Drug sensitivity of Trypanosoma evansi and the use of immunoassays in diagnosing infections with T. evansi in buffaloes in Vietnam.

The biological characteristics of isolates of T. evansi collected from buffalo in different provinces in North Vietnam was determined in terms of their sensitivity to drugs currently used in the treatment of trypanosomosis. Five isolates were collected from buffalo, cloned and then tested against Trypamidium, Samorine, Naganol and Veriben. All isolates were sensitive to Naganol and Veriben. An isolate from a buffalo in Ha bac province (Hb1) was the least sensitive with trypamidium at a CD80 > 128 mg/kg, more than 8 times the CD 100 of the remaining isolates (16 mg/kg). An antigen-detection enzyme immunoassay (Ag-ELISA) based on a T. evansi-specific monoclonal antibody was evaluated for its ability to detect infections with T. evansi in buffalo. The sensitivity of the Ag-ELISA was 63% and the specificity 75%. The positive predictive value of this assay was too low to allow identification of individual infected animals on the results of a single test in the districts investigated. For definitive diagnosis, a serial testing protocol was used, where a more specific test, the card agglutination test (CATT) was used initially and any positive samples was then checked by the Ag-ELISA.

Detection of antibodies to Trypanosoma cruzi in the South American opossum (Didelphis marsupialis).

Stocks of Trypanosoma cruzi belonging to two different zymodemes, one usually associated with silvatic reservoir hosts and the other not normally found in wild reservoir hosts, were used as sources of diagnostic antigens in enzyme-linked immunosorbent assays for the detection of antibodies to T. cruzi in Didelphis marsupialis. Both antigen preparations reacted with antibodies in sera from animals found to be infected by conventional parasitological techniques and also in sera from a proportion of the remaining animals in which it was not possible to detect trypanosomes.

Multiplication of Trypanosoma evansi at the site of infection in skin of rabbits and cattle.

Local skin reactions (chancres) developed at the sites of inoculation with Trypanosoma evansi in rabbits and calves. Trypanosomes multipled in the dermal collagen and, in the rabbit, were present in large numbers by 7 days after infection. Thereafter, however, numbers decreased and few parasites were observed by 11 days after infection. The presence of trypanosomes in the skin caused an extensive inflammatory reaction with disruption of collagen, oedema, necrosis of the skin and increases principally in neutrophils and lymphocytes. In calves, similar changes were observed although there were fewer trypanosomes present in the chancre and the cellular involvement was less extensive than that seen in the rabbit. This early extracellular proliferative phase of development of T. evansi may be of importance in naturally transmitted infections both in the initial establishment of the parasite in the mammalian host and in enabling the parasite to increase the numbers of antigenic variants expressed before the parasites invade the general circulation.

Stability of metacyclic variable antigen types (M-VATs) during the early stages of infection with Trypanosoma congolense.

Expression of nine metacyclic variable antigen types (M-VATs) of Trypanosoma congolense in chancres from infected rabbits was determined using monoclonal antibodies raised against metacyclic forms of trypanosomes. Trypanosomes present in chancres 7-9 days post infection expressed M-VATs present in metacyclic populations of the parasites. The majority of M-VATs expressed showed little proportional change from those observed on metacyclic trypanosomes during this period although expression of one M-VAT increased, and another decreased. Although trypanosomes in chancres continued to express M-VATs, other VATs, not present in the M-VAT repertoire were also expressed and neutralization tests showed that new VATs appeared by 7 days after infection. In infected sheep neutralizing antibodies against M-VATs were detected by day 14 in lymph from efferent lymphatics draining lymph nodes in the region of chancres. Neutralizing antibodies directed against metacyclics were also present in the serum by day 14 and were still detectable for up to 35 days post infection. Hence, it is likely that in the vertebrate host the trypanosomes multiplying in the skin at the site of tsetse bit express all M-VATs characteristic of that particular serodeme, enabling the host to develop immunity to all antigen types present in the M-VAT repertoire.

Variability of in vitro culture characteristics, including metacyclic trypomastigote production, in different stocks of Trypanosoma congolense.

