Trypanosome prevalence in Glossina fuscipes fuscipes (tsetse) and cattle along the shores of Lake Victoria in Tanzania

O Manangwa, J O Ouma^1,2, I Malele, F Mramba³, A Msangi⁵ and G Nkwengulila⁴

Vector and Vector Borne Disease Institute, P O Box 1026, Tanga, Tanzania
okijanga@yahoo.com
¹ Africa Technical Research Centre, Vector Health International (VHI), PO Box 15500, Arusha, Tanzania.
² Trypanosomiasis Research Centre, Kenya Agricultural Research Institute, P.O. Box 362, Kikuyu, Kenya
³ Tanzania Veterinary Laboratory Agency (TVLA) P. O. Box 9154, Dar es Salaam, Tanzania, P O Box 9152, Dar es Salaam, Tanzania
⁴ Department of Zoology, University of Dar es Salaam, Tanzania, P O Box 35064, Dar es Salaam, Tanzania.
⁵ Recent retired scientist from Ministry of Livestock and fisheries development, Tanzania

Abstract

Tsetse flies transmit trypanosomes which affect livestock and human health in sub Saharan african countries including Tanzania.The wide occurrence of this disease in people and their livestock has negatively impacted the development of viable agricultural systems in many areas of great agricultural potential including areas of the Lake Victoria basin in Tanzania.The present study investigated trypanosome infection rate in G. f. fuscipes in Kirongwe, Masonga, Rasi Nyabero and Tobwe River in Rorya district, Msozi village in Ukerewe district and Kemondo in rural Bukoba district during the dry and wet seasons. Trypanosome infection in cattle was also investigated in the same villages except Kemondo village with a view to understand the risk of the disease to livestock and humans in the study area.Tsetse flies were collected using biconical and pyramidal traps in Kirongwe, Masonga, Rasi Nyabero and Tobwe River.Twenty traps were deployed after every 150 to 200 meters during the dry season and wet season. A total of 120 flies were examined microscopically and 215 by Polymerization Chain Reaction (PCR) for trypanosome infection. Trypanosomes infection in cattle was also assessed in order to understand the risk of the disease to cattle and humans in the study area. Blood was collected from 116 cattle and examined under microscope for trypanosome infection. Some blood was stored in FTA cards for trypanosome infection rate analysis using PCR.

No infection was detected microscopically but PCR showed (2%) of G. f. fuscipes was infected by T. b. brucei at Rasi Nyabero village during the dry season. Similarly, for the wet season, one female fly (2.5%) out of 40 was found positive for T. congolense Kilifi in Msozi village. Microscopic examination showed no cattle were infected but PCR indicated one female cow out of 116 cows (0.86%) at Rasi Nyabero village was infected with T. vivax. Generally, trypanosome prevalence in G. f. fuscipes and cattle was low for all seasons. The low infection rate should not be taken for granted, regular surveillance is important in monitoring the problem in the study area since the Lake Victoria shore is known to be a risk area for trypanosomiasis.

Key words: disease, infection rates, transmission

Introduction

Tsetse flies (Diptera: Glossinidae) transmit several pathogenic trypanosome species, which affect livestock and human health in sub Saharan countries including Tanzania. Trypanosomes cause sleeping sickness in humans and nagana in animals. Transmission occurs largely among rural populations, where activities such as agriculture and fishing expose people to the bite of the tsetse fly and increase the risk to infection with trypanosomes (Muturi et al 2011). About 33 percent of the land in Tanzania is tsetse infested (Daffa et al 2013). The losses suffered annually in cattle due to the disease in terms of mortality, morbidity and reduced milk yields have been estimated at US $ 7.98 million in Tanzania (MOAC 1995). About 4 million people are at risk of acquiring sleeping sickness and 400 cases are reported annually (Sindato et al 2008). G. f. fuscipes is one of the important tsetse species in Tanzania that transmit trypanosomes to human and livestock. The species has riverine tendencies, and inhabits shores of lakes and river-banks. In Tanzania this species is distributed from the northeastern shores of Lake Victoria in Rorya district in Mara region to the north western shores of the lake in Rubafu area in Kagera, as well as on the islands in the Lake Victoria (Manangwa et al 2015).

