Glossina fuscipes fuscipes in the trypanosomiasis endemic areas of south eastern Uganda: Apparent density, trypanosome infection rates and host feeding preferences

Acta Tropica 99 (2006) 23–29

Glossina fuscipes fuscipes in the trypanosomiasis endemic areas of south eastern Uganda: Apparent density, trypanosome infection rates and host feeding preferences C. Waiswa a,∗ , K. Picozzi b , E. Katunguka-Rwakishaya a , W. Olaho-Mukani c , R.A. Musoke a , S.C. Welburn b a

Makerere University, Faculty of Veterinary Medicine, P.O. Box 7062, Kampala, Uganda b Centre for Infectious Diseases, University of Edinburgh, EH25 9RG Scotland, UK c Directorate of Animal Resources, MAAIF, P.O. Box 513, Entebbe, Uganda

Received 15 December 2005; received in revised form 13 June 2006; accepted 19 June 2006 Available online 25 July 2006

Abstract A study was undertaken in three districts in south eastern Uganda endemic for human and animal trypanosomiasis, to investigate the status of the vector tsetse fly population. Apparent density (AD) of tsetse was between 2 and 21 flies/trap/day across the three districts, with Glossinia fuscipes fuscipes identified as the predominant species. Trypanosomes were observed in G.f. fuscipes with an infection rate, as determined by microscopy, of 1.55% across the three studied areas. However, trypanosome infections were only identified in female flies giving an infection rate of 2.39% for the female tsetse when this sex was considered in isolation; no male flies were found to be infected. Bloodmeal analysis highlighted 3 principal vertebrate hosts, namely cattle, pigs and monitor lizards (Varanus niloticus). The implication of this, in relation to the cycle of transmission for human infective trypanosomes between domestic animals and man, is discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Tsetse; Trypanosomiasis; Endemic areas; Host; Bloodmeal

1. Introduction The number of reported cases of both animal and human trypanosomiasis, caused by zoonotic Trypanosoma brucei rhodesiense, continues to increase in eastern Uganda (F`evre et al., 2001; Waiswa et al., 2003; ∗

Corresponding author. Tel.: +256 77 501274; fax: +256 41 554685. E-mail addresses: [email protected] (C. Waiswa), [email protected] (K. Picozzi), [email protected] (E. Katunguka-Rwakishaya), [email protected] (W. Olaho-Mukani), [email protected] (R.A. Musoke), [email protected] (S.C. Welburn). 0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.06.005

Picozzi et al., 2005). One of the strategies for the control of the disease is the treatment of domestic livestock with trypanocidal drugs, in order to reduce the size of the animal reservoir (Welburn et al., 2001). The presence of Trypanozoon infections in domestic animals raises serious concerns since livestock are sometimes kept in close proximity to human dwellings (Welburn et al., 2001; Waiswa et al., 2003), and thus presenting a risk for human disease acquistion. In addition to the use of trypanocidal drugs, farmers are encouraged to apply insecticides for the purpose of reducing the tsetse population, thus reducing transmission of the parasites (Eisler et al., 2003). In Uganda, for

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Fig. 1. A map of Uganda with the districts where this work was done: Highlighted in Red (Zone I for Kamuli district), Green (Zone II for Mukono district) and Purple (Zone III for Tororo district).

over a decade now, there has been little published information regarding the tsetse species responsible for the transmission of both animal and human trypanosomiasis and their preferred hosts. It is important to identify the predominant species of the tsetse vector and the animals on which they feed. During this investigation, three sleeping sickness endemic areas were studied to estimate tsetse density, trypanosome infection rates and feeding preferences of tsetse. The findings are discussed in relation to the reservoirs of human infective trypanosomes. 2. Materials and methods 2.1. Study area Three districts of Kamuli (Zone I), Mukono (Zone II) and Tororo (Zone III) given in Fig. 1, endemic for

sleeping sickness in south eastern Uganda were selected for the study on the basis of the ongoing reporting of human sleeping sickness cases that were originating from these areas. The study was carried out between June and December 2000 in villages that had active transmission of sleeping sickness. The livestock populations for these areas are given in Table 1 (based on the 1998 district records). Within each district a list of sub-counties with reported cases of sleeping sickness was made based on records at the Coordinating Office for the Control of Tsetse and Trypanosomiasis in Uganda (COCTTU records January 1998–March 1999) and nine of these (three sub-counties selected by simple random procedure from each of the three district) were chosen for the study. Within each sub-county tsetse trapping was carried out in at least five villages which were chosen

Table 1 Total livestock population and livestock species composition in each of the three districts (based on available District Veterinary Records from 1998) District

Kamuli Mukono Torero a

Total livestock population

535310 405310 425215

Cats, dogs, rabbits and poultry.

