Suspected in utero infection in a boran heifer experimentally infected with Trypanosoma vivax.
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Biomedical subjects
A G Luckins
Publications and source records attributed to A G Luckins.
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.
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Growth rates of suramin-sensitive and resistant Trypanosoma evansi.
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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.
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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.
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Efficacy of Cymelarsan in Friesian Holstein calves infected with Trypanosoma evansi.
Two studies on the efficacy of mel Cy (Cymelarsan, Rhone Merieux, France) for the treatment of cattle infected with Trypanosoma evansi were carried out with groups of 5 Friesian Holstein calves infected with an Indonesian stock of T. evansi and treated 14 days after infection. In the first study 3 groups were injected subcutaneously with Cymelarsan at dose rates of 0.125, 0.25 and 0.50 mg/kg and in the second study 2 i/m at 0.50 and 0.75 mg/kg. The response to treatment was monitored parasitologically by daily microhaematocrit centrifugation technique and weekly mouse inoculation. Enzyme linked immunosorbent assays were used to monitor trypanosomal antibodies and trypanosomal antigens in serum samples collected weekly. Relapse infections occurred in all the groups given the drug s/c whilst all the animals treated i/m remained parasitologically negative up to 80 days after treatment. Results from serological assays, however, suggested the possible persistence of trypanosome infection in the animals treated at a dose rate of 0.50 mg/kg i/m although trypanosomes could not be demonstrated parasitologically. A dose rate of 0.75 mg/kg administered i/m is recommended, therefore, for the treatment of T. evansi infection in Friesian Holstein cattle.
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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.
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Experimental induction of suramin-resistance in cloned and uncloned stocks of Trypanosoma evansi using immunosuppressed and immunocompetent mice.
Suramin-resistance was experimentally induced in cloned or uncloned T. evansi populations using both immunosuppressed and immunocompetent mice by administration of subcurative doses of the drug. The highest level of resistance achieved was 3,000 fold using cloned trypanosomes in immunosuppressed mice. In the absence of suramin, suramin-resistance in T. evansi was observed to be stable for ten passages in mice. The results obtained in this study imply that induction of suramin resistance is by a mutational event followed by selection of resistant mutants by the presence of the drug. Immunosuppression of animals by heavy parasite burden or stressful conditions in conjunction with underdosing may therefore play an important role in the development of drug resistance under field conditions.
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Evaluation of mono- and polyclonal antibody-based antigen detection immunoassays for diagnosis of Trypanosoma evansi infection in the dromedary camel.
Two enzyme-linked immunosorbent assays (ELISA), one based on a mouse anti-Trypanosoma brucei group-specific monoclonal antibody and the other on rabbit anti-Trypanosoma evansi polyclonal antibodies, have been evaluated for their ability to detect circulating trypanosome antigens in camel sera as a means for the diagnosis of T. evansi infections. All 91 sera from a negative control camel herd from Kenya gave negative antigen-ELISA results in the monoclonal antibody-based ELISA and only 2 of them (2.2%) gave false positive results in the polyclonal antibody-based ELISA. In subsequent analyses of sera from infected camels (as determined by mouse inoculation), the monoclonal antibody-based ELISA detected antigens in 90 (83.3%) out of the 108 sera tested. This percentage was lower for the polyclonal antibody-based ELISA which was able to detect antigens in 67 (60.9%) out of the 110 sera tested. The two tests detected probably different antigens and when the results were combined, 99 out of 107 (92.5%) sera were shown to be ELISA positive. In a survey involving 316 camels from the Gao and Nara areas, in Mali, a high proportion of animals tested were antigen positive (43.5 and 42.9%, respectively for the mono- and polyclonal antibody-based ELISA) compared to only 22 (7.0%) diagnosed by the parasite detection techniques. Thus, these immunoassays were at least six times more sensitive than the haematocrit centrifugation technique. As a large proportion of cases may be antigen positive but parasite negative, these two of "surra" immunoassays should be used in routine diagnosis in addition to the parasite detection techniques in the dromedary camel.