There are several methods which are used today in trypanosomes diagnosis in tsetse and domestic animals in the field and laboratory. The methods are grouped into three major categories; Direct methods (parasitological), these include use of microscope, microhaematocrit buffy coat, and mouse inoculation. These methods are simple and rapid, and good for screening large herds of animals in the field though they have lower sensitivity. Indirect methods (serological) include antibody Eliza and antigen Eliza (Connor 1992). These tests measure the interaction between antigen and antibody. The major limitation of these methods is the occurrence of cross-reactions of different trypanosome species (Luckins 1992). Recent developed diagnostic molecular tools in particular PCR have been used for trypanosomiasis diagnosis in domestic livestock and tsetse flies for over 10 years (Picozzi et al 2002). These methods have improved the detection of trypanosome infections over the standard parasitological methods (Cox et al 2010). However the methods are more sensitive and specific, their reagents are very expensive and cannot be done in the field setting.

Studies on trypanosome infection rates in G. f. fuscipes have been carried out in different places in Africa including Sudan, Ethiopia, Uganda and Democratic republic of Congo. Very little is known on the trypanosome infection rate in G. f. fuscipes and in cattle in the Lake Victoria basin in Tanzania. Such information is required for estimating and mapping the local risk of HAT and nagana transmission in the area.The shores of Lake Victoria were first reported to have sleeping sickness at the beginning of the 19^th century. In 1907, sleeping sickness was detected in Shirati in Rorya district and also in Ukerewe Island (Leak 1999). It was revealed that the disease was caused by T. b. gambiense transmitted by G.f. fuscipes in the area (Klein 1909). Since then the disease has not been reported in humans or cattle in the Lake Victoria basin in Tanzania. Understanding the extent of the occurrence of trypanosomes in G. f. fuscipes is important as it indicates the potential transmission of trypanosomiasis to people and livestock (Basheer et al 2012 & Ahmed et al 1989). Furthermore, the information is very useful when planning control activities as it makes it possible to direct control efforts where they are most needed in order to halt the transmission of the disease in a particular area. Therefore, the present study investigated trypanosomes infection rates in G. f. fuscipes and in cattle in order to estímate the risk of the disease for control purposes in the Lake Victoria basin in Tanzania.

Materials and methods

Description of the study area

The study was conducted along the shores of Lake Victoria which included Kirongwe (KW), Masonga (MO), Tobwe River (TW), and Rasi Nyabero (RN) villages in Rorya district; Msozi villange in Ukerewe district (island) and Kemondo in Bukoba district. Sampling sites were: Shirati division, Ukerewe Island and Kemondo division. Generally all these areas experience a bimodal rainfall pattern, the short rains fall between October and December and the long rains from March to May. The average annual rainfall in the basin is estimated to be 1015 mm (Makalle et al 2008).

Source: Yale university GIS unit ------ International boundary ,    Study sites ,      Lake Victoria

Data collection

G. f. fuscipe were collected using biconical and pyramidal traps for three days in each site for three weeks during the dry and wet seasons. Each trap was geo-referenced using a Global Positioning System (GPS) for later incorporation in Geographical Information System (GIS) for map development. Twenty traps (10 biconical traps and 10 pyramidal traps) were deployed after every 150 to 200 meters during the dry season and wet season. Traps were 1- 5 meters away from water level in all sites for both seasons. Collected flies were aged, sexed and dissected for determination of trypanosomes infection.

Determination of Trypanosome infection in flies

Trypanosome prevalence was determined in order to understand the trypanosome infection patterns in flies. Very few flies were dissected for this purpose as more than 90% of the caught flies were found very dry and hence not suitable for dissection. Flies were dissected within 4 h after collection daily. The proboscis, mid-gut and salivary glands were separately investigated for trypanosomes (Lloyd & Johnson 1924). It is important to examine each of these separately as trypanosome species tend to be site specific. Since very few flies were dissected, alternatively infection rate was determined using whole dry flies (stored in ethanol) by PCR.

DNA extraction for the dry and wet season samples

The purpose of DNA extraction was to obtain total genomic DNA from the whole fly for the aim of estimating trypanosome prevalence in G. f. fuscipes. Fifty flies collected from Rasi Nyabero during the dry season and preserved in absolute ethanol in individual 1.5 mL eppendorf tubes were air dried after pouring off the ethanol before grinding. The ground materials were then used to extract DNA. DNA extraction was done following the ammonium acetate precipitation protocol for DNA extraction (Brudford et al 1998). For the wet season, a total of 165 flies from Rasi Nyabero (45), Masonga (40), Bukoba (40) and Ukerewe (40) were analyzed. After decanting the alcohol used for preservation, distilled water was added into individual tubes containing flies and allowed to settle for 5-10 minutes as a way to remove the excess alcohol. Individual flies were each placed in a 1.5 mL individual eppendorf tubes and placed into 96-well racks. Liquid nitrogen was added into each fly (freezing) and immediately crushed using micro pestle to get a fine powder that was used for DNA extraction. Further extraction was carried out using Qiagen DNA extraction kit following a procedure described by the manufacturer.

