Oocyst counts in crossbred ewes under tree-crop plantation in the forest zone of Ghana.

In this paper, the Eimeria oocyst output of two groups, pregnant ewes (group 1) and non-pregnant controls (group 2), which were followed from September 1993 to August 1994, is described. In both groups of animals the level of oocyst output was high during the minor rainy season. However, during the periparturient period the pregnant ewes showed the higher oocyst output. The oocyst output in both group fell to similar levels after weaning of the lambs in March 1994. The species of Eimeria identified in order of dominance were Eimeria parva, E. pallida, E. faurei, E. ahsata, E. ovina, E. intricata, E. granulosa and E. ninakohlyakimovae. There were no differences in the species composition of oocysts in both groups of animals.

Detection of Trypanosoma brucei, T. congolense and T. vivax infections in cattle, sheep and goats using latex agglutination.

A monoclonal antibody-based latex agglutination test for detection of circulating trypanosome antigens in animal serum was evaluated for the ability to detect natural T. brucei, T. congolense and T. vivax infections in cattle, sheep and goats in Ghana. The test detected antigens in 180/422 (42.7%) of cattle, 27/131 (20.6%) of sheep and 14/79 (17.7%) of the goats. By comparison, the microplate-based antigen-ELISA gave similar results (P > 0.01), detecting trypanosome antigens in 41.7% of the cattle, 19.8% of the sheep and 17.7% of the goats. Trypanosomes were demonstrated in the blood of 30 (7.2%) cattle, 7 (5.3%) sheep and 3 (3.8%) goats using the buffy coat technique (BCT). Of these, 26 cattle (86.7%), 6 sheep (85.7%) and all 3 goats (100%) were antigenaemic. The most prevalent single infection in all 3 animal species involved T. vivax, and the most common mixed infection involved all 3 trypanosome species in cattle and sheep. There was no mixed infection in goats. Compared with the antigen-ELISA, the sensitivity of the latex agglutination test was 98.3% in cattle and 100% in both sheep and goats, whilst the specificity was 97.2% in cattle, 99% in sheep and 100% in goats. False positivity with the latex agglutination test was 3.9% in cattle and 3.7% in sheep. There were no false-positive reactions with the test in goats. The latex agglutination assay promises to be ideal for testing small numbers of animals under field conditions.

Characterization of monoclonal antibodies reactive with Schistosoma haematobium-soluble egg and infected human urinary antigens.

Six murine monoclonal antibodies (MAbs) [Sh1/71.7 (IgM), Sh2/15.F (IgG1), Sh3/15.28 (IgG1), Sh3/38.2 (IgM), Sh4/14.3 (IgG1), and Sh5/32.30 (IgM)] produced against S. haematobium were extensively characterized. All the MAbs stained the surface membranes of miracidia as determined by the indirect fluorescent antibody test, yet three of them (Sh1/71.7, Sh3/15.28, and Sh3/38.2) also stained internal cytoplasmic antigens. Proteinase-K digestion and periodate oxidation studies showed that these three MAbs bound glycoprotein antigenic determinants, while the rest detected protein epitopes. Only Sh2/15.F, Sh3/15.28, and Sh4/14.3 could bind antigens with the western immunoblot assay. Sh2/15.F bound a 29-kDa antigen and Sh3/15.28 bound three antigen bands (53, 57, and 66 kDa) all in the soluble egg extract of an Egyptian strain of S. haematobium (SEAEgy), while Sh4/14.3 reacted with a 29-kDa antigen present in SEAEgy, as well as in the adult worm antigen extracts of Ghanaian strain(s) of S. haematobium and S. japonicum. Sh4/14.3 also bound a 78-kDa antigen in the S. japonicum worm. Cross-reactivity studies with S. haematobium, S. mansoni, S. japonicum, and Necator americanus revealed that Sh2/15.F and Sh3/15.28 were S. haematobium species-specific. Each of the remaining MAbs detected the three major human schistosomes without cross-reacting with N. americanus egg antigens. Three MAbs, Sh2/15.F, Sh4/14.3 and Sh5/32.30, could detect S. haematobium antigens in infected human urine.

