Leonard H. Otieno

Detection of trypanosome infections in the saliva of tsetse flies and buffy-coat samples from antigenaemic but aparasitaemic cattle

Relatively simple protocols employing non-radioactive DNA probes have been used for the detection of African trypanosomes in the blood of mammalian hosts and the saliva of live tsetse flies. In combination with the polymerase chain reaction (PCR), the protocols revealed trypanosomes in buffy-coat samples from antigenaemic but aparasitaemic cattle and in the saliva of live, infected tsetse flies. Furthermore, the protocols were used to demonstrate concurrent natural infections of single tsetse flies with different species of African trypanosomes.

Recombinant DNA probes reveal simultaneous infection of tsetse flies with different trypanosome species

The utility of recombinant DNA probes in the detection of natural trypanosome infection of tsetse flies has been assessed in Lambwe Valley, near the shores of Lake Victoria, Kenya. The tsetse flies were surveyed during two different seasons in 1988. Three different probes used each contained highly repetitive DNA sequences specific for a species or subspecies of trypanosomes of the Nanomonas subgenus. A fourth probe contained repetitive sequences common to trypanosome species of the Trypanozoon subgenus. Mixed mature or immature infections were detected in a variety of combinations in different individual tsetse flies. Such infections were detected in both the guts and mouthparts of some tsetse flies. Simultaneous natural infection of tsetse with the savannah type Trypanosoma congolense and Kilifi type T. congolense, T. congolense and Trypanosoma brucei or T. congolense and Trypanosoma simiae were demonstrated. The probes have thus been used to demonstrate the presence of Lambwe Valley, south-western Kenya, of a type of T. congolense first observed among trypanosome isolates obtained from sentinel cattle exposed to natural infection on a ranch at Kilifi on the Kenya coast. This type of T. congolense appears not to be confined to the coastal region nor to any particular species of tsetse flies and may contribute significantly to livestock morbidity in other areas of eastern Africa. In the Kilifi area, T. congolense was found primarily in Glossina austeni; in Lambwe valley, it was found in Glossina pallidipes.

Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya, with a note on the ultrastructure of the affected organ.

Abstract Enlarged salivary gland was found to be widespread among wild populations of Glossina pallidipes in Kenya. The incidence of this abnormality varied from 0.9% in Meru National Park in Central Kenya to 5.4% in the Shimba hills area on the Kenya coast. Ultrastructurally, the enlarged glands were multinucleated with lumen reduced substantially in size. A large number of viruses filled both the lumen and the broken pieces of epithelial cytoplasm. In some cases Trypanosoma brucei trypanosomes were seen in the lumen of the enlarged glands. The epithelial cytoplasm was heavily vacuolated. Comment is made on the suitability of the diseased flies as transmitters of T. brucei .

Gonadal lesions in virus-infected male and female tsetse, Glossina pallidipes (Diptera: Glossinidae).

Abstract Ovaries and testes, dissected out and collected from wild Glossina pallidipes with virus-infected, hypertrophied salivary glands, were processed for routine electron microscopy and examined for pathological changes. Lesions in the male gonads were more severe than in their female counterpart and involved both testes in every case. Examination of the ovaries of the virus-infected females with hypertrophied salivary glands showed that although the occasional germarium appeared normal, the majority were affected by degeneration and severe necrosis. The degenerate ovarioles also showed virus particles within germarial cells. Sections of testicular follicles of adult males with hypertrophied salivary glands revealed a total arrest of spermatogenesis, depicted by complete absence of meiotic and postmeiotic stages. The follicles contained only sheets of undifferentiated premeiotic spermatogenic cells, i.e., spermatogonia and primary spermatocytes. In some testes, degeneration (characterized by diffuse vacuolation and exfoliation of degenerate cells leading to empty lumina) was superimposed on lack of sperm development. Testes of normal adult male G. pallidipes , on the other hand, showed swarms of entangled spermatozoa. The demonstration of virus particles within the germarium of the ovariole of females with hypertrophied salivary glands and the complete sterility observed in males with hypertrophied salivary glands identify the virus of G. pallidipes as a potential candidate for the biological control of the species.

The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae).

Abstract. Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies.The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0.0001) than those of the controls.Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Χ2 = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates.Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells.

The Effects of a DNA Virus Infection on the Reproductive Potential of Female Tsetse Flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae)

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P < 0.0001) and produced pupae that were of significantly less weight in milligrams (P < 0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P < 0.00001).

Ultrastructural evidence for trans-ovum transmission of the DNA virus of tsetse,Glossina pallidipes (Diptera: Glossinidae)

The occurrence of vertical transmission of the DNA virus of tsetse was studied in virus-infected, femaleGlossina pallidipes with hypertrophied salivary glands (HSG). Ultrastructural examination of tissue components of ovaries of these females revealed virus particles within both germ cell cystocyte clusters and in the follicles, sparsely distributed within nurse cells and in the oocyte cytoplasm. The presence of the virus particles within the ooplasm demonstrates the ovum as a vehicle through which theG. pallidipes virus is disseminated in nature.

Population genetics of Trypanosoma brucei and the epidemiology of human sleeping sickness in the Lambwe Valley, Kenya.