Percentage (%) compostion of each animal species Cattle

Goat

Sheep

Pig

Othersa

30 35 40

25 10 15

5 4 8

15 20 5

25 31 32

C. Waiswa et al. / Acta Tropica 99 (2006) 23–29

from a list of villages that had recorded sleeping sickness in the year of the study or had at least one case within the past two years. A list of such villages was made for each of the nine sub-counties and villages in each sub-county constituted the sampling frame from which five villages were selected by simple random procedure. 2.2. Tsetse monitoring 2.2.1. Tsetse capturing method Biconical tsetse monitoring traps were deployed in the selected villages targeting areas of human and animal activity. Twelve traps (at least 200 m apart) were deployed for each square kilometer (Lancien, 1991), covering an area of approximately 5 km2 per sub-county. Traps were set in each study village for two days and tsetse collected every 24 h. During harvesting, each field assistant was assigned two traps and the tsetse were immediately transferred to a waiting van and taken to the field station for dissection. This ensured no losses due to death of the tsetse. 2.2.2. Determination of tsetse density The apparent density (AD) was calculated as “the number of tsetse captured per trap per day”.

in the FAO Training manual for tsetse control personnel (1979). However, from category 5–7, the degree of the wing fray was considered before putting the fly in a given age category as described in the EATRO booklet, 1955 (unpublished). 2.2.4. Determination of infection rates The dissection was carried out as described by the FAO Training manual for tsetse control personnel (1979). All the dissections were carried out in isotonic saline (0.9%). A cover slip was then put on each part of the slide where the proboscis or salivary glands or the midgut (including the proventriculus) were placed and trypanosome infections in the tsetse flies were identified using a compound microscope at a magnification of ×400. Parasites found in the mid-gut, salivary glands and mouthparts were regarded as Trypanozoon; those located in the mouthparts and mid-gut were Nannomonas; while those found in the mouthparts only were put in the group of Duttonella. Infections confined in both the mouthpart and mid-gut were classified as immature Nannomonas or Trypanozoon. The Infection rate (IR) was calculated using the following formulae. Infection rate (IR) =

2.2.3. Ageing of tsetse flies 2.2.3.1. Male tsetse. The age estimation was done according to the degree of wear or fraying observed on the hind margin of the wing. According to the degree of wear, flies were assigned to one or other of the six categories as described by Jackson (1946). After giving the wing fray category, the age was estimated using directions for estimating the mean age of a sample of tsetse flies as given by the East African Trypanosomiasis Research Organisation (EATRO, 1955, unpublished). 2.2.3.2. Female tsetse. This method was used for the aging of the female tsetse flies and the flies were agegraded according to the contents of the uterus, relative development of the four ovarioles, and the presence or absence of follicular relics in these ovarioles. Ageing of the female tsetse flies using this method was done by carrying out tsetse dissection and observing the contents of the uterus and the relative sizes of the follicles in each of the two ovaries and in each of the two ovules that constitute each ovary. It is possible to recognise eight age categories based on the ovarian cycles. The sub-division of each of the age category was done as described by Saunders (1962) and followed as illustrated

25

Number of tsetse flies infected × 100 Total Number of tsetse flies dissected over a given period

2.2.5. Determination of tsetse hosts Tsetse that had undigested bloodmeals, were dissected and the blood smeared directly onto numbered sectors on Whatman filter paper No. 1, which had earlier been soaked in sodium azide 1% (w/v) solution. Samples were dried at room temperature, and then washed in acetone for 5 min in order to kill any pathogens, air dried and stored at −20 ◦ C (Boid et al., 1999). Control preparations of blood from cattle, pigs, dogs, sheep, goats, rat and monitor lizard were similarly made on Whatman filter paper No. 1. 2.2.5.1. Elution. Extraction of the sera was carried out as described by Boid et al. (1999). Preparations were stored at −20 ◦ C until further analysis. 2.2.5.2. Bloodmeal idenficiation by ELISA. Each bloodmeal was screened by enzyme-linked immunosorbent assay (ELISA) against a number of potential host species, namely—cattle, pig, human, sheep, goat, chicken, dog, monitor lizard and rat. The assays were carried out as previously described (Clausen et al., 1998; Boid et al., 1999).

