Behavioural responses of Phlebotomus duboscqi to plant-derived volatile organic compounds.

Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography-mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p-cymene and the benzenoid m-cresol, p-cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose-response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five-component blends, referred to as Blend-f and Blend-m for females and males respectively, were formulated and tested in dose-response assays against 1-octen-3-ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant-derived VOCs can be exploited for sand fly management.

Characterization of Trypanosoma evansi type B.

A distinctive feature of Trypanosoma evansi is the possession of a kinetoplast that contains homogeneous DNA minicircles, but lacks DNA maxicircles. Two major sequence variants of the minicircle have been described and here we have sequenced the type B variant and designed a specific PCR test to distinguish it from type A. Further a test based on maxicircles to distinguish T. brucei brucei from T. evansi was designed and evaluated. Using the designed PCR tests, we detected three type B isolates from camel blood samples collected in northern Kenya, more than 20 years after the first isolation of type B. Comparison of minicircle sequences from all four type B isolates shows >96% identity within the group, and 50-60% identity to type A minicircles. Phylogenetic analysis based on minicircle sequences reveals two clusters, one comprising isolates of type A and one of type B, while random amplification of polymorphic DNA show slight polymorphic bands within type B. Most T. evansi isolates analysed were heterozygous at a repetitive coding locus (MORF2). All type B isolates had one genotype designated 3/5 based on the alleles present. Three camel isolates, which had homogenous type A minicircles, lacked the RoTat 1.2 gene, while another five isolates were T. b. brucei, based on the heterogeneity of their minicircles and presence of maxicircles as demonstrated by PCR amplification of the gene for cytochrome oxidase subunit 1. Our results confirm the existence of T. evansi type B isolates, T. b. brucei and existence of T. evansi type A without RoTat 1.2 gene in Kenyan isolates.

Growth and mortality in sheep and goats under high tsetse challenge in Kenya.

Trypanosomosis is a major impediment to livestock production and economic development in those areas of Africa where it is endemic. Although small ruminants appear to perform better than cattle in various agro-ecological zones, the importance of trypanosomosis has not been extensively investigated in these livestock. This study was designed to investigate the prevalence of trypanosomosis in sheep and goats in an endemic area and to evaluate the performance of different breeds under high tsetse challenge and the potential role of chemoprophylaxis in the control of the disease. The results showed that tsetse flies feed readily on small ruminants, and that these animals are susceptible to trypanosomosis. The Small East African goats acquired fewer infections than the Black Head Persian and Dorper sheep used in the study. In both sheep and goats, chemoprophylaxis with isometamidium chloride (Samorin, Rhone Merieux, Annecy, France) was protective, resulting in fewer infections and higher body weight gain. Trypanosomosis caused anaemia in both sheep and goats, and animals whose PCV fell below 15% rarely recovered, even with trypanocidal drug treatment. The peak transmission period was between 1 and 3 months after the peak tsetse fly density, which raises the possibility of effective strategic prophylaxis.

Financial implications of rearing sheep and goats under natural trypanosomosis challenge at Galana ranch, Kenya.

A study to compare the profitability of rearing sheep and goats under natural trypanosomosis challenge was carried out on Galana ranch in south-eastern Kenya between July 1996 and October 1997. Seventy-nine male weaner sheep and 79 male weaner goats were monitored monthly for weight changes and fortnightly for trypanosomosis. The animals of each species were divided into two groups. Group 1 was an untreated control, while group 2 was treated with isometamidium chloride (Samorin) at 0.5 mg/kg body weight every 3 months. In both groups, trypanosome infections were detected by microscopy and treated with diminazene aceturate (Veriben), at 3.5 mg/kg body weight, when the packed cell volume reached 17% or below. The profitability of each drug regime was expressed as the marginal revenue over the cost of trypanosomosis (MOT). There were greater losses occasioned by trypanosomosis in sheep than in goats. Animals of both species on chemoprophylaxis gave higher MOT values than those that received chemotherapy on diagnosis. However, the MOT values for the chemoprophylactic regime were higher for sheep than for goats, suggesting that the greater weight gain by sheep more then compensated for the higher cost of maintaining them under high trypanosomosis challenge. Thus, a Galana rancher would be better off keeping sheep rather than goats, other things being equal. The marginal revenue per dose of Samorin was lower than that of Veriben for both species, suggesting that strategic use of Samorin timed to precede the peak incidence of trypanosomosis might be a better option to raise the overall profitability in sheep and goats.

