1Prevalence of trypanosome parasites in midgut and proboscis of field-collected Glossina austeni and Glossina brevipalpis. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/353/350
To confirm the infection rate results obtained for the field-collected tsetse flies, laboratory-controlled fly infectivity experiments were conducted. A total of 534 colony-reared G. austeni and 882 G. brevipalpis, fed on experimentally infected parasitaemic cattle, were dissected. Dissection results indicated that all four isolates (BoomerangI, BoomerangII, BmrngK2 and MVU10) became established in the midgut of both G. austeni (20% – 33%) and G. brevipalpis (1% – 4%) (Table 2). Of the infected flies, 22% (n = 121) immature infections and 4% (n = 21) mature infections were found in G. austeni fed on four cattle infected with different T. congolense isolates. Only 2% (n = 17) immature infections were seen in G. brevipalpis and no parasites were detected in the proboscis.
2Trypanosome infection rate in midgut and proboscis of Glossina austeni and Glossina brevipalpis colony flies fed on infected cattle (calculated p-values). http://ojvr.org/index.php/ojvr/article/downloadSuppFile/353/351
Vector competence was also assessed to confirm the results on the infection rates with trypanosomes in G. brevipalpis and G. austeni collected from game parks and communal dip tanks in KwaZulu-Natal. The infectivity of G. brevipalpis fed on susceptible cattle under controlled conditions was not shown and no trypanosome transmission was observed from any of the flies (as many as 180 per animal). On the other hand, transmission with G. austeni was achieved with a small number of feeding flies (i.e. fewer than 10 per animal). There were no significant differences in the immature infection prevalence between the four isolates in G. austeni (p = 0.158). However, there was a significant difference (p = 0.025) in the immature infection prevalence of these isolates in G. brevipalpis. A higher number of midgut infections were observed to have resulted from BoomerangI and II (4.3% and 2.6%, respectively) than from MVU10 and BmrngK2 (0.36% and 0.50%, respectively), as shown in Table 3. In contrast, the number of G. austeni with mature infections from the four isolates differed significantly (p = 0.007). Flies infected with BoomerangI and II isolates had a higher infection prevalence (9.5% and 8.2%, respectively) compared with those infected with isolates MVU10 and BmrngK2 (2.6% and 1.6%, respectively) (Table 3). Mature infections were not detected in the proboscis of any G. brevipalpis flies.
3Infection prevalence in the midgut and proboscis of infected colony Glossina austeni and Glossina brevipalpis flies. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/353/352
After the flies had fed on the animals, parasites were detected in blood samples within 14, 17 and 22 days for cattle and 15 days for the goat. All animals subsequently presented with clinical signs of anaemia (Table 4).
4Infectivity of Glossina austeni and Glossina brevipalpis collected from different field sites and fed on susceptible hosts under controlled conditions. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/353/353
DiscussionIn the present study, only 1% of T. congolense infections were found in the midgut of G. brevipalpis. In contrast, the infection rate in the midgut and the proboscis of G. austeni was significantly higher, with 8% detected in both organs. The age structure of the two Glossina species had not been determined in the field-collected flies. It has been reported that older flies are more likely to be infected than younger flies (Harley 1966; Jordan 1976; Woolhouse et al. 1994). The factors affecting the trypanosome infection rate in flies and contributing to their being refractory to infection have been detailed and discussed by several authors (Jordan 1976; Roditi & Lehane 2008; Welburn & Maudlin 1999). Factors such as age may influence the readiness with which flies can be infected. According to Harley (1967), the longer the female of some fly species, such as G. brevipalpis, lives, the more likely she is to be infected with T. congolense. In contrast, species such as G. austeni can be readily infected when they are 1 day old and less successfully later (Jordan 1976; Ward 1968). In our study, both G. austeni and G. brevipalpis colony specimens were fed a day after emergence and were able to establish infections in the midgut. However, trypanosomes could not develop to maturity in G. brevipalpis. The results showing the poor efficiency of G. brevipalpis as a vector was unexpected based on their higher population densities in areas close to the Hluhluwe–Umfolozi Game Reserve where high infection prevalence in cattle had been reported (Gillingwater, Mamabolo & Majiwa 2010; Van den Bossche et al. 2006). These results further suggest that G. austeni is the major vector of trypanosomes in the area, despite its relatively low population density as reported by