Determination of Trypanosome infection in cattle

The survey for animal trypanosomiasis in cattle was carried out in order to determine the epidemiology of the disease in the study area. A total of 116 cattle were screened for trypanosomes infection in Kirongwe (n =31), Rasi Nyabero (n =35), Masonga (n =30) and Msozi village (n =20) in Ukerewe district. Blood was drawn from the jugular vein, thick and thin blood smears were prepared and stained with Giemsa for examination of trypanosome infection. Some blood from the jugular vein was also preserved in FTA cards for the determination of trypanosome infection by PCR.

DNA extraction from FTA cards for Trypanosome prevalence in cattle

A 6 mm diameter disc was punched from the centre of the dried blood in the FTA card and placed into a 0.5 mills micro centrifuge tube. Two hundred micro liters (200 µl) of water was added into each sample and incubated at 37°C for 30 minutes. Then water was removed after incubation and 60 µl of sterile water was added in the original tube with a filter paper disc and incubated at 100°C for 30 minutes for the second time. After incubation, the supernatant was transferred to a new labeled tube ready to be used for trypanosome infection analysis. Extracted DNA was amplified for the detection of human infective trypanosomes as well as animal trypanosomes following this reaction condition: PCR reaction conditions for the above PCR included sterile distilled water 20µl, forward primer and backward primer1µlfor each, 2µl DNA template and 1µl dry bead making a final volume of 25µl. PCR amplification was accomplished using the following program: initial denaturation at 95^oC for 5 minutes followed by 30 cycles of 95^oC of second denaturation at 1min, 55^oC annealing temperature for 1min, 72^oC for fragment extension for 1min and final extension was done at 72^oC for 5min. The PCR product was viewed by electrophoresis on a 5% agarose gel and the bands observed under UV light.

Results

Trypanosome infection in G. f. fuscipes (Microscopy)

None of the 94 G. f. fuscipes from Rasi Nyabero and Kirongwe village were found infected by trypanosomes during dry season. However, in some sites flies caught were not dissected because of low catches e.g. at Tobwe River and Masonga village. Also, trapped flies at some sites were very dry, and thus not suitable for dissection e.g. Masonga village. Only twenty six G. f. fuscipes were dissected from Rasi Nyabero village which included 16 females and 10 males during the wet season. During this season only a few flies were dissected because most flies caught were found dead and very dry. This observation is contrary to other tsetse species, where a higher number of live flies are usually obtained the next day for dissection. Results indicated that all dissected flies were microscopically negative for trypanosome infection.

Trypanosome prevalence determination by PCR

Results showed that only one male fly (2%) was positive for T. brucei group at Rasi Nyabero village during dry season (table 1). Evaluation by SRA- PCR for the presence of the serum resistance associated (SRA) gene indicated that the T. brucei detected was not human infective. Only one fly (2.5%) from Msozi village was positive for T. congolense Kilifi. None of the flies from Rasi Nyabero, Masonga and Kemondo were infected during wet season.

Blood was analyzed microscopically and by PCR for trypanosome infection. Microscopic examination did not reveal trypanosome infection in cattle. Further analysis of infection was made by ITS - PCR. ITS - PCR indicated a prevalence of 2.85% (one cow out of 35) of cows was infected by T. vivax at Rasi Nyabero village.

Age determination of G. f. fuscipes

More than 50% of G.f. fuscipes were in wing fray category 1 ( see table 2) i.e. flies with less than 12 days and a few flies were in wing fray category 6, 5 and 4. Generally almost all flies caught were young with age ranging from >11 days to 14 days during the dry season. Similarly for the wet season the age of the majority of flies fell in the wing fray category 1 although the percentage, were slightly lower compared to the dry season results but the percentages were higher compared to wing fray category 2 - 6 in all villages (see table 3). The estimated age varied from 11 days to19 days.

Discussion

Generally, microscopy did not reveal trypanosome infection in the present study in both seasons. The method is known for low sensitivity and specificity though it is good when screening large number of animals in the field (Woolhouse et al 1994). Very few flies (120) were dissected from three villages (Kirongwe and Masonga) for both wet and dry season; flies were found very dry for dissection and few tsetse catches were obtained in some site particularly in Tobwe River. A larger sample of dissected flies could probably give a much more realistic picture than the results reported in this study. The shores of Lake Victoria are known to be a focus of trypanosomiasis infection for many years hence regular surveillance is important in monitoring the problem in the area (Leak 1999).