Six cloned stocks of Trypanosoma congolense, isolated from the same area of Eastern Zambia, were maintained in vitro as insect form cultures producing infective metacyclic trypanosomes. Although the same general culture conditions were applied, different handling regimes were required for optimum growth of each stock. During primary isolation, many differences were found in the culture characteristics of the stocks. The time taken for cytoadherence to occur varied from 14 to 62 days, while the interval between attachment and the appearance of infective metacyclic trypanosomes ranged from 9 to 94 days. There was a 10-fold difference in the numbers of metacyclic forms produced by different stocks. Time in culture appeared to have little effect on the production of metacyclic forms, and it is probable that in vitro characteristics of T. congolense depend on the genetic constitution of individual stocks or clones.

Trypanosoma congolense: re-expression of a deleted metacyclic variable antigen type in vivo and in vitro.

The expression of variable antigen types (VATs) was determined among dividing populations of T. congolense growing in vivo in rabbit chancres and in vitro on bovine aorta endothelial cell monolayers. Experiments were performed in which a single metacyclic VAT (M-VAT) was deleted from a cultured metacyclic population by neutralisation with a monoclonal antibody and complement. Subsequent expression of the deleted M-VAT and two unrelated M-VATs was determined by an indirect immunofluorescent antibody test. The deleted M-VAT was re-expressed both in vivo and in vitro and the proportions of unrelated M-VATs were not markedly affected by the neutralisation of this single M-VAT. In addition, an overall similarity was observed between M-VAT expression and re-expression in vivo and in vitro.

Early stages of infection with Trypanosoma congolense: parasite kinetics and expression of metacyclic variable antigen types.

Trypanosoma congolense develops in the skin of sheep at the site of inoculation of metacyclic trypanosomes, forming a chancre containing large numbers of parasites. By cannulating the afferent and efferent lymphatic ducts draining the skin and regional lymph node, the progressive development and migration of trypanosomes from the chancre was monitored and the expression of metacyclic antigen types (M-VATs) was determined. The kinetics of development of parasitosis in the afferent and efferent lymph was similar. Trypanosomes were detected in lymph 5 to 6 days after the inoculation of cultured metacyclic trypanosomes, at the same time as the chancre first appeared in the skin. The numbers of trypanosomes in the lymph reached their peak levels 8 to 10 days post infection and thereafter numbers fell, although there were still parasites in the lymph after the chancre had regressed. Trypanosomes in the afferent lymph expressed mainly M-VATs and the absolute numbers of four M-VATs which were monitored increased up to 9 days post infection. There was a fall in numbers by day 10, but 92% of the trypanosomes in the afferent lymph continued to express M-VATs. In contrast, trypanosomes from the efferent lymph were found not to express M-VATs suggesting that a major switch in VAT expression occurs in the lymph node. Specific antibody responses, measured by neutralization tests, were evident 16 to 20 days after infection in afferent lymph but only low levels of antibodies were found in efferent lymph.

In vitro cultivation of Trypanosoma congolense: establishment of infective mammalian forms in continuous culture after isolation from the blood of infected mice.

Bloodstream form trypomastigotes of four cloned stocks of Trypanosoma congolense from West Africa were successfully adapted to continuous in vitro culture at 28 degrees C using bovine aorta endothelial cell monolayers and Eagle's minimum essential medium supplemented with 20% normal bovine serum or foetal calf serum. The trypanosomes maintained in vitro morphologically resembled bloodstream forms and remained infective for vertebrate hosts. They also induced local skin reactions in rabbits and were therefore designated "mammalian forms", possibly resembling parasites which develop extravascularly in the vertebrate host following introduction of metacyclic trypanosomes into the skin by bites of tsetse flies. Mammalian forms of two stocks were allowed to transform to procyclic trypanosomes in order to obtain cultures producing epimastigote and metacyclic stages of T. congolense. Metacyclic trypanosomes produced in this manner were shown to be neutralized by antiserum raised in rabbits against the homologous trypanosome stock transmitted by tsetse flies.

In vitro cultivation of Trypanosoma congolense: the production of infective forms from metacyclic trypanosomes cultured on bovine endothelial cell monolayers.