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Results were read after 30 min using an automated ELISA plate reader at 450 nm. Positive identification was determined as an absorbance 0.05 nm greater than the mean of the negative control plus five standard errors from the mean of the sample in question. 2.3. PCR analysis for Trypanosoma brucei s.l. DNA Since PCR can be carried out on serum samples and allows a delayed processing of the samples while giving a highly species specific diagnosis (Desquesnes, 1997), the eluates from blood meals in this study were also tested for presence of T. brucei s.l. DNA. Analysis was carried out using primers originally described by Moser et al. (1989). Briefly, a 25 ␮l PCR reaction routinely contained: 1 Units BIOTAQ RedTM DNA polymerase (Bioline), 1 × NH4 buffer with a final concentration of 1.5 mM MgCl2 , 0.2 ␮M of each dNTP and 0.4 ␮M of each primer, with 1 ␮l of eluted serum. Amplification conditions were adapted from Picozzi et al. (2002); an initial denaturation step of 95 ◦ C for 3 min, was followed by 35 cycles of 94◦ for 20 s, 55◦ for 30 s, 72◦ for 30 s, a final extension step of 5 min was preformed to ensure complete amplification. PCR products were separated by agarose (1.5%, w/v) electrophoresis containing Ethidum bromide (0.2 ␮g/ml) and then visualised on a UV transilluminator. 3. Results 3.1. Apparent density of tsetse across the three districts In total, 2447 tsetse were collected from across the three districts. Table 3 gives the apparent density in the study areas. Glossina fuscipes fuscipes was the only species of tsetse recorded from the surveys carried out in Kamuli and Mukono districts. In Tororo district, two Glossina pallidipes (2/660) were also found amongst the fly catches. 3.2. Age and sex of the tsetse caught within the three districts The average age for the trapped male flies in Kamuli was 11 days, for Mukono and Tororo districts the average was less than 11 days old. Trapped female flies were on average aged between 20 and 30 days (Table 2). In Tororo district the proportion of flies aged >30 days old was significantly higher than in the other study areas (χ2 = 31.76, p ≤ 0.001).

Table 2 Age distribution of female G f. fuscipes captured in each of the three districts Age categories (days)

Kamuli

Mukono

Tororo

<8 9–19 20–30 31–40 41–50 50+

74 149 339 119 36 11

94 89 147 68 14 2

147 209 440 275 81 21

Total

728

414

1172

3.3. Trypanosome infection rates within tsetse The trypanosome infection rates for the three districts are given in Table 3. Trypanosomes were observed in G.f. fuscipes with an infection rate, as determined by microscopy, of 1.55% across the three studied areas. Trypanosome infections were only observed in female tsetse, giving an infection rate of 2.39% amongst the female flies (Table 3). Overall 86.8% (or 33/38) of the trypanosome infections, carried by the female tsetse, were identified as belonging to the Duttonella group; these were classified as Trypanosoma vivax and the 13.2% (5/38) were T. brucei s.l. None of the dissected male flies (857 in total from the three study areas) was found infected. There was no significant difference in the proportion of tsetse infected with trypanosomes between the three districts (χ2 = 1.987, p = 0.37). However, there was an age related effect in the number of trypanosome infections detected by microscopy with the number of infected Table 3 Tsetse apparent density (AD) and infection rates (IR) in the three districts Parameters No. of Tsetse/trap/day (AD) Highest Lowest Total number of tsetse dissected Total number of tsetse infected Overall infection rate (IR%) Number of male flies dissected Number of male flies infected Number of female flies dissected Number of female flies infected Infected female flies (<30 days) Infected female flies (>30 days+) IR% amongst female flies

Kamuli

Mukono

Tororo

20.66 10.00

14.17 1.46

9.17 4.33

1086 19 1.75 358 0 728 19 7 12 2.61

701 7 1.00 287 0 414 7 3 4 1.7

660 12 1.82 212 0 448 12 1 11 2.68

The combined IR amongst females flies from across the three study areas = 2.39%.

