Development of a non-destructive PCR method for detection of the salivary gland hypertrophy virus (SGHV) in tsetse flies

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Abstract

A PCR based diagnostic method to detect salivary gland hypertrophy virus (SGHV) in tsetse flies is described. Two sets of primers GpSGHV1F/GpSGHV1R and GpSGHV2F/GpSGHV2R were selected from a virus-specific sequence. Both primer sets can detect specifically the virus in individual tsetse flies by generating an amplicon of 401 bp. Attempts were made to develop a simple and reliable non-destructive virus detection method in live flies. PCR reactions were performed on either crude or purified tsetse DNA from saliva and legs. While saliva can be an indicator for the presence of the virus in flies, the method is laborious. Crude extract from an excised middle leg resulted in a positive PCR reaction equivalent to crude extract from whole fly. However, sensitivity could be significantly increased when purified DNA was used as the template. In conclusion, PCR using a purified DNA template from a single tsetse leg represents an efficient, non-destructive method for virus diagnosis in live tsetse flies.

Introduction

Tsetse flies (Diptera: Glossinidae) are the sole vectors of cyclical pathogenic trypanosomes in tropical Africa, Trypanosoma brucei rhodesiense and T. b. gambiense causing Human African Trypanosomiasis, or sleeping sickness and T. b. brucei, T. congolense and T. vivax causing a wasting and fatal disease in cattle, domestic pigs and other farm animals, known as nagana. It is conservatively estimated by the World Health Organization (WHO, 2001) that there are currently 300,000–500,000 cases of Human African Trypanosomiasis with 60 million people at risk in 37 countries covering approximately 40% of Africa (11 Mkm2), while the animal disease has restricted agricultural development and nutritional resources in sub-Saharan Africa, profoundly impacting the economy of much of the continent (Jordan, 1986, Steelman, 1976). Control of vector populations is one of the main methods used for controlling the disease. Following the eradication of Glossina austeni from the island of Unguja, United Republic of Tanzania, using the sterile insect technique (SIT) (Vreysen et al., 2000), new programmes incorporating the sterile insect technique are being developed for application on the African mainland including one in the Southern Rift Valley of Ethiopia which targets Glossina pallidipes (Feldmann et al., 2005).

The sterile insect technique requires the successful colonisation and rearing of large numbers of the target species and in support of the programme in Ethiopia, the Entomology Unit of the FAO/IAEA Agriculture and Biotechnology Laboratory Seibersdorf was tasked with the establishment of a colony from the target population in the Southern Rift valley. Shipments of G. pallidipes pupae from the target area were received regularly and by July 2000 the colony reached a size of 15,000 females. Subsequently, the colony experienced a steady decline and became extinct by 2002. During this period substantial efforts were made to identify the cause of the decline, and environmental conditions, blood quality, handling protocols and pupation conditions were all excluded as possible causes. During dissection of females to assess if poor insemination could be a factor, an extremely high proportion of individuals, over 85% in some samples, displayed salivary gland hypertrophy (SGH) (Fig. 1A).

Hypertrophy of the salivary glands has long been known to occur in wild tsetse flies (Burtt, 1945, Whitnall, 1934) and a virus infection was later identified as the cause not only of salivary gland hypertrophy (Jaenson, 1978), but also of testicular degeneration and ovarian abnormalities (Jura et al., 1988, Kokwaro et al., 1990, Sang et al., 1998, Sang et al., 1999). Salivary gland hypertrophy virus (SGHV) was subsequently observed in many tsetse species from different African countries (Ellis and Maudlin, 1987, Gouteux, 1987, Jura et al., 1988, Jura et al., 1989, Minter-Goedbloed and Minter, 1989, Odindo et al., 1982, Otieno et al., 1980, Shaw and Moloo, 1993). The presence of the salivary gland hypertrophy virus has also been shown to affect the development, survival, fertility and fecundity of naturally (Feldmann et al., 1992) or artificially (Jura et al., 1993, Sang et al., 1997) infected flies. Odindo et al. (1986) characterized the virus particles, which appeared to be rod-shaped and containing a double-stranded, most likely linear, DNA genome. In the wild, the salivary gland hypertrophy virus is thought to be maternally transmitted (Jura et al., 1989). Under colony conditions where membrane feeding is used (Feldmann, 1994), as in Seibersdorf, horizontal transmission during feeding may be significant.

The collapse of the G. pallidipes colony in Seibersdorf and the potential risk of repeated failures during subsequent colonization attempts encouraged more studies on this disease in tsetse and prompted the development of sensitive specific PCR assay to unequivocally identify individuals that carry the virus.

The development of a PCR based assay for the virus would in theory enable virus free individuals to be selected in order to initiate a healthy colony. However, in tsetse with its viviparous reproduction and the labour intensive nature of rearing single families, there was a further requirement; i.e. the assay had to be non-destructive. This would enable flies that tested negative to be used immediately for colony maintenance and eliminate the need for progeny testing of individual families.

In the present report, a PCR diagnostic assay for salivary gland hypertrophy virus in tsetse is described. The development of a non-destructive assay is also reported which enables the virus status of live flies to be determined.

Section snippets

Strain

All experiments were carried on a G. pallidipes colony, originating from pupae collected in Tororo, Uganda in 1975 and initially colonized in The Netherlands. It was transferred to the Seibersdorf laboratory in 1982 and has been subsequently maintained on an in vitro rearing system. The adults were fed on heated, defibrinated, bovine blood by membrane feeding for 10–15 min three times per week. The deposited larvae pupated and were incubated at 24 °C until emergence (Feldmann, 1994, Gooding et

Virus purification, and viral DNA extraction

The virus was purified from hypertrophied salivary glands and viral DNA was extracted as described in Section 2. Electron microscopy observation of PTA-stained (phosphotungstic acid) purified virus preparation revealed rod-like particles 700–1000 nm in length and 50 nm in diameter (Fig. 1B). This observation concurs with viruses previously described in G. pallidipes (Jaenson, 1978, Odindo et al., 1986). The migration of purified viral DNA on an agarose gel revealed a high molecular weight,

Discussion

All previous studies to detect the salivary gland hypertrophy virus in both field and laboratory colonies relied on observations of salivary gland hypertrophy symptoms in dissected tsetse flies, electron microscopy examinations of thin sections or crude extracts of infected organs (Jaenson, 1978, Odindo et al., 1986, Ellis and Maudlin, 1987, Jura et al., 1993, Sang et al., 1999). This paper reports the first sensitive and reliable diagnostic method for the salivary gland hypertrophy virus based

Acknowledgments

We thank Abdul Hasim Mohamed, Musie Kiflom Gebretensae and Rudolf Boigner for their help with dissections and tsetse fly rearing.

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