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We conducted an open-label safety and immunogenicity bridging study that compared liquid and lyophilized formulations of the candidate malaria vaccine RTS,S formulated in AS02A in 34 healthy, malaria-naïve adults at WRAIR. Volunteers received two doses of either formulation on a 0, 1-month schedule. Both vaccines were well tolerated and similarly immunogenic. Nineteen of 25 subjects who received the lyophilized formulation and six infectivity controls underwent sporozoite challenge to assess vaccine efficacy. All six controls had parasitemia detectable by thick blood smear by day 13 (mean pre-patent period 12.3 days; range 11-13). In the vaccine group, 8 of 19 vaccinees did not develop malaria and were completely protected (i.e., 42%). Among the 11 vaccinees who did become infected, the mean pre-patent period was delayed (14.4 days; range 13-18). The two formulations of RTS,S were equally safe and immunogenic, and the lyophilized formulation showed similar levels of efficacy against sporozoite challenge to that conferred by the liquid formulation in previous studies.

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.

Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites. Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P. falciparum infection and drug cure. Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment. Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo. The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection. In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro. Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P. falciparum challenge.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Different strains of Plasmodium vivax vary in their sensitivity to primaquine, the only drug that prevents relapses. Described are the clinical data and relapse pattern for 75 soldiers treated for vivax malaria since returning from Somalia. Following their initial attack of malaria, 60 of the 75 cases received a standard course of primaquine (15 mg base daily for 14 days). Twenty-six of the 60 soldiers subsequently relapsed for a failure rate of 43%. Eight soldiers had a second relapse following primaquine therapy after both the primary attack and first relapse. Three of these soldiers had received a higher dosage of primaquine (30 mg base daily for 14 days) after their second attack. The apparent ineffectiveness of primaquine therapy in preventing relapses suggests the presence of primaquine-resistant P. vivax strains in Somalia.

Background: The candidate vaccines against malaria are poorly immunogenic and thus have been ineffective in preventing infection. We developed a vaccine based on the circumsporozoite protein of Plasmodium falciparum that incorporates adjuvants selected to enhance the immune response., Methods: The antigen consists of a hybrid in which the circumsporozoite protein fused to hepatitis B surface antigen (HBsAg) is expressed together with unfused HBsAg. We evaluated three formulations of this antigen in an unblinded trial in 46 subjects who had never been exposed to malaria., Results: Two of the vaccine formulations were highly immunogenic. Four subjects had adverse systemic reactions that may have resulted from the intensity of the immune response after the second dose, which led us to reduce the third dose. Twenty-two vaccinated subjects and six unimmunized controls underwent a challenge consisting of bites from mosquitoes infected with P. falciparum. Malaria developed in all six control subjects, seven of eight subjects who received vaccine 1, and five of seven subjects who received vaccine 2. In contrast, only one of seven subjects who received vaccine 3 became infected (relative risk of infection, 0.14; 95 percent confidence interval, 0.02 to 0.88; P<0.005)., Conclusions: A recombinant vaccine based on fusion of the circumsporozoite protein and HBsAg plus a potent adjuvant can protect against experimental challenge with P. falciparum sporozoites. After additional studies of protective immunity and the vaccination schedule, field trials are indicated for this new vaccine against P. falciparum malaria.

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

Owl monkeys (Aotus lemurinus griseimembra) were immunized against Plasmodium falciparum by infection and drug cure. After challenge, 3 of 4 monkeys developed extended prepatent periods and low grade parasitemias followed by self cure. The fourth monkey did not develop a patent infection. Immune monkey serum passively transferred at the time of challenge conferred immunity to 20 naive monkeys. Immunity was characterized by extended prepatent periods in 19 monkeys, low levels of parasitemia (< or = 1%) followed by self cure in 12 animals, and lack of detectable infection in 3 recipient monkeys. Immune serum collected from monkeys undergoing repeated challenges afforded more protection than serum from singly infected monkeys. However, single doses of hyperimmune serum appeared to be as effective as multiple doses. Normal serum had no effect on the course of infection in 12 monkeys. These studies confirm that owl monkeys can be immunized by infection and cure and demonstrate that this immunity can, in large part, be transferred to nonimmune recipients with serum from immune donors.

