THE EFFECTS OF SAMORIN TREATMENT AND FIRST BLOOD MEAL ON THE ESTABLISHMENT OF TRYPANOSOMES IN GLOSSINA PALLIDIPES

EFFET DU TRAITMENT AU SAMORIN ET DU PREMIER REPAS DE SANG SUR L’APPARITION DE TRYPANOSOMES CHEZ GLOSSINA PALLIDIPES

S. Okoth, G. Tinega, P. Mukiria & G. Murilla                       

Trypanosomiasis Research Centre,
 Kenya Agricultural Research Institute (TRC -KARI),
Box 362 Kikuyu, Kenya

Résumé

            Cette étude visait à déterminer l’effet du Samorin et de l’exposition différée sur l’acquisition par Glossina pallidipes de l’hyporéactivité à l’infection de Trypanosoma brucei rhodesiense. Six groupes de 50 mouches ténérales chacun ont été nourris de sang bovin contenant 0(Ca), 6, 8, 10, 12 et 14µg de Samorin/ml de sang au jour 0. L’on a donné au septième groupe (Cb) du sang infecté de parasite comme premier repas au jour 0. Les mouches ont été nourries in vitro de sang infecté de T. brucei rhodesiense (2,5 x 107/ml) le 3ème jour, et maintenues in vitro sous régime de sang bovin pendant 30 à 35 jours, avant d’être disséquées. Les taux de survie, d’infection et les charges parasitaires ont été enregistrés. Les mêmes procédures ont été suivies avec les groupes nourris de sang infecté de parasite les 5ème et 10ème jours. Plus les mouches passaient de temps à se nourrir de sang infectieux, plus les taux d’infection augmentaient considérablement (r = 0,527 ; P<0,014). De plus fortes concentrations de Samorin (12 & 14) ont pu réduire considérablement la charge de trypanosomes et les taux d’infection (r = - 0,666 ; P<0,001 ; et r = -0,550 ; P<0,01) respectivement. Les traitements au Samorin ont accru les chances de survie des tsétsé, même si leur effet n’est pas important. Ces résultats indiquent que le traitement au Samorin à des concentrations supérieures à 12µg/ml et l’exposition prolongée des mouches à un premier repas infecté réduisent les taux d’infection chez les mouches, et peuvent par conséquent servir à conférer une résistance aux mouches destinées au lâcher en masse dans le cadre des programmes de TIS.

            A study was carried out to assess the protective role of Samorin, when given with the blood meal, and the effect of delayed exposure to infective blood meal on the establishment of Trypanosoma brucei rhodesiense in Glossina pallidipes. Five groups, each consisting of 50 teneral flies, were fed on fresh bovine blood containing 0(Ca), 6, 8, 10, 12 and 14µg of samorin/ml of blood on day 0.  The flies were then fed in vitro on T. brucei rhodesiense infected blood (2.5 x 107/ml) on day 3. A sixth group (Cb) was offered parasite-infected blood at first feed on day 0. Similar procedures were followed when offering parasite-infected blood on days 5 and 10. Dissection was carried out 25 days post infection and survival, infection rates and infection load observed.  Delayed exposure to infective feed significantly increased infection rates in flies (r = 0.527; P< 0.014). Higher samorin concentrations, (12 & 14), significantly reduced trypanosome load and infection rates (r = -0.666; P < 0.001; and r = -0.550; P< 0.01) respectively. However, though the treatments enhanced tsetse fly survival, the effect was not significant. These results indicate that samorin treatment at above 12µg/ml and delayed infection may be useful in reducing transmission pressure by flies destined for mass released in SIT programmes.

hntroduction

            Agricultural production throughout Africa is severely constrained by human and livestock diseases such as trypanosomosis. African trypanosomosis is transmitted by tsetse flies, which infest a total area of 10 million Km2 in 36 African countries. Annual losses incurred directly and in control programmes are estimated to be between US $ 600 million and 1.2 billion (FAO, 1994 quoted by Feldman, 2001). Control and or eradication of tsetse flies will therefore considerably reduce the impact of the disease.

            The recent eradication of Glossina austeni from Zanzibar using the Sterile Insect Technique (SIT) (Vreysen et al 2000; Saleh et. al. 2001) demonstrated the potential of the technique in an integrated intervention campaign against tsetse and trypanosomosis. Consequently, International Atomic Energy Agency (IAEA) in collaboration with the Government of Kenya (GOK) has launched one such programme targeting eradication of Glossina pallidipes from Lambwe valley.

            Use of SIT technique in the eradication campaign requires that vectorial capacity of the flies produced in the laboratories for release in the field severely reduced since sustained release of sterile males increases the trypanosomosis challenge in the wild. To avoid upsurge in challenge following large-scale releases, released flies need to be made refractory to trypanosome infection. Leak (1999) reported that establishment of trypanosome infection in tsetse was limited in subsequent feeds if the first blood meal was uninfected. Moloo (1987) also showed that maturation of pathogenic trypanosomes in Glossina morsitans morsitans was completely suppressed when their first blood meal was spiked with between 8 to 12 µg Samorin/ml of blood. This study investigated the effect of samorin treated first blood meal and that of delaying the infective feed on the establishment of trypanosome infection in the G. pallidipes colony flies destined for mass release in Lambwe Valley.

Materials and methods

Blood spiking with Samorin

            Freshly collected bovine blood was defibrinated by gently swirling in a 250 lm conical Pyrex beaker containing glass beads for 10 minutes. It was then decanted and spiked with samorin. A stock of samorin-spiked blood was aliquoted and diluted to final concentrations of 6, 8, 10, 12 and 14µg samorin per millilitre of blood.

