Ultrastructure of the chorioallantoic placenta and chorionic vesicles of the lesser bush baby (Galago senegalensis).

The ultrastructure of the chorioallantoic placenta of the lesser bush baby (Galago senegalensis) has been studied. The placenta was shown to be of the diffuse, epitheliochorial and adeciduate type. The trophoblasts of the chorionic villi, other than those lining the chorionic vesicles, were characterized by the presence of many lipid droplets. In the later stage of gestation, the fetal capillaries indented the trophoblastic epithelium reducing the distance between fetal and maternal circulations. In addition chorionic vesicles were observed. The trophoblasts lining the chorionic vesicles have outward bulging apical surfaces. There are clefts between these cells and this region is occupied by microvilli of adjacent cells. Several layers of fusiform cells that did not extend up into the cores of the chorionic vesicle villi formed the outer component of the vesicular wall. Granulated cells were observed within the maternal connective tissue and their possible role is discussed.

Intracellular antigens (microtubule-associated protein copurified with glycosomal enzymes)--possible vaccines against trypanosomiasis.

African trypanosomes are motile unicellular eukaryotes that can cause diseases such as sleeping sickness in humans and nagana in animals, debilitating millions of people and livestock. All members of the Trypanosomatidae family contain subpellicular microtubules cross-linked to each other and to the plasma membrane by unique trypanosomal microtubule-associated proteins (MAPs). These MAPs may serve as specific intracellular target sites for therapeutic attack against trypanosomiasis. A trypanosomal MAP (p52) copurifies with two glycosomal enzymes (aldolase and GAPDH) on phosphocellulose columns. Rats and mice vaccinated with antigen preparation p52 containing the glycosomal enzymes were protected against a potentially fatal Trypanosoma brucei infection. Sera of protected animals caused in vitro aggregation of trypanosomes, and immunoelectron microscopy of these aggregates located antibodies in the cytoplasm of the trypanosomes.

Comparison of antibody- and antigen-detection enzyme immunoassays for the diagnosis of Trypanosoma evansi infections in camels.

A total of 183 camels from Kenya were examined for circulating trypanosomal antigens by four methods: (1) a monoclonal antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) and circulating anti-trypanosomal antibodies; (2) antibody-detection enzyme-linked immunosorbent assay (Ab-ELISA); (3) buffy-coat examination (BCE); (4) mouse subinoculation (MI). Thirty-seven camels (20%) were parasite-positive by BCE and 60 camels (33%) were parasite-positive by MI. Sixty-three camels (34%) tested positive on Ag-ELISA. Of the 24 camels which could not be detected by BCE, Ag-ELISA detected 18 (75%). Ab-ELISA detected 90 (49%) positive camels. Of all the parasite-positive camels (61), Ag-ELISA detected 93% and Ab-ELISA 95%. Based on the results of 55 camels, there was a significant statistical difference (P < 0.0001) in Ag-ELISA optical density (OD) values (of either serum or plasma antigen analysis) between parasite-positive and parasite-negative camels. No significant difference was observed in Ab-ELISA OD values between parasite-positive and parasite-negative camels. Diagnosis of T. evansi infection in camels by the use of Ag-ELISA alone or in combination with BCE could therefore be a more preferred approach in assessing patient infection than the use of Ab-ELISA.

Trypanosomal antigen and antibody levels in field camels following treatment with two trypanocidal drugs.

The efficacy of treatment in 61 naturally trypanosome-infected camels was evaluated by antigen and antibody detection. Following treatment of 14 infected field camels with an arsenical drug (RM110) no trypanosomal antigens could be detected in the animals which were treated with 0.6 mg/kg body weight and 1.2 mg/kg body weight, 90 days thereafter. In two out of three camels treated with 0.4 mg/kg body weight no trypanosomal antigens could be detected by day 90 post-treatment. However, there was evidence of trypanosomal antigens in camels treated with 0.2 mg/kg body weight and untreated positive controls. Antibody levels were still high in all the 14 camels, 90 days post-treatment. In another group of 55 field camels, of which 47 camels were parasite-positive and eight parasite-negative, trypanosomal antigens could not be detected in 42 camels, 28 and 48 days post-treatment with Quinapyramine Prosalt. However, antigen levels were still high in five parasite-positive camels, 48 days post-treatment. In all the parasite-positive camels, antibody levels were still high 48 days after treatment. In the eight parasite-negative camels, antigens were detected in four camels before treatment. By day 48 post-treatment, all the four camels were antigen-negative. However, four of the eight parasite-negative camels were still antibody-positive by day 48 post-treatment. These observations indicated that antigen-detection could be used to evaluate the success of therapeutic trials where trypanosome detection tests may fail to pick low patent infections.

An enzyme-linked immunosorbent assay (ELISA) for the detection of trypanosomal antigens in goat serum using a monoclonal antibody.