Amplified restriction fragment length polymorphism in parasite genetics.

The amplified restriction fragment length polymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: glossinidae).

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

A repetitive DNA sequence specific for Trypanosoma (Nannomonas) godfreyi.

The satellite DNA sequence of Trypanosoma (Nannomonas) godfreyi, a recently described parasite of Suidae, was determined. The sequence is 373bp in length, and contains two imperfect internal repeats of approximately 170bp. Like other trypanosome satellite DNAs, it has no extensive open reading frames and is probably non-coding. There is no significant homology with other major repetitive DNAs within subgenus Nannomonas. We have developed a PCR test that is specific for T. godfreyi and used it to identify the parasite in natural tsetse infections from Zimbabwe and Côte d'Ivoire. This test shows no cross reaction with non-target trypanosomes, even within subgenus Nannomonas, and will be invaluable in studies of the prevalence and distribution of T. godfreyi.

A high prevalence of mixed trypanosome infections in tsetse flies in Sinfra, Côte d'Ivoire, detected by DNA amplification.

The prevalence of various species and subgroups of trypanosomes in the Sinfra area of Côte d'Ivoire was determined using the polymerase chain reaction (PCR). Using this technique to amplify specific satellite DNA targets, it was possible to identify developmental-stage trypanosomes in the midguts and the proboscides of tsetse without expansion of parasite populations. The predominant tsetse species in the area was Glossina palpalis, while G. pallicera and G. nigrofusca were also present. Microscopical examination of 811 non-teneral flies revealed an infection rate of 14% in midguts and/or proboscides. Three subgroups of Trypanosoma congolense (Savannah, Forest & Kilifi), T. simiae, T. godfreyi, West African T. vivax and T. brucei ssp. were identified using PCR. T. congolense Forest was the most abundant of the Nannomonas trypanosomes. Approximately 40% of all infections were mixed, and there was a significantly higher prevalence of apparently mature T. brucei ssp. trypanosomes than has previously been reported. The present study demonstrates that PCR facilitates the easy identification of mature trypanosome infections in tsetse, providing a reliable estimation of trypanosomiasis challenge.

Multiple trypanosome infections in wild tsetse in Côte d'Ivoire detected by PCR analysis and DNA probes.

Trypanosomes were isolated from the midguts of Glossina palpalis palpalis, G. pallicera pallicera and G. nigrofusca nigrofusca captured around the village of Guediboua, South West of Daloa in Côte d'Ivoire. Seventy of the 124 isolates, obtained from 688 flies, were examined for four different kinds of trypanosome using the Polymerase Chain Reaction (PCR). Prevalences were: Trypanozoon 46%, riverine-forest T. congolense 86% and savannah T. congolense 54%. Only 29 samples were examined for T. simiae but it was not detected. Just 30% of the infections involved a single kind of trypanosome; the remainder were mixtures either of two (37%) or all three (27%) of the target organisms. 30 of the 70 isolates examined by PCR were successfully amplified to provide material for DNA probe hybridization. To a large extent, DNA probes confirmed the PCR results; all (28/28) of the riverine-forest and 82% (18/22) of the savannah T. congolense infections were identified. However, only 8% (1/13) of the PCR positives for Trypanozoon hybridized with the appropriate DNA probe. No T. simiae or T. godfreyi infections were identified using DNA probes but a large proportion (97%) (29/30) of the probed midguts were shown to contain Kilifi T. congolense. Four isolates out of 70 could not be identified by any method. There was no obvious association between the different species of flies and the infecting trypanosomes.

Sensitive detection of trypanosomes in tsetse flies by DNA amplification.

African trypanosome species were identified using the Polymerase Chain Reaction (PCR) by targeting repetitive DNA for amplification. Using oligonucleotide primers designed to anneal specifically to the satellite DNA monomer of each species/subgroup, we were able to accurately identify Trypanosoma simiae, three subgroups of T. congolense, T. brucei and T. vivax. The assay was sensitive and specific, detecting one trypanosome unequivocally and showing no reaction with non-target trypanosome DNA or a huge excess of host DNA. The assay was used to identify developmental stage trypanosomes in the tsetse fly. The use of radioisotopes was not necessary and mixed infections could be detected easily by incorporating more than one set of primers in a single reaction. The use of crude preparations of template made the process very rapid. The methodology should be suitable for large-scale epidemiological studies.

Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles.

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.