Trypanosoma vivax isolated from dairy cattle undergoing a haemorrhagic disease was inoculated into Ayrshire steers. Five of six infected animals experienced brief periods of diarrhoea and sublingual and gastro-intestinal haemorrhage. Gastro-intestinal bleeding coincided with markedly reduced numbers of thrombocytes and a high level of parasitaemia in the peripheral blood. Prothrombin times were extended and fibrinogen levels were elevated in infected animals. Plasma paracoagulation tests were positive for the presence of fibrin monomers and/or clottable early fibrin degradation products during the course of infection.

Sixty four Kenyan patients with visceral leishmaniasis were treated with sodium stibogluconate (Pentostam) (40 patients) or various combinations of Pentostam and allopurinol (24 patients). Three patients, initially considered cured after Pentostam, relapsed but responded to further treatment. Sixty two were cured and two patients died. The treatment and the clinical, haematological and parasitological response to treatment are described in detail. If follow up is impossible or unlikely it is advised to continue treatment until parasitological cure is obtained. Prolonged courses of Pentostam, which were required in some patients, resulted in cures and apparently were non toxic. Consideration is to be given to extended treatment with Pentostam before more toxic drugs such as pentamidine and amphotericin B are given.

Trypanosoma brucei brucei, derived from the salivary glands of infected tsetse flies (Glossina morsitans morsitans) and maintained in culture for over 4 years, were infective to both albino rats and tsetse flies. Virulence was markedly enhanced during the first passage in albino rats or tsetse flies. Irradiated cultured trypanosomes induced immunity to homologous challenge but not to tsetse fly or blood-induced challenge with the same stock.

Further studies of the complement fixation test using homologous parasite antigen in an endemic area for visceral leishmaniasis have showed that 82% of individuals with proven visceral leishmaniasis were positive initially whilst 92% were positive at some stage during their illness. Titres fell slowly following effective treatment and anticomplementary activity, confined to confirmed visceral leishmaniasis, was usually lost during treatment. Individuals with alternative causes of hepatosplenomegaly from the same population were negative apart from those with a presumptive diagnosis of visceral leishmaniasis but without parasitological confirmation. Less than 1% of people in the same endemic area without visceral leishmaniasis were positive, suggesting that preliminary serodiagnostic investigation would limit the need for invasive investigation under field conditions.

Five crossbred cattle infected with Trypanosoma vivax (Likoni) by Glossina morsitans developed capillary haemorrhages at the onset of parasitaemia, followed by the presence of occult blood in faecal samples and eventually melena. Two animals required treatment to survive, on days 13 and 38 respectively. The other three animals cleared their parasitaemias without treatment. Packed cell volume (PCV) levels decreased in all animals to levels ranging from 7.5 to 17%. Relapse in a treated animal initiated marked haemorrhage and a loss of 14 PCV units during a six-day period. Thrombocytopenia was common to all animals, and thrombocytes decreased to levels of 4000/microliters of blood. All animals developed increased levels of fibrinogen and fibrin monomer. Prolonged prothrombin times were found in all animals, and activated partial thromboplastin times were also extended in the two animals with high parasitaemias.

The complement fixation test using homologous parasite antigen was evaluated in 60 individuals with confirmed visceral leishmaniasis and compared with the results obtained in individuals with other parasitic and infectious diseases; 88% of the confirmed cases of visceral leishmaniasis were positive whilst no positive reactions were observed in individuals with other infectious and parasitic diseases. The specificity was partially compromised by anticomplementary activity in 5%, limited to individuals with visceral leishmaniasis, and doubtful (2%) or sero-negative (7%) results.

Surveys in Zebu cattle in the Lambwe Valley in 1980 indicated that many (up to 70%) were infected with trypanosomes. The predominant parasite was Trypanosoma brucei sspl followed by T. congolense. Cerebrospinal fluid (CSF) analysis showed a high proportion of animals with pleocytosis and elevated total CSF protein. Trypanosomes were detected in CSF and signs of a central nervous system (CNS) disease were observed. Histopathological lesions in the CNS were identical to those found in experimentally-infected cattle and consisted of perivascular infiltrations, swollen endothelium of vessels, infiltration of the vascular wall, and perivascular oedema. The severest cases showed rarefaction, astrocytosis and areas of necrosis. Infected cattle transported to the Veterinary Research Laboratory were studied for up to four years. Absence of trypanosomes from the peripheral blood was common, and even subinoculation of lymph node aspirates and CSF were usually negative. Death was preceded by a period of weight loss and the development of severe CNS signs. An attempt to cure animals with Mel-B treatment failed. Serum from naturally-infected cattle neutralized T. b. rhodesiense stocks collected in the same area.