An IgM murine monoclonal antibody (MAb) TEA 1/23.3.4.6 raised against circulating trypanosome antigens was used in a sandwich ELISA to assay trypanosomal antigens in a trypanosome lysate preparation and in sera from goats infected with Trypanosoma brucei evansi. As little as 1.25 ug/ml of trypanosomal antigen could be detected by this assay. Following infection, trypanosomal antigens were first detected in goat serum 24 hours after the intravenous (i/v) or 6 days after the intramuscular (i/m) inoculation of trypanosome parasites. Antigen levels remained detectable during the course of infection. After treatment with diminazene aceturate, antigens dropped to undetectable levels between day 12 to 41, suggesting that this assay offers a promising approach to the diagnosis of African Trypanosomiasis.

Investigation of the efficacy of flumethrin pour-on for the control of high tsetse and trypanosomiasis challenge in Kenya.

The effects of bi-weekly flumethrin pour-on treatments at 1 mg kg bodyweight on tsetse fly population and trypanosome infection rates were monitored over a one-year period (2/89-2/90) in 2000 head of cattle on a trial farm, located in the Lamu District in East Kenya, an adjacent control farm and a transsecting road for additional fly monitoring. The tsetse fly population on the trial farm dropped from pretreatment counts of 118 flies/trap/week (Feb. 1989) to 13 in June 1989 and 32 in Jan. 1990. During the same period and months the fly population in the control farm was 90.34 and 87 flies/trap/week. Fly counts on the transsecting road, however, increased from 72.53 to 163 flies/trap/week. The impact of tsetse fly control is clearly reflected in the reduction of trypanosome infection rates on the trial farm, e.g. 37% (pre-treatment infection rate), 10% and 11% in January, June and December 1989 respectively. On the control farm the infection rates remained at distinctly higher levels of 34%, 17% and 24% during the same period. Mean weekly weight gains were 66% higher in the treated herd as compared to the untreated control herd.

Differential susceptibility to DL-alpha-difluoromethylornithine in clinical isolates of Trypanosoma brucei rhodesiense.

DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense, is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of 16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or combinations of established agents may be needed for chemotherapy of East African disease.

Homidium bromide as a chemoprophylactic for cattle trypanosomiasis in Kenya.

Homidium bromide was used in a strategic chemoprophylactic regime to control trypanosomiasis in Boran cattle in Kenya. Trypanosome infection rates in cattle receiving homidium bromide prophylaxis were compared with those in control cattle which received no prophylaxis but were treated with diminazene aceturate when infected. Homidium bromide was administered twice during the year after which no infections were detected for periods of nineteen weeks and seventeen weeks respectively. The drug sensitivity of the infecting trypanosomes is believed to be a major factor in determining the duration of prophylaxis.

Review of tsetse control measures taken in the Lambwe Valley in 1980-1984.

During an outbreak of Rhodesian sleeping sickness in the Lambwe Valley in 1980 initial tsetse control measures consisted of applications of dieldrin to the periphery of the Ruma National Park. This activity had a marked effect on the prevalence of sleeping sickness. Concern about the use of dieldrin caused the cessation of this programme and justified an aerial spray programme using endosulfan. Although the Lambwe Valley did not appear to be a good candidate for aerial spray, the endosulfan had a marked effect on tsetse fly levels and on the prevalence of sleeping sickness. Sleeping sickness cases were detected in decreasing numbers for eight months following the endosulfan programme, but the subsequent five months yielded no cases of sleeping sickness in the area. Some flies persisted, however, and they had regained high levels in about a year. As the prevalence of sleeping sickness increased another aerial spray programme was initiated in 1983, using pyrethrum as insecticide. The pyrethrum aerial spray programme did not make significant reductions in the Glossina pallidipes population or in the prevalence of sleeping sickness. A subsequent ground control programme using insecticides (dieldrin and cypermethrin) and bush clearing, conducted primarily within the National Park, has subsequently limited the prevalence of sleeping sickness to low levels.

Trypanosoma vivax: disseminated intravascular coagulation in cattle.

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.

Natural and acquired resistance to Trypanosoma vivax in cattle.

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.

Isolation of a subpellicular microtubule protein from Trypanosoma brucei that mediates crosslinking of microtubules.

The cell body of Trypanosomatidae is enclosed in densely packed, crosslinked, subpellicular microtubules closely underlying the plasma membrane. We isolated the subpellicular microtubules from bloodstream Trypanosoma brucei parasites by use of a zwitterion detergent. These cold stable structures were solubilized by a high ionic strength salt solution, and the soluble proteins that contained tubulin along with several other proteins were further fractionated by Mono S cation exchange column chromatography. Two distinct peaks were eluted containing one protein each, which had an apparent molecular weight of 52 kDa and 53 kDa. (Mr was determined by SDS-gel electrophoresis). Only the 52 kDa protein showed specific tubulin binding properties, which were demonstrated by exposure of nitrocellulose-bound trypanosome proteins to brain tubulin. When this protein was added to brain tubulin in the presence of taxol and GTP, microtubule bundles were formed with regular crosslinks between the parallel closely packed microtubules. The crosslinks were about 7.2 nm apart (center to center). Under the same conditions, but with the 53 kDA protein or without trypanosome derived proteins, brain tubulin polymerized to single microtubules. It is thus suggested that the unique structural organization of the subpellicular microtubules is dictated by specific parasite proteins and is not an inherent property of the polymerizing tubulin. The in vitro reconstituted microtubule bundles are strikingly similar to the subpellicular microtubule network of the parasite.