Esterhuizen et al. (2005) and Hendrickx et al. (2003). The relatively low numbers of G. austeni collected in this study indicate either that the natural population density is low or that the H-trap is not effective for the collection of this species. Similar observations were reported by Gaturaga, Maloo and Loehr (1989) when they collected only 33 flies representing two Glossina species over a period of one year, despite a high trypanosome infection rate (22%) amongst cattle. They attributed the low numbers of G. austeni collected, assumed to be the major vector, to the inefficiency of the biconical trap used. Interestingly, flies infected with isolates BoomerangI and II exhibited more midgut infections in G. brevipalpis and more mature infections in G. austeni than those infected with isolates MVU10 and BmrngK2. Both fly species seemed to be more susceptible to infection with Boomerang I and II isolates; however, G. brevipalpis is refractory to subsequent parasite maturation occurring in the proboscis. Goossens et al. (2006) found a very low prevalence of T. congolense in cattle (0.8%) on Mafia Island, Tanzania, where G. brevipalpis is the only tsetse fly species encountered and widely distributed. They attributed the low prevalence to a combination of factors, such as frequent use of prophylactic treatment of cattle with trypanocidal drugs, a low feeding frequency of G. brevipalpis on cattle and the low vectorial capacity of the fly. In contrast, Wilson, Dar and Paris (1972) found the infection rate with T. congolense in field-collected G. brevipalpis to be about 2% in their study in Uganda. The injection of the infected proboscis collected from these flies produced patent infection in mice and thus demonstrated the ability of T. congolense to mature in the proboscis.The present data do not support results from two recent studies on the trypanosomes infection rates in tsetse flies conducted in the same area using only polymerase chain reaction (PCR) analysis (Gillingwater et al. 2010; Mamabolo et al. 2009). Mamabolo et al. (2009) detected trypanosome DNA in 89% of the flies examined but the results did not specify the species origin. Furthermore, this molecular test does not distinguish between mature and immature infections. In the study of Mamabolo et al. (2009), the injection of a suspension of macerated fly proboscis did not produce any viable infections in mice that were monitored for the development of trypanosome parasitaemia. Similarly, Gillingwater et al. (2010) reported a higher percentage (20%) of flies testing positive for trypanosome DNA in the midgut with only 1.6% of mature infections. Again, the results were not separated according to species. The higher infection rates detected by the PCR analysis may be related to recent feeding of the flies on infected animals although these trypanosomes would not necessarily develop successfully in the midgut or the proboscis. ConclusionA wealth of entomological data have been collected over the years in KwaZulu-Natal, which were used by Hendrickx et al. (2003) to produce distribution and prediction maps. However, parallel data on the epidemiology of the disease have not been generated to support the intention of the veterinary authorities to control or eradicate tsetse flies from South Africa. The results from the current study support the findings of Goossens et al. (2006) that G. brevipalpis is not the main vector of T. congolense in KwaZulu-Natal, despite its higher abundance, whereas G. austeni has been shown to have a higher vector competence. Focus should, therefore, be directed towards the control of G. austeni in the province whilst more research is still needed to develop more efficient traps to monitor the population dynamics of this species before, during and after control operations. AcknowledgementsThe Department of Science and Technology and the ARC are thanked for financial support Dr Gert Venter provided valuable comment on an earlier version of the manuscript. Mr Dannie de Klerk assisted with field collections and feeding on susceptible animals. Mr Jerome Ntshangase and Mr Gazu are thanked for deploying, and maintaining traps and assistance with field collections. Competing interests The authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. Authors’ contributions M.M. (ARC-Onderstepoort Veterinary Institute) was the project leader and performed most of the experiments and wrote the manuscript. J.M. (Universite Pedagogique Nationale) was a student mentor who assisted in the execution of some experiments and also edited the manuscript. A.L. (ARC-Onderstepoort Veterinary Institute) was the project supervisor and also contributed to revision and editing of the manuscript. B.M. (ARC-Onderstepoort Veterinary Institute) was the co-supervisor of the study and contributed to revision and editing of the manuscript. P.v.d.B. (Institute of Tropical Medicine) was a co-supervisor to J.M. 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