C. Waiswa et al. / Acta Tropica 99 (2006) 23–29

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Table 4 Origin of bloodmeal of G.f. fuscipes captured in each of the three districts and number of flies harbouring a T. brucei infection in their residual bloodmeal Animal species

Study districts Kamuli

Avian Cattle Dog Human M. lizard Pig Rat Sheep/goat Totala

2 (2%) 19 (18%) 9 (9%) 2 (2%) 23 (22%) 41 (40%) 0 7 (7%) 103

Total Mukono 0 30 (32%) 1 (1%) 6 (6%) 32 (34%) 17 (18%) 0 8 (9%) 94

Positive for T. brucei by PCR

Tororo 0 55 (54%) 3 (3%) 6 (6%) 26 (26%) 11 (11%) 0 0 101

2 (1%) 104 (35%) 13 (4%) 14 (5%) 81 (27%) 69 (23%) 0 15 (5%) 298

15/104 (14%)

8/81 (10%) 13/69 (19%)

36/254 (14%)

a

Two hundred and ninety-eight (76%) of the 394 blood spots were identified; 16 (4%) were in the mixed detection category and were not classified while the remainder 80 (20%) could not be identified and therefore remained unclassified.

flies older than 30 days being significantly higher than those aged <30 days (χ2 = 33.12, p ≤ 0.001).

brucei irrespective of the host from which the bloodmeal had been taken (χ2 = 5.13, p ≤ 1.0).

3.4. Bloodmeal identity

4. Discussion

A total of 394 blood meals were collected from G.f. fuscipes during the study (Table 4). Hosts were identified in 76% of the bloodmeals, while 20% could not be associated with any of those species tested for. The remaining 4% of the samples cross-reacted with more than one species among those tested and were therefore left unclassified. Cattle, pigs and monitor lizards were the main hosts for tsetse in all the three districts and the percentage contributions are given in Table 4. At 40%, the pig was the predominant host for tsetse in Kamuli, while cattle and the monitor lizard were the predominant hosts in Mukono, accounting for >60% of all identified bloodmeals. In Tororo district, cattle were identified as the main host for tsetse with 54% of all feeds being bovid in origin (Table 4). Linear regression analysis showed that the number of tsetse feeds on a cattle was proportional to the number of cattle within the domestic herd (R2 = 0.9838).

This paper reports on tsetse fly populations in south eastern Uganda. 2447 tsetse were collected from across Kamuli, Mukono and Tororo districts. G.f. fuscipes was the prominent species of tsetse, accounting for 99.9% of trapped flies. However, in Tororo district a small number of G. pallidipes were also trapped. G. pallidipes used to be a major vector of sleeping sickness in south east Uganda but had not been recorded for three decades (Magona et al., 2005). It is possible that the Ugandan side of the border is experiencing a re-invasion of this tsetse species from Kenya where it has been always reported (FITCA records). G. pallidipes has however been identified as the major species infesting ranches in the Luwero district of central Uganda (Okello-Onen et al., 1994). The apparent densities of tsetse varied, with the number of flies/trap/day ranging from 2 to 21. Our reported AD of tsetse flies in Mukono and Tororo districts were similar to previously reported levels (Okoth et al., 1991; Katunguka-Rwakishaya and Kabagambe, 1996). However, there are no published data for Kamuli district with which to compare our results. Using parasitological techniques, our investigation revealed an overall trypanosome infection rate of 1.55% which was similar to those recorded during the 1976–1989 sleeping sickness epidemic in south eastern Uganda (Okoth and Kapaata, 1986) but significantly higher than the 0.5% recorded by Okoth et al. (1991). As expected a high proportion Duttonella infections was

3.5. Detection of T. brucei DNA within fly bloodmeals All bloodmeals were screened by PCR for the presence of T. brucei s.l. DNA. It was found that 14% (36/254) of bloodmeals contained detectable levels of T. brucei s.l. genomic material (Table 4). There was no significant difference between the detectable levels of T.