Eight game animals representing seven species in the Ruma National Park in South Nyanza, Kenya, were examined for the presence of blood protozoa, ectoparasites, and helminthic and coccidian endoparasites using standard parasite-identification methods. Haematological parameters were also determined. The oribi was positive for Trypanosoma brucei ssp. and the reedbuck for T. congolense. No other blood protozoans were found. Strongyle eggs were found in the faeces of all species except the water buck. Five of eight animals harboured liver flukes and five were parasitized by ticks of the genus Amblyomma. One roan antelope was anaemic, but the other animals had haemoglobin levels within the normal range and appeared to be in a good state of health.

Early during the course of Trypanosoma congolense infection in cattle decreases in PCV occurred and coincided with increases in both MCV and MCH. The indices reached highest levels between eight and 12 weeks post-infection. By week 20 of infection MCV and MCH had decreased to pre-infection levels even though a substantial anemia persisted. Serum iron levels were elevated at eight weeks postinfection. (Infected 271 mg dl-1 v. control 140 mg dl-1) but decreased to low levels in infected animals by Week 28 (63 mg dl-1). At eight weeks post-infection PITR was elevated in infected animals (infected 2.6 mg dl-1 day-1 v. control 0.82 mg dl-1 day-1). By Week 28, however, PITR had fallen in infected animals to 0.62 mg dl-1 day-1 indicating a severe dyshaemopoesis since PCV levels averaged only 22%. Cattle which were treated with Berenil or that self-cured the infection had normal iron parameters when tested 61 weeks post-infection. Cattle treated early during the course of infection showed a significantly greater PCV response that those treated later during infection.

The isolation and characterization of a new serodeme of Trypanosoma brucei rhodesiense is described. A clone of organisms derived from a human infection produced chronic infections in mice. Additional clones of differing antigenic specificities were isolated from peaks of parasitemia which occurred in these mice. The variable antigen types (VATs) of these clones were determined by agglutination, immunofluorescence, and protection of actively immunized mice. Thirteen distinct VATs were isolated and designated Walter Reed Army Trypanozoon antigenic types. The described methodology and reagents, together with the chronicity of the infection produced in mice by this serodeme, provide a model for further study of immunopathology and antigenic variation in African trypanosomiasis. The use of these reagents in determining the incidence of VATs in an endemic area may allow an assessment of the feasibility of immunoprophylaxis.

Monoclonal antibodies have been made against clones of Trypanosoma brucei rhodesiense from the WRATAR 1 serodeme and analyzed by indirect immunofluorescence assay for specificity against homologous and heterologous clones. These antibodies were shown to be variable antigen type (VAT)-specific as they identified the majority of parasites in the homologous clones but few parasites in other clones. The reactivities of these VAT-specific monoclonal antibodies with uncloned human trypanosome isolates from Kenya were compared with the reactivities of polyvalent, VAT-specific rabbit sera on the same isolates. Different reaction patterns were obtained with the two sets of reagents and concordant reactions were less than 20%. Our data indicate that single monoclonal antibodies are not interchangeable for sera in the typing of antigenic variants of African trypanosomes, and multiple monoclonal antibodies for each antigenic variant will probably be needed.

A bisquinaldine, 1,6-bis-(6-amino-2-methyl-4-quinolylamino) hexane, was tested against Trypanosoma brucei ssp. in goats and against T. brucei, T. congolense and T. vivax in mice. At doses of 25 and 100 mg kg-1, the drug protected goats for at least 90 days against blood challenge with T. brucei ssp. Fifty to sixty per cent of goats challenged 180 days after treatment were protected, but all goats challenged 270 days after treatment became infected. In mice, bisquinaldine also had a marked effect on T. brucei, but only a minimal effect on T. vivax and no apparent effect on T. congolense. No drug toxicity was noted in mice even at doses of 2000 mg kg-1. Both a short-term (25 and 100 mg kg-1) and long term (100 mg kg-1) toxicity was apparent in goats treated with bisquinaldine.