Field evaluation of a dot-ELISA for the detection and differentiation of trypanosome species in infected tsetse flies (Glossina spp.).

A rapid, visually read, dot-ELISA developed for the detection and differentiation of trypanosome species in tsetse flies (Glossina spp.), was field tested alongside the standard fly dissection method on a range in south eastern Kenya. Of 104 G. pallidipes dissected, 2 were found to be infected with trypanosomes in their midguts. By the dissection method the infecting trypanosome species could not be identified, as both flies had no salivary gland infections. However, using the dot-ELISA, the 2 flies were shown to be infected with Trypanosoma congolense in their midguts. The midguts of an additional 6 (5.8%) of the 104 G. pallidipes tested positive for T. congolense int he dot-ELISA, even though no trypanosomes were seen on dissection. The infection rate for this fly species as determined using the dot-ELISA, therefore, was 7.7% for T. congolense in midgut infections compared to 1.9% identified by fly dissection. The salivary glands and mouthparts of the 6 additional tsetse flies identified by dot-ELISA were all negative as determined by the 2 techniques. None of 390 G. longipennis flies dissected and examined for trypanosomes in the midgut, salivary glands and mouthparts was shown, by this method, to be infected. Using the dot-ELISA, however, 17 (4.4%) of the flies tested positve for T. congolense in the midgut, whilst the salivary glands and mouthparts of the same flies were negative. Thus, the dot-ELISA appears to be more sensitive than the fly dissection method under field conditions. Moreover, the dot-ELISA can be performed in the field without electricity. It is simple to perform, and was not affected by high ambient temperatures (22-32 degrees C), or by contamination of reactants with dust.

Differentiation between culture-derived insect stages of T. brucei, T. vivax, T. congolense and T. simiae using a monoclonal antibody-based dot-ELISA.

A sensitive and specific nitrocellulose (NC) membrane-based dot-ELISA, utilizing a panel of monoclonal antibodies (mAbs), was developed for differentiation between in vitro-derived procyclic forms of Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the aforementioned trypanosome species in both single and artificially mixed preparations. Ten T. brucei, 4 T. vivax, 7 T. congolense and 3 T. simiae procyclic stocks and clones from different geographical areas were tested and identified using the specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could detect as few as 10 trypanosomes/dot, whilst 1 T. vivax mAb was able to detect a minimum of 100 trypanosomes/dot in monospecies preparations. A concentration of 1 x 10(4) trypanosomes/microliters/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 degrees C under desiccated conditions did not show any loss in activity for up to 90 days. However, when stored under similar conditions at room temperature (17-26 degrees C), the T. congolense-specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90.

Production and characterization of a monoclonal antibody specific for Trypanosoma simiae.

Monoclonal antibodies (MoAb) were produced against invariant antigens of vector forms of Trypanosoma simiae. X63/AG8.653, NSI/1AG401 and Sp20Ag14 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized with various preparations of T. simiae procyclics. A T. simiae-specific MoAb [KNS7/14.X (IgG1)] was detected in the hybridoma culture supernatants, which were screened for antibody activity by micro-plate and dot ELISA. Immunofluorescence studies showed that KNS7/14.X stained cytoplasmic antigens in T. simiae procyclics. Proteinase-K digestion and periodate oxidation studies revealed that KNS7/14.X binds to a carbohydrate antigenic determinant in glycoprotein or glycolipid. Cross-reactivity studies using vector forms of T. brucei, T. vivax, T. congolense, T. simiae and T. grayi showed that KNS7/14.X only reacted with T. simiae. Attempts to generate other T. simiae-specific MoAb, using 107-, 75- or 41.7-43.6-kDa peptides selected by western blotting analysis, did not yield positive results.

Hydrogen peroxide destaining: a new method for removing non-specific stains in nitrocellulose membrane-based dot-ELISA for the detection of trypanosomes in tsetse flies (Glossina spp.).

Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto nitrocellulose (NC) membrane were found to stain the membrane with differing coloration and intensity. The stains were, predominantly, either reddish to brown or blackish-brown to black and occasionally greenish to almost colourless, depending on the stage of digestion of the bloodmeal in the fly. NC membrane strips applied with tsetse gut samples from T. brucei infected and uninfected control flies were tested with the standard antigen detection dot enzyme-linked immunoassay (dot-ELISA), using a T. brucei specific monoclonal antibody (MoAb) and horseradish peroxidase goat anti-mouse conjugate. The stains in both infected and uninfected sample dots persisted through the assay. Furthermore, the staining intensity of some assayed uninfected sample dots were enhanced as a result of non-specific reactivity, making it difficult to distinguish between the infected and uninfected flies. This necessitated the development of a simple technique by which the non-specific stains and reactions could be removed. Sample 'dotted' NC membrane strips were destained by incubation with 5% hydrogen peroxide (H2O2) diluted in 5% skimmed milk in Tris buffer, pH 8.0. After washing, the destained strips were tested in the dot-ELISA. This method gave satisfactory reproducible results, since the most intense stains could be removed, and it had no effect on trypanosome antigens detected by a panel of four T. brucei species-specific, three T. vivax species-specific, four T. congolense species-specific and four Nannomonas subgenus-specific MoAbs. Using the destaining process in a modified dot-ELISA, 86 out of 95 (90.5%) of Glossina morsitans centralis flies experimentally infected with T. brucei, were identified. The destaining method was also used successfully to decolorize NC membrane bound tsetse faecal material.

Detection and differentiation between trypanosome species in experimentally infected tsetse flies (Glossina spp.) using dot-ELISA.

A modified NC membrane-based dot-ELISA was used to detect and differentiate between Trypanosoma brucei, T. congolense and T. simiae procyclics in the midguts of experimentally infected tsetse flies. The modification of the assay consisted of (a) the lysis of T. congolense or T. simiae in NC membrane applied sample dots using Triton X-114, and (b) treatment of sample applied NC membrane strips with hydrogen peroxide to remove non-specific stains. Also, T. brucei was detected in the salivary glands, and T. congolense and T. vivax were detected in the mouthparts, however, in dot-ELISA without modification. In all the assays, T. brucei and T. congolense parasites were detected directly using MoAbs specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-specific MoAbs. The sensitivity of the assay for detecting midgut infections was 90.5%, 84.6% and 94.4% in detecting T. brucei, T. congolense and T. simiae, respectively. Sample dots stored at room temperature (19-26 degrees C) under desiccated conditions did not show any loss in activity in 90 days. However, after 7 days of storage, a ring-pattern reaction appeared on some sample dots that were tested with T. brucei specific MoAb, irrespective of whether T. brucei antigens were present or not. These ring reactions, however, did not interfere with correct interpretation of the assay results. The specificity of the assay for detection of T. brucei in the salivary glands was 100% and the sensitivity was 90%. Also, T. vivax and T. congolense organisms were each detected in the mouthparts of infected tsetse flies, with 100% specificity. The sensitivity was, however, lower, 43.8% for T. vivax and 55.6% for T. congolense.

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle.

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.

Haemorrhagic lesions resulting from Trypanosoma vivax infection in Ayrshire cattle.

Infection of Ayrshire cattle with a stock of Trypanosoma vivax from the Galana Ranch, Kenya, resulted in an acute disease characterised by profound anaemia and haemorrhage, which reached maximum severity between 3 and 5 weeks after infection. Bleeding from the ears, nose and rectum occurred. At necropsy, petechial and ecchymotic haemorrhages were widespread, but were particularly severe in the gastrointestinal tract. In confirmation of the gross findings, congestion, haemorrhage and degenerative changes in most tissues and organs were found histologically. Thrombi were seen in the lymphatic vessels and clots of fibrin were present in the ventricles of the brain. The anaemia was a consequence of frank blood loss through haemorrhaging, exacerbated by erythrophagocytosis of deformed red blood cells, whose occurrence was indicative of microangiopathic changes. Animals were euthanised between 23 and 36 days after infection when they became recumbent with PCV values as low as 9%. There is no doubt that animals affected by this syndrome in the field would die within a few weeks of infection, if left untreated.