Mice infection and recovery of parasites

            Swiss white mice were infected intraperitonially with T. brucei rhodesiense isolate KETRI 3083. The infected mice were maintained on mice pellets and water ad libitum. Parasitaemia was monitored daily and at peak parasitaemia the mice were anaesthetized using CO2 and blood aseptically collected by cardiac puncture. The blood was dispensed into a vacutainer tube coated with 10% EDTA, and then diluted with uninfected bovine blood to a final concentration of 2.5 x 107 trypanosomes / ml of blood.

In vitro infection of tsetse with T. brucei rhodesiense

            Five experimental groups of 50 teneral Glossina pallidipes were fed on fresh bovine blood containing 6, 8, 10, 12 and 14µg of samorin per millilitre of blood on day 0.  Two control groups were not offered samorin treated blood.  One group (designated Control a (Ca) was offered parasite-free blood, and the other (Cb) was offered parasite infected blood as the first blood meal. The Ca group and the samorin fed group flies were fed on T. brucei rhodesiense infected blood (2.5 x 107 parasites/ml blood) in vitro on day 3.  Flies that were subjected to but failed to feed on the infective feeds were excluded from the experiment.  Mortality was checked daily and the final survivals recorded per group. The flies were subsequently maintained in vitro on uninfected-unspiked bovine blood for 35 days. Similar procedure was repeated in groups that obtained their first infective feeds days 5 and 10 post first blood meal.

Infection rates and trypanosome load

            All flies were dissected at the mouthparts, midgut and salivary gland day 35 after the infective meal and infection status determined by microscopy. For each infected fly, infection load was estimated on the basis of mean concentration of parasites counts from 20 fields. Organs of uninfected flies were pooled into 1.5 ml ampoule and preserved at –20 o C for analysis using PCR.

Data analysis

            Data was analysed in SPSS 9.0 for Windows. Data on survival and infection rates were compared among the groups using analysis of variance, and differences separated using Least Significance Difference (LSD). Effects of delayed infection and dosage level were determined from log10 transformed infection rates. Risks of infection attached to each dosage level were assessed as individual probability of acquiring infection within a group. This was compared to that of the control group using student-t-test. The combined probability of a fly acquiring infection when subjected to delay infective feed and samorin treatment was calculated from the individual probabilities as independent events.

Results

Generally samorin treatment did not significantly affect the survival of the flies (F = 1.002, DF6, 14; P=0.462) (Table 1). However, analysis of within group variation showed that samorin treatment affected survival significantly only in the day 3 group as the following graphs ( Fig. 1 – 3 ) show.

Figure 1. Percent survival for flies infected 3 days post samorin treatment

Table 1. Effect of samorin concentration on fly survival, infection rate and  infection load 30 days post infection

            Samorin treatments did not have significant effect on infection rates (P=0.265) ( Table 2 ). However, the day of infection had a strong significant correlation effect on infection rates in that the rates increased with delayed exposure (r = 0.527; P< 0.014). Correlation analysis indicated that increasing concentration of samorin reduced trypanosome load and infection rates (r = -0.666; P < 0.001 and r = -0.550; P< 0.01) respectively. Trypanosome load was significantly influenced by treatments (F=2.845; DF14, 6; P=0.05).

            Table 2. Effect of samorin on fly survival, infection rate and infection load

Discussion

             Strategies to interfere with the ability of tsetse to transmit trypanosomes are of interest since the vectors transmit trypanosomes to both man and his livestock.

            In this study, samorin treatment enhanced the fly survival. Kibugu, et al., (2003) (unpublished) reported similar findings in which treatment of flies with Ethidium resulted in increased longevity. It is believed that trypanocidals have antimicrobial activity and therefore protect the flies from entomophagous microbes thus increasing their survival. Higher survival will improve the competitiveness of release flies and benefit the control operation.

            The highest infection was noted in the group that fed on infective meal as tenerals and there was a decrease in infection rates with increasing day of infection. Similar trend was also observed between infection rates and concentration of Samorin though the difference between the groups was not significant.  These observations compare well with that of Moloo (1987) who showed that when Glossina morsitans were offered Samorin treated blood as a first blood meal, their ability to be infected with T. brucei and T. congolense was significantly depressed when compared to those fed on untreated blood. Welburn and Maudlin (1999) also reported that for T. brucei and T. congolense infections, the age of the tsetse fly at the time of the infective feed is an important determinant of infection rates.

            When infection loads were compared, the flies that managed to develop infection in spite of the presence of the drug developed a lower load with increasing concentrations of the drug. In terms of the SIT project this is a favourable observation because such flies, however few, go on to develop mature infections in the field. The few fly numbers that will pick infections will be counteracted by the over-flooding ratio. Not too much premium should be placed on this unless it can be demonstrated in repeat experiments. During repeat experiments this observation is going to be monitored carefully.

            Infection rates with trypanosomes are traditionally determined by dissection and microscopic examination of the gut and mouthparts. Infection rates in flies ranged between 0 and 6.45%. Sensitivity of microscopy as a tool for detection of infection is known to be low. Salivary gland infection prevalence less than 1% is typical when determined by microscopic analysis. PCR-based studies have revealed a high frequency of T. brucei positive proboscides, often in flies without detectable salivary gland infections (Masiga et al., 1996). There is now a compelling case that estimation of infection rates with T. brucei based solely on dissection and microscopy underestimate trypanosome challenge and conclusions based solely on the above should be treated with caution. We propose to analyse the negative flies using PCR

References

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