Of 16 patients with kala-azar treated with sodium stibogluconate (0.1 ml/kg body weight a day), one died on the 12th day of treatment and nine were cured by a 30-day course, although two subsequently relapsed. Extending the course cured a further five patients, and in one patient allopurinol was used in addition before a cure was achieved. Clinical and hematological recovery began within a few days of the start of treatment, but parasites continued to be seen in splenic aspirates for 3 weeks or more.

In a study of 269 sleeping sickness patients treated with Mel-B, 14 (5.2%) died during treatment. With total dosages of at least 30 ml (1.08 g), 1.4% relapsed and another 6.4% died, mostly of unknown causes, within three years of treatment, giving a success rate of 92.1% over the three years. Mel-B was used to treat 55 relapses after suramin therapy with 1.8% deaths during treatment, 3.6% relapses, and 92.7% success over at least three years. Apparent drug resistance to Mel-B was found in three patients who continued to relapse after repeated treatments. During 1980, 51 patients were treated with suramin on the basis of clinical condition without benefit of cerebrospinal fluid (CSF) analysis. Subsequently 49% of these patients relapsed within three years of treatment. When 29 patients were treated on the basis of CSF evaluation only two (7%) relapsed.

Clinical and laboratory findings of a series of 64 consecutive patients with visceral leishmaniasis are presented. The diagnosis was established by demonstration of parasites in splenic aspirates. Findings are discussed in comparison with those of former reports from Kenya and elsewhere.

Gambian sleeping sickness entered what is now Kenya from Uganda in about 1901 and quickly spread along the Kenyan shores and islands of Lake Victoria, reaching Tanzania in 1902. By 1910 the disease had spread 25 miles inland along the Kuja and Migori rivers and their tributaries. Sleeping sickness waxed and waned in these areas despite attempts to control tsetse fly populations by various methods. It was not until 1950, when the use of insecticides (DDT) applied by backpack sprayer proved successful against Glossina fuscipes at Kibigori, that eradication of G. fuscipes and Gambian sleeping sickness seemed possible. Subsequently the Kuja-Migori endemic area was cleared of flies and disease, as well as the South and Central Nyanza lake shores and islands. By 1965 Gambian sleeping sickness had virtually disappeared from Kenya. A more virulent form of the disease, Rhodesian sleeping sickness, may have also spread to Kenya from Uganda, although its appearance in diverse areas of the Gambian disease suggest that local ecological factors may have played a role in enhanced virulence of trypanosomes stocks. The Rhodesian form of sleeping sickness appeared in the Lambwe Valley, South Nyanza, in about 1959, and despite attempts to eradicate this disease it still persists as the only remaining endemic area in Kenya at this time. The usual transmission of Rhodesian sleeping sickness by G. pallidipes in Kenya was altered when an outbreak occurred at Alego, in Central Nyanza, in 1964. It was discovered that G. fuscipes was the vector and that domestic cattle were an important reservoir of infection. Glossina fuscipes was also the vector of Rhodesian sleeping sickness in an outbreak in Samia in 1976 and another along the lakeshore in South Nyanza in 1981. Sleeping sickness has been restricted primarily to the Western and Nyanza Provinces of Kenya (Fig. 1).

Fourteen cerebrospinal fluid (CSF) samples obtained from Rhodesian sleeping sickness patients from the Lambwe Valley at relapse were positive for the presence of anti-trypanosomal antibody by both IFAT and ELISA. The mean optical density (o.d.) in the ELISA test was 0.804 +/- 0.362 and ranged from 0.258 to 1.363. CSF from five patients from the same area without evidence of meningoencephalitis were all negative by ELISA (mean o.d. 0.023 +/- 0.016, range 0.011-0.051). Control CSF samples from U.K. patients without Rhodesian sleeping sickness but with elevated levels of CSF total protein were also negative. Antibody detected by ELISA declined after Mel-B treatment of relapse and most samples had returned to negative within two years of treatment. We present evidence that serological evaluation of the CSF by ELISA and/or IFAT can provide supportive evidence of the trypanosomal origin of the infection. This is especially important at the time of relapse, when parasitological diagnosis may be impossible and records of treatment for the primary infection may not be available.