After transfer to bovine endothelial cell monolayers cultured in Eagle's minimal essential medium at 28 degrees C or 37 degrees C metacyclic trypanosomes of three cloned stocks of Trypanosoma congolense became morphologically similar to parasites found in the bloodstream of the vertebrate host. The trypanosomes resumed division and grew in close association with the mammalian cells, which were essential for growth. These dividing infective forms had the ability to cause local skin reactions and systemic infections when inoculated intradermally into rabbits. Trypanosomes grown in medium supplemented with foetal calf serum (FCS) eventually differentiated into procyclic forms. No such change occurred in medium supplemented with normal bovine serum. If procyclic forms in FCS were allowed to continue their differentiation at 28 degrees C they eventually produced epimastigotes which gave rise to infective metacyclic trypanosomes once more. It was thus possible to grow and maintain several different developmental stages of T. congolense by varying culture conditions.

In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks.

Glossina morsitans were infected with two cloned stocks of T. congolense. The proboscides, foreguts and midguts of infected flies were then used as sources of trypanosomes in vitro at 28 degrees C in the presence of bovine dermal collagen explants. Cultures were established in which trypanosomes differentiated into adhering colonies of epimastigote forms which could then be maintained and subcultured in Eagle's Minimum Essential Medium supplemented with foetal calf serum for over 40 weeks. Within 2-4 weeks of establishment of each culture or subculture the epimastigote trypanosomes differentiated into metacyclic trypanosomes which could be harvested from supernatant medium at concentrations of 1 X 10(5)-3 X 10(6) parasites/ml. These organisms were used to induce the formation of local skin reactions in rabbits. Successful cultivation of infective trypanosomes appeared to depend on the initial adhesion of the parasites to the surface of the flask where they subsequently differentiated first into epimastigote and then to metacyclic forms.

Increase in CD5+ B cells and depression of immune responses in sheep infected with Trypanosoma evansi.

The effects of Trypanosoma evansi on the cellular and humoral immune responses of sheep to Pasteurella haemolytica vaccine were studied. Peripheral blood lymphocytes (PBLs) from the sheep were analysed using single and double-colour indirect immunofluorescence staining and flow cytometry to monitor changes in circulating B and T cell subsets. Serum antibody responses were assayed using the enzyme-linked immunosorbent assay technique (ELISA), in addition to measuring local skin reactions at the site of vaccine administration. Results showed significant increases in circulating B cells in all sheep after the primary (p < 0.01) and secondary (p < 0.001) vaccinations although the increases were much more dramatic in the T. evansi-infected sheep. In addition, infection induced significant increases (p < 0.004) both in proportions and numbers of CD5+ B cells with more than 70% of circulating B cells expressing the CD5 antigen and showed significant differences (p < 0.01) from those of control sheep in which vaccination alone failed to induce similar increases. Also, infection resulted in significant decreases in CD5+ (p < 0.003), CD4+ (p < 0.03) and CD8+ (p < 0.03) T cell subsets in contrast to their increases in all control animals after vaccination. Moreover, there were significant suppression of both local skin reaction (p < 0.005) and serum Ig and IgG1 (p < 0.001) antibody responses to the vaccine antigen.

Effects of the depletion of CD8(+) T cells and monocytes on the proliferative responses of peripheral blood leucocytes from Trypanosoma evansi-infected sheep.

Sheep peripheral blood mononuclear cells and those depleted of CD8(+) T cells and/or monocytes were stimulated with polyclonal mitogens and specific antigens, and analysed by means of cell proliferation assay procedure to examine whether these cell populations are involved in Trypanosoma evansi-induced immunosuppression. The removal of CD8(+) T cells failed to normalize the proliferative responses of peripheral blood mononuclear cells from infected sheep to concanavalin A stimulation while the depletion of monocytes resulted in full and enhanced response, showing that macrophages are mainly responsible for the suppression. Although the depletion of CD8(+) T cells, monocytes or both restored the responses of the cells to lipopolysaccharide stimulation, the responsiveness of the undepleted cells to this mitogen was significantly higher from day 24 post infection (p<0.01). The results were discussed in relation to current known mechanisms of depressed lymphocyte proliferation in tsetse-transmitted African trypanosome infections.

Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones.

Four Trypanosoma evansi stocks with sensitivity to suramin in mice ranging from 0.05 to 160 mg kg-1 were cloned and sub-cloned and the sensitivity of the clones determined. The results suggest that it is easier to clone and sub-clone trypanosome stocks which are sensitive to suramin than those that are resistant to the action of the drug. The clones obtained from the four stocks had sensitivities to suramin which were similar to or different from the parent stocks. These results are important in view of the development of resistance for, in the presence of suramin, these resistant yet heterogeneous populations would provide the material from which selective processes could operate. These observations also suggest that the maintenance and spread of suramin-resistant trypanosomes might be curtailed by their comparative inability to establish themselves in a new host.

Trypanosoma congolense infection in sheep: ultrastructural changes in the skin prior to development of local skin reactions.

Events occurring in the skin of sheep prior to development of Trypanosoma congolense-induced local skin reactions (chancres) were studied using electron microscopy. Three days after infection, few trypanosomes were present in the dermal collagen. However, these parasites were more abundant 5 days after infection, and were also found in dermal lymphatics and in the connective tissue matrix between collagen bundles. Mast cells in the skin obtained 5 days after infection showed evidence of degranulation. These events may play a role during the induction phase of trypanosomal chancres.

Interference in the establishment of tsetse-transmitted Trypanosoma congolense, T. brucei or T. vivax superinfections in goats already infected with T. congolense or T. vivax.

An interference phenomenon that delays superinfection with a trypanosome species different from that used for the initial infection has been found to occur in goats. Following tsetse transmission of Trypanosoma brucei to goats already infected with T. congolense, there was a delay in chancre development, as well as in the appearance of T. brucei and anti-T. brucei antibodies in the blood when compared to previously uninfected goats. However, there was no delay in the establishment of a tsetse-transmitted superinfection with T. vivax in goats already infected with either T. congolense or in animals already infected with a different serodeme of T. vivax.

The use of enzyme linked immunosorbent assays to investigate the prevalence of Trypanosoma equiperdum in Ethiopian horses.

A field study involving 309 horses was undertaken in the provinces of Arsi and Bale in the Ethiopian highlands to investigate the prevalence of Trypanosoma equiperdum infections using enzyme linked immunosorbent assays (ELISAs) for the detection of both trypanosomal antigen and antibody. Adult horses of both sexes were examined for clinical signs of T. equiperdum infection and serum samples were collected for the assays. One hundred and one horses showed the presence of trypanosomal antibodies in their serum and 70 animals showed typical clinical signs of dourine. Nineteen horses showed the presence of trypanosomal antigen. Eight horses were positive for both T. equiperdum antibody and antigen. Blood and genital washes from seven antigenaemic horses were inoculated into mice and rabbits in an attempt to isolate trypanosomes but none became infected. Statistical analysis of the results of antibody assays indicated that there were significant differences in the distribution of serologically positive horses in the different clinical groupings, with seropositivity increasing with the severity of the observed clinical signs (P < 0.001). There was also a positive correlation between the presence of circulating trypanosomal antigen and clinical evidence of infection. Although it was not possible to obtain direct parasitological evidence of infection, the results of the serological assays, together with the clinical signs of disease observed in many of the horses, provide strong circumstantial evidence that T. equiperdum occurs in Arsi and Bale provinces of Ethiopia. Furthermore, in view of the large number of horses in Ethiopia and the unrestricted movement of animals throughout the country it is likely that dourine may be more widespread in Ethiopia than is currently realised. The assays used show potential for diagnosis of dourine, but to be widely applied in field situations for the diagnosis and control of dourine in Africa they require validation of their specificity and sensitivity.

Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine-biotin immunohistochemistry.

