 hrs. In contrast, other studies have shown that the clearance rates of trypanosomes by different trypanocides such as diminazene aceturate and isometamidium chloride are influenced by the duration of infection (Peregrine et al 1991; Murilla et al 1993). It is worth noting that Cymelarsan was able to cure trypanosomosis in horses when the trypanosomes had penetrated the central nervous system as indicated by the partial paralysis observed in most of the horses by day 25 after infection. In addition, the drug was able to reverse the observed symptoms and the effect of infection on packed cell volumes and body temperatures and weight. However, the recovery of PCVs to pre treatment values was slower than the recoveries observed on body temperatures and weight.
In conclusion, this study has demonstrated the effectiveness and the very good tolerance of Cymelarsan® administered at the lowest recommended dose rate in horses. The advantage of use of this drug in the field is that unlike the other trypanocides used in livestock, induction of resistance occurs at a slower rate (Barrett and Fairlamb, 1999). No resistance to the drug has been reported in the field. However, resistance to the drug has been induced in T. evansi and T. brucei population in laboratory using immuno-suppressed mice (Osman et al., 1992; Pospichal et al., 1994). Cymelarsan® may therefore be useful in areas where drug resistance to quinapyramine has been reported. A field evaluation for the drug is therefore recommended.
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