Characterisation of a monoclonal antibody against Trypanosoma evansi and its application for detecting circulating antibodies

Monoclonal antibodies were obtained against Trypanosoma evansi. The 2-4F6 IgM monoclonal antibody (Mab) was chosen for the study because of its ability to detect antigens and its specificity (as it did not recognise T. cruzi, T. equiperdum, Babesia equi or B. caballi). The immunoblot test revealed that the 2-4F6 IgM Mab recognises epitopes in two antigenic bands, one measuring 85 kDa and the other 122 kDa. An immunoassay for antigen detection in serum using polyclonal antibodies for capture, the Mab 2-4F6 as primary antibody and an antimouse IgM as secondary antibody gave positive results in 10 of the 11 equidae infected with T. evansi, whereas 20 controls gave negative results. These research results show that the Mab 2-4F6 and the antigen it recognises are useful in identifying equidae infected with T. evansi.     

Variant-specific trypanolytic antibodies in sera from patients infected with Trypanosoma brucei rhodesiense

Infections with salivarian trypanosomes are characterized by the successive development of parasite populations of distinct variable antigen types (VATs), the corresponding antibodies accumulating in the blood of the host. Sixty VATs had been cloned during previous studies on variable antigen repertoires of Trypanosoma brucei rhodesiense; 12 of these were selected for immunolysis tests against 85 sera from T.b. rhodesiense patients in Busoga (Uganda). One variant, ETat 1/1, reacted with 59 out of 65 sera that contained detectable lytic antibodies. ETat 1/1 in combination with two other variants ETat 1/14 and Utat 1/1, covered all the seropositive sera; other VATs showed varying degrees of reactivity. The results suggest that sera from T.b. rhodesiense patients contain easily detectable VAT-specific antibodies and that their corresponding antigens might be used in the preparation of serodiagnostic reagents for the disease.     

Validation of an indirect immunoenzyme assay for the detection of antibodies against Trypanosoma evansi in horses in Argentina

An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Trypanosoma evansi was evaluated using 90 different sera, obtained from naturally-infected horses. As negative controls, 218 sera from the T. evansi-free zone of Argentina, and 90 uninfected sera from the enzootic zone were used. The results of the ELISA were expressed in terms of percent positivity (PP) when compared with a positive primary reference serum, obtained from a horse experimentally-infected with T. evansi. The inter-assay coefficient of variation (CV), expressed as PP, was 44.7% for the negative control serum, 8.8% for the mildly positive reference serum, and 9.2% for the secondary positive control serum, while the intra-assay CV for each of the above sera was 6%, 2.8% and 5%, respectively. Positive and negative serological results were differentiated using a histogram of the distribution of the results obtained using sera from infected and uninfected animals from the enzootic zone (expressed in PP). A PP of 50 indicated a sensitivity of 95.5% for a confidence interval (CI) of 91.3% to 99.7%, and a specificity of 98% for a CI between 95% and 100%. Positive and negative predictive values were established for each rate of prevalence between 0.01% and 25%. The use of reference control sera in each assay enabled reproducible results to be obtained. The author recommends that this methodology be used whenever certification of the T. evansi status of horses is required, and particularly when animals are to be moved from an infected to a disease-free area.     

Absence of a surface coat from metacyclic Trypanosoma v/vax: possible implications for vaccination against vivax trypanosomiasis

Trypomastigotes attached to the wall of the hypopharynx in tsetse flies infected with Trypanosoma vivax are believed to represent the true metacyclic stage of this trypanosome. Electron microscopy demonstrates that attachment is mediated by hemidesmosome-like junctions along the flagellar membrane and that none of the trypomastigotes, either attached or free in the hypopharynx lumen, possesses a surface coat comparable with that on the metacyclics of T. brucei and T. congolense and on the bloodstream stages of all salivarian trypanosomes. As the variable antigen of bloodstream and metacyclic T. brucei is located in the surface coat, the absence of the coat from metacyclic T. vivax suggests that the mechanism of antigenic variation in this species may be somewhat different from that of antigenic variation in T. brucei, and that vaccination of cattle against T. vivax may prove a simpler proposition than vaccination against T. brucei.     

A pattern in the development of agglutinogenic antigens of cyclically transmitted isolates of Trypanosoma gambiense.

Three isolates of T. gambiense, belonging to 2 serologically different substrains of the species, were cyclically transmitted to rabbits and monkeys by G. morsitans or G. tachinoides. Sera collected from the infected animals were tested for agglutinating antibodies to 17 different serotype antigens prepared from rodent-adapted isolates of T. gambiense. The first antibodies detected in sera from all of the animals agglutinated the same serotype antigen L2, indicating that it possibly consisted of trypanosomes bearing a basic antigen of T. gambiense. In 2 animals infected with 1 isolate the development of antibody to the common serotype L2 was accompanied by the development of antibody to a second serotype U1. Comparisons of the patterns in which antibodies were produced to other serotype antigens in different animals indicated that there were many similarities in the sequences in which they developed in different rabbits infected with (a) any 1 isolate (b) 2 different isolates of 1 substrain and (c) isolates of 2 serologically different substrains. There were also similarities in the sequences in which the antigens developed in rabbits and monkeys infected with an isolate by the same fly or by different tsetse flies. The ordered sequence in which these 3 cyclically transmitted isolates of T. gambiense, of significantly varied origin and laboratory history, produced variant antigens in hosts of different species provides further evidence to support previous suggestions that antigenic variation in trypanosomes is an adaptive process, rather than one involving selection of mutants.     