Zebu x European (Z x E) crossbred cattle suffered a more severe course of disease than Boran cattle when infected with Trypanosoma vivax (Likoni) by Glossina morsitans. All Z x E animals in this study required Berenil treatment while all Borans self-cured the infection without treatment. The more severe disease in Z x E animals was characterized by longer periods of patent infection and fever, more severe anaemia and greater likelihood of haemorrhage. Cattle previously infected and cured with Berenil showed resistance and self-cured challenge infections. After self-cure cattle remained immune to tsetse fly challenge with the homologous trypanosome stock for long periods. Immunity induced by infection and drug or self-cure appeared to be specific for the homologous stock, since cattle immune to T. vivax (Likoni) showed no resistance when challenged with stocks of T. vivax isolated in Lugala, Uganda or Galana, Kenya. Severe haemorrhages, most prominent in the digestive tract, were seen in some infected cattle before treatment.

A stock of Trypanosoma brucei ssp. isolated from a naturally-infected goat in the Lambwe Valley, Kenya, induced cerebral trypanosomiasis in experimentally-infected goats. Six of nine goats with cerebral trypanosomiasis induced by this stock were cured by a single high dose of suramin (50 mg kg-1). Two other goats appeared to be cured with this dosage of suramin but later developed abnormal central nervous system (CNS) signs and parasitaemia. Parasites first appeared in the cerebrospinal fluid (CSF) and then in the blood and lymph nodes. Mel-B was also effective against primary and relapse cerebral trypanosomiasis in goats.

By 1936 the Lambwe Valley, which had been heavily populated in the early years of this century, was nearly devoid of people. Population since that time has increased markedly as a result of a settlement scheme and efforts made to control and eradicate Glossina pallidipes and trypanosomiasis. The formation of a game reserve (now a National Park) prevented the completion of a tsetse eradication programme and has provided an unmolested habitat for both G. pallidipes and large numbers of game animals which act as a reservoir for trypanosomiasis. Rhodesian sleeping sickness, as well as animal trypanosomiasis, have been severe problems for the local farmers who live around the boundaries of the National Park.

Infection of cattle with various stocks of Trypanosoma brucei rhodesiense indicated that 49% developed a fatal CNS disease comparable to that found in man. Duration of disease ranged from 85 to 1613 days post infection. All eight stocks of T. b. rhodesiense tested, including those from Ethiopia and Tanzania, induced CNS disease. Blood became positive three to five days after inoculation, and after an initial peak of parasitaemia remained positive for three to five months. Subinoculation of blood into rodents subsequently became negative, although trypanosomes persisted in the lymph nodes for at least 56 to 1613 days. Only animals with CNS disease had detectable parasites in the CSF, usually after the animals had undergone severe deterioration. At post mortem examination trypanosomes could usually be found in the lymph nodes and CSF, and occasionally in the blood. Clinical signs included fever, hyperkinesia, weight loss, cerebellar ataxia, tremor, salivation and hyperaesthesia. A mild to moderate anaemia accompanied a transient thrombocytopenia and leucopenia. Animals subsequently developed leucocytosis. A pleocytosis and elevated total protein in the CSF was found, which persisted in some animals for long periods. Histopathological examination of the brain showed prominent generalized perivascular infiltrates consisting mainly of lymphocytes and plasma cells. Mott's cells were regularly observed. Vascular changes were characterized by swollen endothelium, infiltration of the vascular wall by inflammatory cells, and in some instances perivascular oedema. In the most severe cases evidence of ischaemia consisted of large numbers of astrocytes, rarefaction of the parenchyma, and areas of necrosis with loss of normal architecture. Demyelination was limited to perivascular areas. Occasionally a moderate to severe pancarditis was found.

