Sensitivity of animal-derived Trypanoroon stocks from sleeping sickness endemic foci of Nigeria to trypanocides and human plasma

Twelve Z’rypanozoon stocks isolated from semi-nomadic cattle in known sleeping sickness foci of central and northern Nigeria were studied in terms of susceptibility to two trypanocides, diminazene aceturate (Berenil) and isometamidium chloride (Samorin) and human olasma. In infected small ruminants, three of the stocks were resistani to diminazene aceturate at does of 7.0 -14.0 mglkg body weight (b.w.) while isometamidium chloride at doses of 1.0 mglkg b.wr or higher failed to effect parasitological cure of infections with two of the diminazene-resistant stocks. The two isometamidiumresistant stocks were also consistently resistant to the trypanolytic action of human plasma. It is suggested that cattle are reservoirs of Trypnosoma brucei subspecies potentially infective to man and resistant to the therapeutic action of the known sanative pair (diminazene and isometamidium).


INTRODUCTION
In West Africa, Ttypanosoma vivax, T. congolense and T. * b. brucei are the most important trypanosome species for livestock. The last two enjoy a wide host range, infecting also laboratory animals, wildlife and pigs as reviewed by Losos and Ikede (18) and Anosa (3). T. brucei gambiense, the main cause of sleeping sickness in the subregion has been isolated from pigs (10, 19, 27), sheep (26), dogs (lO), game animals (20) and domestic chickens (31).
Spot surveys, abattoir samples and occasional outbreaks have provided the main sources of epidemiological studies of trypanosomosis in Nigerian livestock over several decades (2). Although the study of the prevalence and distribution of the disease in ruminants is currently benefiting from a European grant (4) knowledge of the role of domestic animals and wildlife in its transmission in man and livestock is limited to the report of Joshua et a/. (14) on the potential of migratory cattle to harbour humaninfective trypanosomes.
Also, despite reports on drug resistance among haematic trypanosome species viz T. vivax and T. congolense (2, 5) few attemps have been made to study the phenomenon among Trypanozoon species under Nigerian field conditions. This study was designed to assess the sensitivity to trypanocides of T. brucei subspecies isolated from ruminants and pigs in sleeping sickness zones of Nigeria and to test the potential infectivity of the stocks to man.

MATERIALS and METHODS
Sleeping sickness foci, survey areas The sleeping sickness endemic areas surveyed included the primordial foci in Tiv Province of Benue State. This area has been regarded as one of the oldest permanent foci of sleeping sickness in Nigeria. Others were the more northern areas of Jema'a Local Government (Kaduna State) and Plateau State. The areas lie within 7"lO' -10'25' Not-th and 8"OO' -9"45' East and extend from the Southern to the Not-thern Guinea vegetation zones ( fig. 1).

The livestock
Samples were taken from bovine and porcine hosts reared semi-intensively. The pigs were housed in piggeries in the vicinity of owners' houses, while the cattle were either provided with shelter or housed in open enclosures in front of owners' residence, after the day's grazing within a mean of 5 km radius, as described by Kalu et a/. (16).   Isolates were maintained by stabilation in liquid nitrogen at -196°C till required. Mixed populations were differen-tially eluted through a DEAE cellulose (DE 52, Whatman Chemical Ltd, UK) anion exchange column with PSG according to Lanham and Godfrey (17), passaged, through trypanosome naive suckling mice, and cloned ', derivatives were stabilized. All trypanosome stabilates i irrespective of source were passaged twice through mice ~ l and their Trypanozoon morphology was confirmed by differentiation on Giemsa-stained thin films prior to the studies.

Experimental animals
Adult Wister mice (18.2 i 1.5 kg) were obtained from the Laboratory Animal Unit of the Parasitlogy Division, NITR, Vom. Red Sokoto goats (mean weight 14.6 i 2.2 kg) and West African dwarf sheep (12.5 f 1.8 kg) were purchased at various times from the local markets in Mangu LGA on the tsetse-free Jos plateau. They were screened for trypanosomes and other haemoparasites, dewormed with thiophanate (Nemafax, May & Baker, U.K.) and given doses of iron dextran (Myofer 100, Farbweke Hoechst, Germany) during a 3 month acclimatization period. The animals were fed concentrates supplemented by a grass/legume mixture and Acha hay (Digitaria exilis) ad libitum. Only parasite-free animals were used in the experiments.