Recently, more sensitive and specific molecular biology tools such as PCR have been developed and applied (Picozzi et al 2002). Studies in Uganda have shown that the detection rate by PCR was two times higher than parasitological techniques (Clausen et al 1998). Similarly, in the present study the PCR method detected infection that was not detected by microscope. The low trypanosome infection rates observed in the present study by PCR corroborate results reported in southeastern Uganda in which low trypanosome infection rate (1.55%) was observed in G. f. fuscipes in the study area (Waiswa et al 2006). However, findings in southwest Ethiopia reported higher infection rate of 22.8% in adult G. f. fuscipes (Bitew et al 2011).The discrepancy in the trypanosome infection in G. f. fuscipes between the study conducted in Ethiopia and the present study might be due to differences in the average age of the flies dissected. Field data suggest that older flies have higher chances of becoming infected with trypanosomes than younger flies (Tarimo et al 1985). Most flies caught in the present study during the dry and wet season were young, falling into the wing fray category one and two (Table 3 & 4). Moreover studies has shown that infections are easily acquired, particularly of the brucei-type when the first blood meal is infected, while chances of transmitting infections is higher in adult flies, especially those old enough to have allowed the development of the parasites to the infective stages ( Kuzoe and Schofield 2004). Often therefore, natural populations of tsetse show relatively low infection rates; generally less than 0.1% in the case of human infective forms, but can be as high as 10 -15% in the case of animal trypanosomes ( Leak 1999). Another possible reason for the higher trypanosome prevalence obtained in Ethiopia could be the differences in the available hosts between Ethiopia and Lake Victoria in Tanzania (Bitew et al 2011). Different tsetse species have different host preferences; for example flies feeding on bovids have higher trypanosome prevalence than those feeding on other hosts like bushpig and warthogs (Jordan 1974). Tsetse hosts like suids have less infection due to their refractoriness to some trypanosome species like T. vivax (Leak 1999). One other reason could be low trypanosomes infection in animal and human populations in the study area. G. f. fuscipes are not as good vectors of trypanosomes like other tsetse species (Abila et al 2008). This could also be a reason for lower infection reported in the present study. Report by Ikeda et al 1986 in Nigeria indicated some riverine and forest species of tsetse flies were poor vectors of trypanosomes than the savanna species.

Trypanosome infection rate in cattle was also low in all surveyed villages in the study area for both methods used. ITS- PCR revealed an infection rate of 0. 86 (T. vivax) at Rasi Nyabero village and none was detected by microscopy. In the present study, DNA was extracted from blood samples that were stored on FTA cards, whereby a single punch of FTA card was used in the DNA extraction. This could be one reason for the low trypanosome prevalence in cattle. Multiple PCR samples taken from FTA cards with a single punch has been reported to be insufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card (Cox et al 2010). The sample volume contained on the punched-out material represent only a small fraction (often < 1%) of the total blood sample captured on the FTA card, which is itself extremely small in relation to the volume of blood within a host. It has been shown that taking several punches increases the detection rate of Trypanosoma species from 9.7% to 86% (Cox et al 2010).This practice was not adopted in the present study because the information was not available when the present study was conducted. Trypanosomiasis infection is characterized by fluctuation of parasite numbers in the blood, if the sample is taken when the parasitemia was low in the animal the sampling effect will result in underestimation of prevalence based on single punch PCR testing. The result of trypanosome infection in the present study corroborate results obtained in Uganda where low prevalence of T. brucei (0.91%), T. congolense (1.58%), and T. vivax (1.05%) were observed in zebu cattle with no mixed infection (Cox et al 2010). A study in western Kenya based on parasitological methods reported low prevalence (1.2%) despite screening a larger number of cattle (n = 402) than we screened in this study (Ng`ayo et al 2005).

Conclusion

• Generally, the prevalence of trypanosomes in G. f. fuscipes in the present study was low as revealed by both microscopy and PCR. Flies from Rasi Nyabero (2%) and Msozi (0.61%) villages were found infected with T. brucei and T. congolense cliffi, respectively. Likewise for cattle, Rasi Nyabero village was found positive for T. vivax (0.86%).
  
  
• It is recommended that care should be taken when doing interpretation of this data particularly for parasitological infection rate as they are based on few dissections of G. f. fuscipes.The low trypanosome infection reported in the present study should not underestimate the epidemiological significance as it is an indicator of on going local transmission. Therefore, regular surveillance is important in monitoring trypanosomiasis disease in the area.

Acknowledgement

We are very grateful for WHO for funding this study. I wish to thank the Manager of Vector and Vector-Borne Disease Institute for logistical support which enabled our field to be conducted smoothly. I wish to thank my fellow colleagues Benedict Kimbisa and Godfrey Mashenga for their help on data collection in the field.

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Received 2 July 2016; Accepted 7 July 2016; Published 1 August 2016

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