Numerical taxonomy was used to review isoenzyme variation in isolates of Trypanosoma brucei obtained from cattle, tsetse, humans and wildlife from the Lambwe Valley, Kenya. From isoenzyme information alone, it was possible to classify isolates as to source through the use of linear discriminant functions analysis, with an error rate of only 2% in humans, and 14% over all groups. Differentiation was mostly dependent on patterns in the enzymes ASAT, PEP1, and ICD. Parasites from non-human sources, especially tsetse, were characterized by high isoenzyme diversity, and many unique zymodemes. Observed frequencies of genotypes for ICD, ALAT, and ASAT did not agree with expected frequencies based on random mating of a diploid organism. Deviations were particularly large for tsetse isolates, and were mostly due to a deficiency of one homozygote. Cluster analysis revealed complex relationships among isolates, with patterns evolving through time. Major human zymodemes from the 1970s clustered together with most wildlife isolates from East Africa. This chronic human-wildlife transmission cycle was characterized by ASAT pattern I. Other, minor human zymodemes were associated with a human-cattle transmission cycle characterized by ASAT pattern VII. These original chronic transmission cycles appeared to change in 1980 with the appearance of two new zymodemes in humans. These zymodemes involved changes in ALAT and/or PGM to patterns typical of tsetse and cattle isolates. A resultant epidemic was halted with repeated aerial spraying of endosulfan in 1981. Since then, a variety of new zymodemes of unknown human infectivity have appeared. The origins of these changes are discussed in terms of genetic exchange in tsetse, adaptation to human and cattle transmission cycles, and selection resulting from chronic use of insecticides.

A SIMPLE METHOD FOR ARTIFICIAL INFECTION OF TSETSE, GLOSSINA MORSITANS MORSITANS LARVAE WITH THE DNA VIRUS OF G. PALLIDIPES

Newly deposited Glossina morsitans morsitans larvae were chilled over ice and inoculated with 1 μl of either virus suspension derived from Glossina pallidipes salivary gland homogenate or sterile tsetse physiological saline. They were allowed to pupariate and then maintained at 25°C, 70% r.h. until soon after emergence when their salivary glands were examined for enlargement and presence of virus particles. Teneral G. m. morsitans which received the virus inoculum (n = 135) as larvae all became infected as revealed by gross hypertrophy of their salivary glands and ultrastructural manifestation of virus particles within the glandular epithelial cells and lumina. In the control group, which received the tsetse physiological saline (n = 91), only 1.1% of the flies showed the salivary gland enlargement, a level equivalent to the prevalence of virus infection normally detectable in the G. morsitans colony. This technique opens the way for testing the biocontrol potential of this virus. The DNA virus from G. pallidipes is clearly infective to G. morsitans morsitans, suggesting that the hypertrophied, chalky-white salivary glands, reported in various Glossina spp., are a manifestation of infection by one and the same virus.RésuméLes larves de Glossina morsitans morsitans nouvelement déposées étaient refroidies sur la glace et inoculées avec 1 μl soit d’une suspension de virus provenant d’homogenat de glandes salivaires de Glossina pallidipes ou d’une solution physiologique sterile de tsétsé. Les larves se sont transformées en pupes puis maintenues à une temperature de 25°C et une humidité relative de 70% jusqu’à l’emergence lorsque leur glandes salivaires étaient examinées pour la présence des virus. Tous les 135 jeunes G. m. morsitans provenant des larves ayant été inoculées par des virus ont subit l’infection comme révélé par l’hypertrophie des glandes salivaires et la manifestation ultrastructurale des particules des virus au niveau des cellules épitheliales glandulaires et du lumen. Quant aux mouches tsétsé qui n’ont reçu que la solution physiologique (et utilisés comme contrôle), seulement 1, 1% de 91 jeune G. m. morsitans ont montré l’agrandissement des glandes salivaires, un niveau équivalent à l’incidence de l’infection du virus normalement rencontrée dans la colonie de G. morsitans. Le succès de cette technique ouvre une voie pour tester, l’utilisation de ce virus comme agent potentiel dans la lutte biologique. Le virus ADN provenant de G. pallidipes sans doute infecte G. m. morsitans. Ceci suggère que les glandes salivaires hypertrophiées observées chez différentes espèces de Glossines sont des manifestations d’infection causées par le même virus.

Influence of d(+)-glucosamine on infection rates and parasite loads in tsetse flies (Glossina spp.) infected with Trypanosoma brucei

Teneral Glossina morsitans centralis, G. m. morsitans and G. pallidipes were infected with three different clones of Trypanosoma brucei in blood containing D(+)-glucosamine, an inhibitor of tsetse midgut lectin. On average, 5 days of D(+)-glucosamine treatment tripled infection rates, without affecting the proportion of infections that matured. Total infection rates were equal in males and females, but twice as many infections matured in males. Counts of parasites in the guts and salivary glands of 277 flies revealed order of magnitude differences among flies, with females consistently having 2-3-times as many parasites as males. Parasite numbers varied in a sex-specific manner among tsetse-clone combinations, but these differences were not correlated with similar large differences in infection rates. D(+)-glucosamine treatment had no significant effect on parasite loads.