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carried by flies, when compared to T. brucei s.l. (Jordan, 1974). According to Waiswa et al. (2003), the overall trypanosome prevalence among cattle in the area was 13.25%, with the prevalences of 4.99%, 5.44%, 2.06% for T. brucei s.l., T. vivax and T. comgolense, respectively. Of all the infected cattle, more than 44% carried T. vivax infections carried as single or mixed infections (Waiswa et al., 2003) and during our investigation, T. vivax was recorded at 86% which could be partly explained by the high prevalence of the parasite in the animals and by the fact that since it has the simplest life cycle (Jordan, 1974). Only female flies were found to carry these infections, increasing the infection rate to 2.39% of female flies. The failure to find infected male flies can be explained by the low average age of trapped male flies (11 days or less) as infections take 5–53 days to mature. G.f. fuscipes is acknowledged as a non-preferential feeder, with bloodmeals taken from the most available hosts (Clausen et al., 1998). During this investigation the origins of 76% of the bloodmeals were identified, with cattle being the preferred host for G.f. fuscipes in south eastern Uganda. The absence of wild game in present day south eastern Uganda could be the main factor for the high percentage of blood taken from livestock. Previous studies in south eastern Uganda have also highlighted the importance of reptiles, in particular monitor lizards, as a food source for tsetse (Okoth and Kapaata, 1986; Clausen et al., 1998); however, the percentage of monitor lizard feeds recorded during this investigation is much lower than the 73–98% recorded along the shores of Lake Victoria in Kenya (Mohamed-Ahmed and Odulaja, 1997). The identification of avian as a source of food (1% of the bloodmeals) for G.f. fuscipes is consistent with previous reports (Okoth and Kapaata, 1986). Although chicken have been reported to be susceptible to infection with T.b. brucei and T.b. rhodesiense under experimental conditions (Joshua et al., 1982), no epidemiological significance was attached to this finding in our study. Cattle as the major blood source for tsetse in Tororo district reflects the important role that these animals play as reservoirs for sleeping sickness within this area (Waiswa et al., 2003; F`evre et al., 2005; Welburn et al., 2001). Moreover, linear regression analysis relates the proportion of feeds on cattle to the proportion of this species within the domestic herd. Similarly, in areas that keep many pigs for example Kamuli district the pig was the predominant tsetse host, accounting for 40% of all feeds. A similar finding was reported in Mukono district in 1998 (Clausen et al., 1998). In light of the recent identification of pigs as a previously overlooked reservoir of potentially human infective trypanosomes,

this feeding preference has serious implications for the implementation of disease control (Waiswa et al., 2003). Currently most programmes aimed at reducing the transmission cycle of trypanosomes focus on cattle (F`evre et al., 2005); as pigs too are an important tsetse food source within these areas and carry T. brucei infections (Waiswa, 2005), control programmes need to consider these animals. Selective screening of bloodmeals identified T. brucei DNA in 14% of fly feeds; none of these flies had mature salivary gland infections by microscopic analysis. The presence of trypanosome DNA is therefore due to either an immature gut infection or, less likely, carry-over from a previous infected feed. In a previous study the T. brucei s.l. infections in tsetse were inoculated in White Swiss mice and thereafter tested and some were found to be potentially human infective by the human serum resistance test (BIIT) (Waiswa et al., 2003). Taken together with the feeding preference of tsetse flies sampled suggests that active transmission of T.b. rhodesiense is occurring within these areas. As 5% of bloodmeals were identified as human in origin, action should be taken to break the cyclic spread of sleeping sickness from “cow/pig to fly to man”. 5. Conclusion From the study, it is apparent that hosts for G.f. fuscipes vary; the feeding patterns of these flies seem to depend on the availability, abundance and behaviour of the host animals. In addition to cattle, other animal species that are major hosts for tsetse in given sleeping sickness foci, particularly pigs, should be considered in bait and other tsetse and trypanosomiasis control strategies. Acknowledgements This Investigation received financial assistance from UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical diseases (TDR) (C.W.), DAAD (C.W.), The Cunningham Trust (K.P.), DFID Animal Health Programmed (S.C.W., K.P., C.W.), although the views expressed are not necessarily those of the sponsors. We thank Eric F`evre for advice on the manuscript. References Boid, R., Jones, T.W., Munro, A., 1999. A simple procedure for the extraction of trypanosome DNA and host protein from dried blood meal residues of haematophagous Diptera. Vet. Parasitol. 85, 313–317.

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