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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.
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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.
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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.
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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.
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Immunohistology of lymph nodes draining local skin reactions (chancres) in sheep infected with Trypanosoma congolense.
Marked enlargement of lymph nodes draining local skin reactions (chancres) occurred in sheep following intradermal inoculation of cultured metacyclic forms of Trypanosoma congolense. Histologically, these lymph nodes were characterized by follicular hypertrophy and hyperplasia, compression and relative reduction of the paracortical areas and expansion of the medullary regions. Immunohistochemical staining with monoclonal antibodies to ovine lymphocyte subsets and Fc receptor (FcR) bearing macrophages, revealed increased expression of B cells (CD45R+), major histocompatibility complex (MHC) Class II, FcR+ macrophages, and CD1+ cells in the cortical and paracortical areas. The paracortical areas were found to be sparsely populated by CD5+, CD4+ and CD8+ cells, while the medullary areas contained numerous CD8+ cells and FcR+ macrophages. FcR+ macrophages were also present in cortical trabecular and subcapsular sinuses. As the chancre regressed, lymph node reactivity also subsided and fewer B cell follicles were observed and there was decreased expression of CD45R+ and MHC Class II+ cells.
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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.
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Characterization of Trypanosoma congolense serodemes in stocks isolated from Chipata District, Zambia.
Six stocks of Trypanosoma congolense were cloned from 17 stocks isolated from Eastern Zambia and used to initiate insect-form in vitro cultures producing metacyclic trypanosomes. Serological assays were then developed using these in vitro-derived metacyclics as a reference collection of antigens. Monoclonal antibodies recognized 8 metacyclic variable antigen types (M-VATs) of one stock, T. congolense TREU 1885, representing 70-80% of that stock's M-VAT repertoire, and in an indirect fluorescent antibody test (IFAT) there were no cross-reactions between them and the metacyclic trypanosomes of the other 5 stocks. Cross-protection assays between the 6 stocks in mice showed that the stocks cultured in vitro were serologically distinct. In order to facilitate serological typing for serodeme characterization, an IFAT was developed using formalin-fixed metacyclic trypanosomes to identify VAT specific immune responses using 21 day post-infection antisera. The cultured stocks reacted only with their homologous antisera thus confirming the results obtained in the cross-protection assays. No cross-reactions were observed with the 6 cloned stocks and antisera against the 11 stocks of T. congolense isolated in the same area at the same time suggesting that these stocks were different from the reference collection of cultured metacyclics. Hence, at least 7 serodemes of T. congolense have been identified from the 17 stocks isolated.
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Cellular phenotypes in Trypanosoma congolense infected sheep: the local skin reaction.
Mononuclear cell subpopulations in local skin reactions (chancres) in sheep infected with metacyclic forms of Trypanosoma congolense were studied by indirect immunoperoxidase staining using a panel of monoclonal antibodies (MoAbs) specific for ovine leucocyte subsets. Morphometric analysis revealed significant increases in numbers of cells expressing CD5, CD4, CD8, CD45R (mainly B cells), major histocompatibility complex (MHC) class II antigens, Fc receptors (FcR) on macrophages (VPM32) and FcR on B cells and macrophages (VPM33) from five days post-infection. B cells which also expressed MHC class II were found mainly in dense aggregates. The CD4/CD8 ratios were raised over pre-infection levels at 5-7 days post-infection. In sheep which had been infected, treated with trypanocidal drugs and then challenged with an heterologous serodeme of T. congolense, changes in cellular phenotype kinetics were similar to those seen in the skin in primary infections. Sheep superinfected with either an homologous or an heterologous, T. congolense serodeme showed only mild cellular infiltration and slight increases in various cellular phenotypes at the sites of inoculation.
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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.
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