Serodiagnosis of African sleeping sickness in vervet monkeys by detection of parasite antigens.

Sera of vervet monkeys experimentally infected with T. b. rhodesiense were examined using a double antibody sandwich ELISA and Procyclic Agglutination Trypanosomiasis Test (PATT) for the presence of circulating trypanosomal antigens and anti-procyclic surface antibodies, respectively. Trypanosomal antigens were detected at 7 days post infection and remained at a detectable level thereafter during the infection. Antigens were not detected in sera prior to experimental infection or at 26 days after trypanocidal drug treatment. Although both the PATT and the sandwich ELISA results correlated with the infection status of the animals, the sandwich ELISA gave a better indication of the disease progression than the PATT, especially during trypanocidal drug therapy. The results illustrate the potential utility of the double antibody sandwich ELISA for diagnosis of African sleeping sickness.

Assessment of sensitivity of Trypanosoma congolense to isometamidium chloride: a comparison of tests using cattle and mice.

The sensitivities of 3 strains of Trypanosoma congolense to isometamidium chloride (Samorin) were determined in mice and cattle, with the objective of evaluating sensitivity testing in mice as a means of predicting curative doses in cattle. Comparison of mouse effective dose 80% (ED80) or curative dose 80% (CD80) values with cattle minimum curative dose (MCD) values demonstrated a wide variation between trypanosome strains. Although a mouse test may give a broad indication of the sensitivity of a strain, it cannot be used to predict curative doses for cattle. It was concluded that care should be exercised in extrapolating the results of a mouse test to cattle.

Use of procyclic trypanosomes for detection of antibodies in sera from vervet monkeys infected with Trypanosoma rhodesiense: an immunodiagnostic test for African sleeping sickness.

Uncoated procyclic culture forms of African trypanosomes were used in immunofluorescence and simple agglutination assays to detect antibodies in the sera of vervet monkeys infected with T. b. rhodesiense. Antibodies to procyclic surface antigens were found in sera from animals with active, untreated infections or sera taken soon after treatment with trypanocidal drugs. The antibodies were detectable within 7 days of infection. No specific antibodies were detected in sera prior to infection or long after drug cure. The results indicate that antigens expressed on the surface of procyclic culture forms of T. brucei spp. are useful for the detection of antibodies produced in response to infection with T. b. rhodesiense and may allow the development of a simple immunodiagnostic test for African sleeping sickness. In addition, the use of a form of the trypanosome of a different differentiation state from the infecting organism illustrates the utility of this approach for detection of antibodies to common antigens.

A simple epidemiological method for animal trypanosomiasis to provide relevant data for effective financial decision-making.

The epidemiology of bovine trypanosomiasis on Galana Ranch, Kenya was studied for one year (July 1980--June 1981), using measurements from an experimental population of 3 types of Boran cattle. The results were applied to the estimated ranch population at risk, and an attempt was made to measure the potential economic loss due to trypanosomiasis. The potential losses in beef production due to trypanosomiasis in the ranch population at risk at Galana were estimated at around K.Shs. 8900/km2, when the stocking rate was 14.2 Tropical Livestock Units per km2. The estimated potential loss in the total population at risk was approximately K.Shs. 5 million (around US$ 700,000 at 1981 values). These losses can be minimised by good management techniques based on accurate epidemiological information. Results indicated that Orma-type Boran steers are more resistant to trypanosomiasis than Galana-type Boran steers. Although both types showed similar mortality rates, untreated Orma animals which survived showed a similar growth performance to animals maintained under 3-month Samorin prophylaxis. Untreated Galana Borans lost 14% of their body weight when compared with animals maintained under 3-month Samorin. Also, 30% of the untreated Orma Borans never showed trypanosomes in their blood over the study period, while all corresponding Galana Borans showed parasites; this resulted in a lower measured attack rate in Orma cattle. In 1982, a combination of treating only those animals under trypanosome attack, relating the timing of chemotherapy to measured increased trypanosome attack, and the increased utilisation of the trypano-tolerant Orma Borans resulted in an annual saving of around US$ 110,000 in control costs and an increased land usage of approximately 5%.

Trypanotolerance in East African Orma Boran cattle.

Comparative studies on two types of large East African zebu (Bos indicus) Boran cattle, on a beef ranch in Kenya, have indicated that a Boran type bred by the Orma tribe has a superior response to tsetse fly challenge. The Orma Boran when compared with an improved Boran was found to have lower trypanosome infection rates and, when untreated, better control of anaemia and decreased mortality.