THE VECTORIAL CAPACITY OF TSETSE FLIES REVISITED
REEXAMEN DE LA CAPACITE VECTORIELLE DES TSETSE
P. Van den Bossche1,* C. Kubi1, R. De Deken1, P. Dorny1 & J. Van den Abbeele2
Institute of Tropical Medicine, Animal Health Department1 and Parasitology Department2, Nationalestraat 155, 2000, Antwerpen, Belgium.
3University of Pretoria, Faculty of Veterinary Science, Department of Veterinary Tropical Diseases, Onderstepoort, South Africa.
Résumé
L’épidémiologie des trypanosomoses humaine et animale dépend de la proportion de tsétsé infectées, qui est surtout déterminée par la capacité vectorielle intrinsèque de la mouche. Cette capacité varie d’une espèce et d’une population de tsétsé à l’autre, et au sein d’une même population ; elle semble être influencée en grande partie par l’âge de la mouche au moment de l’infection. Les mouches ténérales sont généralement considérées comme la tranche d’âge la plus sensible, tandis que les mouches adultes sont jugées réfractaires. Cependant, des études de terrain sur la prévalence des infections trypanosomiennes chez des tsétsé de différentes tranches d’âge indiquent qu’un grand nombre de populations de tsétsé adultes contractent l’infection trypanosomienne.
Pour déterminer le rôle des mouches adultes dans l’épidémiologie de la maladie, des études ont été menées sur l’infection expérimentale des tsétsé. On a utilisé à cet effet des Glossina morsitans morsitans élevées en laboratoire. On a d’abord examiné la sensibilité des mouches non ténérales, âgées de 3 à 20 jours, à l’infection de Trypanosoma congolense ou T. brucei brucei ; par la suite, l’impact de la faim sur la capacité vectorielle des mouches ténérales et adultes a été étudié. Enfin, une étude a été conduite pour déterminer la sensibilité des mouches infectées à une autre infection due à une espèce différente de trypanosome.
Les résultats de ces études expérimentales ont montré qu’une grande partie de la population de tsétsé adultes peut s’infecter de T. congolense et de T. brucei. Leur sensibilité à l’infection de trypanosome varie considérablement selon leur état d’infection et de nutrition. Les conséquences épidémiologiques de ces résultats ont fait l’objet de discussion.
Summary
The epidemiology of human and animal trypanosomiasis depends on the proportion of infected tsetse flies which is determined significantly by the fly’s intrinsic vectorial capacity. This intrinsic vectorial capacity differs between tsetse species and tsetse populations and, within a population, seems to be largely affected by the fly’s age at the time of infection. Teneral flies are generally assumed to be the most susceptible age group whereas adult tsetse are considered to be refractory. However, field studies on the prevalence of trypanosome infections in different age categories of tsetse flies suggest that a substantial amount of the adult fly population acquires a trypanosome infection.
To elucidate the role of adult flies in the epidemiology of the disease, experimental tsetse fly infection studies were conducted. Use was made of laboratory reared Glossina morsitans morsitans. First, the susceptibility of non-teneral flies, aging between 3 and 20 days, to infection with Trypanosoma congolense or T. brucei brucei was examined. Subsequently, the effect of starvation on the vectorial capacity of teneral and adult flies was studied. Finally, a study was conducted to determine the susceptibility of infected flies to an additional infection with another trypanosome species.
The outcome of these experimental studies showed that a large proportion of the adult tsetse population can acquire a T. congolense or T. brucei infection. Depending on their infection status and nutritional status their susceptibility to trypanosome infections varies substantially. The epidemiological repercussions of those findings are discussed.
Introduction
The proportion of infected vectors is an important determinant in the epidemiology of vector-borne diseases. In tsetse-transmitted trypanosomiasis also, the proportion of the tsetse population that is infected with trypanosomes and effectively transmits the parasite is a major factor in disease challenge. The majority of tsetse flies is refractory to infections with trypanosomes. Furthermore, the tsetse’s susceptibility to infection varies between trypanosome species and is associated with the complexity of the trypanosome’s cycle in the fly (Leak, 1998). Trypanosoma vivax, having a simple cycle in the tsetse’s mouthparts is easily transmitted whereas the complex cycle of T. brucei s.l. results in low infection rates. Moreover, some tsetse species seem to be less susceptible to trypanosome infections than others. Within a species various extrinsic and intrinsic factors have been described that affect the tsetse’s susceptibility to infection. Origin of host blood or composition of the diet is known to significantly affect the fly’s vectorial capacity (Mihok et al., 1993). The innate susceptibility of tsetse flies is governed mainly by the fly’s immune system. Over the past years much research has gone into identifying the various components of the immune systems of insects (Lehane et al., 2004). In tsetse also various components linked to the fly’s immune system have proven to have an effect on the development and maturation of trypanosome infections (i.a. Hao et al., 2001). Generally speaking, it is accepted that tsetse acquire the majority of their T. congolense or T. brucei s.l. infections during the first bloodmeal. Nevertheless, several field studies have shown an increase in the age-specific infection rates (Woolhouse and Hargrove, 1998) and a high proportion of mixed trypanosome infections (i.a. Masiga et al., 1996). To clarify those observations that are of considerable epidemiological importance, experiments were conducted.
Materials and methods
Tsetse flies
Male G. morsitans morsitans (less than 32h old), from the colony maintained on rabbits (in vivo) at the Institute of Tropical Medicine, were used in all the experiments. The origin of this tsetse colony and the rearing technique are described by Elsen, Van Hees & De Lil (1993).