Infection of goats and sheep
Cardiac blood of donor mice infected with Trypanozoon stocks was pooled and the trypanosomes counted in a Neubauer haemocytometer.
Each experimental small ruminant was then syringe-inoculated, via the intramuscular route, with approximately 1 x 107 bloodstream trypomastigotes contained, after dilution in phosphate buffered saline (PBS; pH 7.4), in 2 to 3 ml of the inoculum.

Drug susceptibility trials
Drug treatment started at different days following prepatency (table 1), depending on the pathogenicity of the strains. Diminazene aceturate (Berenil, Farbweke Hoechst AG, Germany) and isometamidium chloride (Samorin, May & Baker Ltd, U.K.) were used at the recommended doses for ruminants of 3.5 mg and 0.5 mg/kg body weight, respectively or higher (tables 1, Il). Cures and relapses of infection following treatment were monitored daily by detection of parasites in ear vein blood using the dark ground buffy coat examination (23) and the haematocrit centrifugation technique (29), as described by Kalu et a/. (15).

Blood incubation infectivity test (BIIT)
The potential infectivity of Trypanozoon isolates to man was evaluated by the blood incubation infectivity test.
The technique employed was that described by Rickman (24). Each incubated sample was inoculated into 5 test mice as recommended by ILCA's manual (22). Experimental animals were housed in separate cages to prevent oral transmission (21). Stocks were designated sensitive or resistant to human plasma according to the criteria of Hawking (12).

Susceptibility to trypanocides
Twelve T. brucei isolates were each tested for susceptibility to Berenil and Samorin. Out of these, 3 were consistently resistant to Berenil at 7.0 mg/kg b.w. or higher doses (table 1). Also 2 T. brucei stocks were resistant to Samorin at 1.0 mg/kg b.w. (or more) (table Il). The two Samorin-resistant stocks (ICS/CT 40 and GBS 2/CT 18) were among the Berenil-resistant ones. All the stocks susceptible to Berenil were also cleared from small ruminant hosts by Samorin at 0.5 mg/kg b.w.

Sensitivity
to human plasma Two of twelve stocks tested were repeatedly resistant to normal human plasma (table Ill). Morphologically, the Trypanozoon stocks were pleomorphic like the others, susceptible to human plasma, had longer prepatent periods and also exhibited lower parasitaemia in caprine/ovine hosts (table Ill).

DISCUSSION
Drug resistance among trypanosomes has been reported mostly among the haematic trypanosomes (T. vivax and T. congolense) which are more pathogenic and prevalent among ruminants in Nigeria (3, 4, 18). Trypanosomes of the brucei group require higher doses to effect a cure (especially with Berenil) as they invade tissues and cause relapse infections from their locations e.g. the brain (3, 4, 13). They may also become more pathogenic under stress conditions and in the areas where other trypanosome species have been effectively reduced by chemotherapy. The finding of a high proportion of stocks of this species resistant to trypanocides indicates the necessity of more judi; cious drug use and suggests that! adequate diagnosis should be made prior to any therapy under field conditions.
It has long been believed that human infective T. brucei gambiense and T. brucei rhodesiense also occur in animal hosts (8, 30). Direct evidence using human volunteers (9) and indirect evidence by the blood incubation infectivity test (6, 20, 25) support this belief. Also, the role of domestic animals as resewoirs of trypanosomes infective to man has been reviewed by Mehlitz et a/. (20)    confirmed by Brun and Jenni (7). This study provides evidence that semi-nomadic cattle, in close proximity to herd-owners, harbour trypanosomes potentially infective to man in sleeping sickness endemic areas of Nigeria. The Nigerian control Trypanozoon strains were either inactivated by the normal human plasma (T. brucei brucei 8/18) in line with the trypanolytic action of human plasma, serum or blood, or resistant to it (T. brucei gambiense Kwa) (6, 9).
In addition to being resistant to human plasma and trypanocides, the low parasitaemia of the 2 stocks in experi-mental ruminants, close contact between livestock and their owners in known sleeping sickness endemic foci is highly suggestive of the reservoir status of cattle in this area. G. tachinoicfes, a riverine tsetse fly species with high vectoral capacity for the sleeping sickness parasite, has recently been reported to be the only vector of hyperendemic ruminant trypanosomosis in Gboko, Benue state (16) -one of the areas covered by this study. These findings, under conditions where transmission could be essentially from animal to man (i), are of epidemiological importance for human sleeping sickness.