Twenty-four percent of hog deer (Cervus porcinus) that ranged free on a farm in Samut Prakarn province, Thailand, died showing nervous signs between September 1997 and February 1998. The nervous signs shown by most of them included ataxis, paresis of hind limbs, lateral recumbency, excitation and convulsion. Six animals and one carcass were submitted for diagnosis at the National Institute of Animal Health, Bangkok. Trypanosoma evansi was detected in blood and cerebrospinal fluid of four and five animals, respectively. Antibodies to T. evansi were found in all the hog deer by indirect enzyme-linked immunosorbent assay. Histopathological observation revealed a generalised non-suppurative meningoencephalitis affecting the white and grey matter at all levels of the brain. Typically, there were broad perivascular cuffs of mononuclear inflammatory cells, including lymphocytes, and some Mott cells. No trypanosomes were found in any tissue examined by conventional histopathology. However, numerous T. evansi were demonstrated by streptavidine-biotin immunohistochemistry in neuropil and Virchow-Robin spaces of brain in three animals.

Trypanosoma evansi infection in cattle, buffaloes and horses in Indonesia.

Cattle, buffaloes and horses in several areas of Indonesia were examined for evidence of infection with Trypanosoma evansi by the microhaematocrit centrifugation technique (MHCT) and an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to T. evansi. Evidence of infection was found in animals at each sampling site although differences were seen in prevalence rates between sites. Prevalence rates in buffalo were usually higher than in cattle in the same area while in horses they were much lower than in cattle or buffalo. An age-dependent prevalence rate was seen in buffalo and cattle with the highest rates seen in animals older than 2 years. These results concur with the view that T. evansi infection is widespread throughout most of the livestock-producing areas of Indonesia. The apparent lack of any obvious disease owing to T. evansi infection in the sampled animals suggests that a form of stability exists in most endemic areas which serves to ameliorate the effect of T. evansi infection and has an immunological basis linked to the parasite's limited antigenic diversity.

Adaptation and validation of antibody-ELISA using dried blood spots on filter paper for epidemiological surveys of tsetse-transmitted trypanosomosis in cattle.

The indirect enzyme-linked immunosorbent assay (ELISA) for the detection of anti-trypanosomal antibodies in bovine serum was adapted for use with dried blood spots on filter paper. Absorbance (450 nm) results for samples were expressed as percent positivity, i.e. percentage of the median absorbance result of four replicates of the strong positive control serum. The antibody-ELISA was evaluated in Zambia for use in epidemiological surveys of the prevalence of tsetse-transmitted bovine trypanosomosis. Known negative samples (sera, n = 209; blood spots, n = 466) were obtained from cattle from closed herds in tsetse-free areas close to Lusaka. Known positive samples (sera, n = 367; blood spots, n = 278) were obtained from cattle in Zambia's Central, Lusaka and Eastern Provinces, diagnosed as being infected with Trypanosoma brucei, T. congolense, or T. vivax using the phase-contrast buffy-coat technique or Giemsa-stained thick and thin blood smears. For sera (at a cut-off value of 23.0% positivity) sensitivity and specificity were 86.1 and 95.2%, respectively. For bloodspots (at a cut-off value of 18.8% positivity) sensitivity and specificity were 96.8 and 95.7%, respectively. The implications of persistence of antibodies following treatment or self-cure are discussed.

Trypanosoma congolense infection in sheep: cellular phenotypes in lymph and lymph nodes associated with skin reactions.

Intradermal inoculation of sheep with culture-derived metacyclic forms of Trypanosoma congolense resulted in the development of localized skin reactions (chancres) and enlargement of the draining lymph nodes 7 days after infection. Changes in the expression of surface antigens of lymphocytes in lymph leaving the affected skin reactions and in the associated lymph nodes were monitored by cannulating the afferent and efferent lymphatic ducts. Trypanosomes appeared in afferent and efferent lymph 3 to 5 days after infection and persisted even as the chancres regressed. The cellular output in both afferent and efferent lymph increased markedly after the onset of parasitosis. Sequential analysis of the phenotypes of lymphocytes by immunofluorescent staining and flow cytometry revealed that in afferent lymph draining the chancre there was an early response which was due to an increase in T cells, particularly CD4+ and CD8+ cells; however, as the chancres-regressed there was an increase in lymphoblasts and surface immunoglobulin-bearing cells. In contrast, in the efferent lymph, the increase in lymphocytes was due predominantly to a higher number of cells bearing surface immunoglobulins.