Trypanosomes
Trypanosoma brucei brucei stock EATRO 1125 isolated from a bushbuck in Uganda (Van Meirvenne, Janssens & Magnus, 1975) and three isogenic clones of T. congolense IL 1180 were used in the experiments.
Experimental designs
A total of 3 separate experiments was conducted.
Age-specific susceptibility - In a first experiment the age-specific susceptibility of G. m. morsitans for T. congolense and T. b. brucei was determined. Batches of teneral and non-teneral flies were fed once on mice infected with one of the trypanosome species. Only the fully engorged flies were retained and maintained on rabbits and offered the opportunity to feed three times weekly. The rabbits were replaced weekly to avoid cyclic transmission of the trypanosomes.
Infection of teneral tsetse with mixed trypanosome infections
In a second experiment teneral tsetse were infected concurrently with T. congolense and T. b. brucei. Infections were conducted using cattle that were infected through bites of infected tsetse. In a first phase, all animals were infected with T. b. brucei. Sixty days after the animals became parasitaemic, five of them were challenged with tsetse infected with T. congolense. The remaining two animals were kept as (T. brucei) controls. Once the animals challenged with T. congolense had developed a mixed (T. b. brucei/T. congolense) infection in the peripheral blood, batches of teneral tsetse were offered a single bloodmeal on the flanks of one of the seven experimental animals. Only engorged flies were retained. After the infective bloodmeal, flies were maintained on uninfected rabbits. The rabbits used for feeding were changed at weekly intervals.
Infection of infected tsetse flies
In the third experiment groups of tsetse were infected once or twice (once as teneral with either T. congolense or T. b. brucei and afterwards when 20 days old with either T. congolense or T. b. brucei depending on the species at the first infection) by feeding on infected mice. After offering an infected meal, only fully engorged flies were retained. Infected flies were maintained on rabbits and offered the opportunity to feed three times weekly. The rabbits were replaced weekly to avoid cyclic transmission of the trypanosomes.
Dissections
To determine their infection status, flies were dissected 3 weeks after infection using the method described by Lloyd and Johnson (1924). Flies of which only the midgut was infected were considered to have an immature infection. Flies with infections in the midgut or salivary glands were considered to have a mature infection. The mature infection rate was calculated as the proportion of dissected flies that developed a mature trypanosome infection. Differences in infection rates were compared using the Fisher Exact Test. In the case of mixed infections, species identification was conducted using a PCR-RFLP (Geysen et al., 2003)
Results
Age-specific susceptibility
The uptake of a single bloodmeal resulted in a steep decline in the susceptibility to infections with T. congolense or T. b. brucei. The vectorial capacity even declined further once flies reached the age of about 10 days. It remained low but constant thereafter (Figure 1).
Infection of teneral tsetse with mixed trypanosome infections
Teneral tsetse flies were capable of infecting themselves with two trypanosome species during their first bloodmeal. Of the total of 140 flies that were dissected, 4 developed a mature T. b. brucei infection, 29 had a T. congolense infection and 13 had a mixed T. b. brucei/T. congolense infection. The remainder were negative of were only infected in the midgut (immature infections). The presence of one trypanosome species did not hinder the development of a second species. Hence, the T. b. brucei infection rate of flies infected on animals with a single T. b. brucei of mixed T. b. brucei/T. congolense did not differ significantly. The same applied to infections with T. congolense.
Infection of infected tsetse flies
The presence of a mature T. congolense or a mature T. b. brucei infection did not hinder the development of a secondary infection with either T. b. brucei or T. congolense. Two flies of the 69 (2.9%) that were infected with a mature T. congolense infection also developed a T. b. brucei infection when infected on day 20. Six flies of the 27(22.2%) that were infected with a mature T. b. brucei infection also developed a T. congolense infection when infected on day 20. The infection rates of adult flies infected with T. b. brucei or T. congolense were 5.2% and 9.2% respectively.
Discussion
The results from the age-specific susceptibility experiment show unequivocally that the susceptibility to trypanosome infections decreases with age. The susceptibility is highest in teneral flies, reduces substantially in young adult flies and is low but constant in mature flies (about > 10 days old). Generally speaking, however, adult flies can still infect themselves with both trypanosome species. The impact of those findings on the infection rate of tsetse populations in the field is not clear but studies of the age-prevalence relationship of tsetse field populations have already suggested that adult also acquire infections but at a much lower rate than teneral flies (Woolhouse and Hargrove, 1998). Woolhouse and Hargrove (1998) suggested that a factor that may explain this age-specific increase in infection rates of tsetse is variations in the development period of the infection. Although such variations may result in the observed increase in prevalence, our experimental infections suggest that such increases are best explained by the infection of adult flies.
Figure 1: Mature infection rate of teneral and non-teneral male G. m. morsitans infectd with T. congolense or T. b. brucei.
The high proportion of mixed infections observed in the field is explained by the outcome of the two other experiments. Indeed, according to our findings tsetse are as susceptible to a single as to a double infection. The presence of one trypanosome species in a tsetse’s bloodmeal does not hinder the development of another species. Since mixed trypanosome infections develop easily in, for example, cattle (Van den Bossche et al., 2003) and a large proportion of potential hosts are infected with mixed trypanosome it is expected substantial proportion of teneral tsetse flies must ingest bloodmeals that contain several trypanosome species. Furthermore, adult infected tsetse are at least as susceptible as adult non-infected flies. Hence, a proportion of mixed infections observed in field populations of tsetse are a result of sequential ingestion of infected bloodmeals and subsequent maturation of the infection.
Acknowledgement
The work presented in this manuscript received financial support from the Directorate General of the Belgian Development Co-operation.
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