Immunosuppression in experimental African trypanosomiasis. Polyclonal B-cell activation and mitogenicity of trypanosome-derived saturated fatty acids.

Changes in antibody responses in adult mice infected with Trypanosoma congolense and subsequently challenged with unrelated antigens (sheep red blood cells and pneumococcal polysaccharide) were studied. Immune responses were significantly depressed within 1 week of infection, and complete suppression of both IgM and IgG responses to both types of antigen was established 15 days after immunization. Coincidentally with the development of high parasitaemias, background IgM plaque-forming cell responses to sheep red cell antigen significantly increased in non-immunized T. congolense-infected animals. Autolysates of T. congolense and chloroform-soluble extracts of the autolyzed trypanosome were found to be mitogenic in vitro for the spleen cells of normal mice. Fractionation of these extracts by thin-layer chromatography indicated that the mitogenic activity migrated with the free fatty acids. Substitution of the relevant saturated and unsaturated free fatty acids in the autolyzed trypanosome extracts with commercial pure fatty acids in the mouse spleen cultures indicated that the mitogenicity was due to palmitic and stearic acids. It is suggested that the general immunosuppressing effect of trypanosomes may be attributed, at least in part, to the polyclonal activation, and subsequent depletion and/or clonal exhaustion of B-cells as a result of blastogenic stimulus from the parasites. This may operate, at least in part, through the generation of B-cell mitogenic saturated fatty acids.

Experimental induction of an immunohaemolytic anaemia in the chicken with Salmonella gallinarum endotoxin.

The possibility that the extreme refractoriness of the chicken to injected endotoxin would permit the buildup of sufficient free endotoxin in the circulation to immunologically modify the erythrocytes in vivo and thereby cause them to be eliminated from the circulation was investigated. It was shown that a moderately severe immunohaemolytic anaemia accompanied by a mild spenomegaly could be induced in chickens by single (large) or multiple injections of Salmonella gallinarum endotoxin (SgE). Varying degrees of reticulocytosis, with corresponding decreases in haemoglobin and haematocrit values, were observed from the tenth day after the injection of the SgE. All the injected animals also showed significant immune responses to SgE, peak haemagglutinin titres occurring coincidentally with the peaks of the haemolytic episodes. A similar injection schedule in specifically immune chickens also showed the anaemia appearing much earlier, persisting longer and being much more profound in intensity. It is considered that the in vivo erythrocyte sensitization induced by SgE injection initiates the erythrocytic changes observed and that the underlying mechanism responsible for the subsequent development of the haemolytic anaemia is immunologically mediated.

Mitogenicity of autolysates of Trypanosoma congolense.

Autolysates of Trypanosoma congolense, in subcytotoxic amounts, were found to be highly mitogenic in vitro for the spleen cells of normal mice. Significant amounts of [3H]-thymidine were also incorporated by the responding spleen cells of nu/nu (athymic) mice. In contrast, the spleen cells of cyclophosphamide-treated mice were unresponsive. The findings suggest that a potent B-cell-mitogen is generated by the autolysing T. congolense organism.

Free fatty acids, complement activation, and polyclonal B-cell stimulation as factors in the immunopathogenesis of African trypanosomiasis.

Human and animal forms of African trypanosomiasis are characterised by sustained hypocomplementaemia, gross hypergammaglobulinaemia M, and profound immunosuppression. It is suggested that this hypocomplementaemia is probably due to the action of a trypanosome-derived complement-activating factor and that the elevated IgM levels may be the combined result of this decomplementation, together with a subsequent failure of the normal IgM-to-IgG antibody switch mechanism and polyclonal B-lymphocyte activation by a trypanosome-generated mitogen. The immunosuppression in this disease may be a result of the collective immunosuppressive effects of trypanosome-derived immune-modulating free fatty acids, polyclonally stimulating B-cell mitogen, and complement-activating factors.