Interrupted feedings of teneral, laboratory-reared Glossina morsitans morsitans were used to study mechanical transmission of Trypanosoma brucei rhodesiense. Intervals between exposure of individual flies on parasitaemic rats and refeeding on clean rats were varied from five minutes to 24 hours. Direct transmissions were demonstrated at each interval up to 160 minutes after exposure. Proboscis dissections showed that active trypanosomes were present up to 320 minutes after exposure. No mechanical transmissions from bovine to bovine occurred in 39 attempts, when groups of 20-120 flies exposed on parasitaemic bovines were transferred immediately to uninfected cattle, but two of 40 individual flies exposed on parasitaemic bovines mechanically transmitted trypanosomes to clean rats. Proboscis dissections made immediately after flies were exposed to a bovine with a parasitaemia of 4.8 x 10(-4) trypanosomes/microliters of blood showed that 11 of 20 (55%) had active trypanosomes in the food canal. The mean number of trypanosomes per proboscis was 29.4 (+/- 20.5). Of 20 flies exposed on a bovine with a low parasitaemia, however, only one trypanosome was seen in proboscis dissections. The parasitaemia of the infected donor was an important factor in mechanical transmission. The mechanical transmission of trypanosomes from one host to another may largely depend on the infectivity threshold of the recipient host, and individual mechanically-infected tsetse flies may not transmit an infective dose.

A total of 912 cases of sleeping sickness have been recorded from the Lambwe Valley from 1959 to 1984. After a period of decreasing prevalence in the 1970s an outbreak of disease occurred between 1980 and 1984. The incidence of disease for this five-year period was highest in areas adjoining the Ruma National Park, reaching 54% in Area I. Attack rates were highest in the 50+ age group (125) and children had significantly lower attack rates (8%) in this area of peridomestic transmission. Sex ratios of patients (M/F) were near 1.0 in areas in closest proximity to the thickets in the National Park, while in distant areas the ratios rose to 6.0. The distribution of the number of patients within different households was studied; fewer households than expected had 0 or one patient, and more than expected had three or more patients. No difference in attack rates were found between Nilotic and Bantu groups. Twelve different zymodemes were found in 136 stocks of Trypanosoma brucei rhodesiense. Four new zymodemes appeared in 1980 in the latest outbreak and accounted for 73% of the stocks isolated from man during this outbreak. Neutralization tests indicated that each trypanosome zymodeme may also represent a different serodeme.

220 Trypanosoma (Trypanozoon) brucei sp. stocks isolated between 1969 and 1983 from the Lambwe valley sleeping sickness focus in South Nyanza, Western Kenya, were characterized by isoenzyme electrophoresis using 12 enzymes. 12 different zymodemes of T. (T.) b. rhodesiense were isolated from patients during the 13-year period and identical stocks were also found in cattle, reedbuck (Redunca redunca) and tsetse (Glossina pallidipes). Cattle may have played an important role in maintaining and increasing peridomestic transmission of trypanosomes during the 1980 outbreak of sleeping sickness in the valley, even though they themselves suffered heavy mortality. Sleeping sickness in Lambwe valley is unlikely to have been introduced from elsewhere, since T. (T.) b. rhodesiense stocks isolated from the valley were different from those from neighbouring epidemic areas. Alternatively, the recent outbreak may have been caused by the increased transmission associated with an expanding tsetse population. The possibility that genetic exchange contributed to the biochemical diversity of the trypanosomes examined is discussed.

During a recent outbreak of Rhodesian sleeping sickness in the Lambwe Valley no asymptomatic Rhodesian sleeping sickness patients were found although 54% of the primary patients had mild symptoms and 9% were stuporous or comatose at presentation. The duration of symptoms was three months or less in 90% of the patients. Headache, weakness, joint and back pains and weight loss were claimed by at least 75% of the patients, while 82% of the females reported amenorrhoea and 70% of the males claimed impotency. Physical examination revealed lymphadenopathy in 86% but fever in only 36% of the patients, while chancres were found in only 16%. Patients had significantly lower levels of haemoglobin and thrombocytes than controls and their erythrocyte sedimentation rates were elevated. A comparison of both blood group and haemoglobin type between patients and controls yielded no significant differences. Fifty-seven per cent of the primary patients reporting mild symptoms had abnormal levels of leucocytes in their CSF. All relapse patients had abnormal CSF parameters. Levels of serum urea nitrogen were significantly elevated in patients, but SGOT, SGPT and total bilirubin were not. Levels of albumin and beta-globulin in patients were significantly lower than controls while gamma-globulin was elevated. Mean serum IgM levels in patients were elevated to nearly three-fold those of controls, but 35% of the individual patient values fell within the 95% range of control values. Some patients had extended prothrombin and thrombin times while fibrinogen levels were significantly elevated. No patients reported haemorrhage, and none was seen.