Trypanosomiasis in domestic animals: the problems of diagnosis

Animal trypanosomiasis presents special problems with regard to diagnosis. The clinical signs are not pathognomonic and the standard techniques for the detection of trypanosomes are not sufficiently sensitive. Although significant improvements have been made in diagnosis, a high proportion of infections still remain undetected as the chronic, more common form of the disease, is often aparasitaemic. In the face of these constraints, alternative methods of diagnosis have been developed, most of which are for the detection of antibody responses to the antigens of the infecting trypanosomes. The most useful of these tests, in view of their sensitivity and specificity, are the indirect immunofluorescent antibody test, enzyme immunoassay (ELISA) and the card agglutination test for trypanosomiasis (CATT) which is used for the diagnosis of Trypanosoma evansi infections. However, there are several shortcomings in antibody detection tests: the antigens used are ill-defined, thus making standardisation of the tests rather difficult with regard to sensitivity and specificity. Furthermore, some of the tests are not applicable to the field. Moreover, the presence of antibody in the serum does not necessarily reflect an existing infection, as antibodies may persist for several months following recovery. Recently, development of assays for the detection of circulating trypanosomal antigens in the blood of infected animals has circumvented this problem since antigen-positivity indicates existing infection. These new assays have not yet been fully evaluated in the field, but the data generated so far do indicate that the diagnostic strategy for the future is likely to be a combination of one of the more sensitive standard trypanosome detection techniques with antigen-trapping ELISA.     

Evaluation of antigen detection and antibody detection tests for Trypanosoma evansi infections of buffaloes in Indonesia.

Two Ag-ELISAs, an IgG-specific antibody detection ELISA (IgG ELISA) and a card agglutination test (CATT) for the detection of Trypanasoma evansi infections in buffaloes in Indonesia, were compared. Diagnostic sensitivity estimates were obtained by testing sera from 139 Indonesian buffaloes which had been found to be infected by parasitological tests. Diagnostic specificity was estimated by testing sera from 263 buffaloes living in Australia. Response-operating characteristic curves were constructed, and optimal ELISA cut-off values, which minimized the number of false-negative and false-positive results, were chosen. The IgG ELISA had the highest sensitivity (89%) and the CATT had the highest specificity (100%). There was a significant difference between the sensitivities (71 and 81%), but not between the specificities (75 and 78%), of the two Ag-ELISAs. The four tests were further compared by calculation of post-test probabilities of infection for positive and negative test results using a range of prevalence values, and likelihood ratios. The results suggested that the CATT was the best test to 'rule-in' infection (i.e. the highest probability of infection in test-positive animals) and the IgG ELISA was the best test to 'rule-out' infection (i.e. the lowest probability of infection in test-negative animals).     

Trypanosoma cruzi isoenzyme variability in Ecuador: first observation of zymodeme III genotypes in chronic chagasic patients

We have analysed by multilocus enzyme electrophoresis (MLEE) at 21 genetic loci 10 Trypanosoma cruzi stocks isolated from chronic chagasic patients and 3 stocks isolated from Triatoma dimidiata collected in human habitats from the coastal part of Ecuador (all stocks isolated in August-December 1998). Isoenzyme profiles were compared to those of 4 laboratory-cloned stocks representing the major phylogenetic subdivisions of T. cruzi. This parasite's genetic variability in Ecuador proved to be considerable, even in this limited sample, since all main isoenzyme genotypes were recorded. Four stocks from patients were identical at all loci to the reference stock MNcl2 ('major clonet #39'; T. cruzi II) isolated in Chile. The 3 stocks isolated from T. dimidiata were closely related to the formerly described zymodeme I (T. cruzi I). Finally, 3 stocks from chronic chagasic patients (one with an asymptomatic form, 2 with a cardiac-digestive form) were closely related to the formerly described zymodeme III (presently not classified in either T. cruzi I or T. cruzi II). This is the first observation of this category of T. cruzi genotypes in chronic chagasic patients. In the past it was recorded only in acute patients, wild mammals and wild triatomine bugs. The epidemiological implications of these results are discussed.     

Evaluation of a synthetic tripeptide as antigen for detection of IgM and IgG antibodies to Trypanosoma cruzi in serum samples from patients with Chagas disease or viral diseases

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.     

Antigenuria in chronic chagasic patients detected by a monoclonal antibody raised against Trypanosoma cruzi

A monoclonal antibody (B2/5) raised against Trypanosoma cruzi was able to immunoprecipitate a major 100 kDa polypeptide in 84% of the urines collected from chronic chagasic patients. Other polypeptides were also detected. The antibody recognized polypeptides on the surface of epimastigotes (150-25 kDa) and metacyclic trypomastigotes (150-50 kDa), suggesting that the antigens share a common epitope.     

CpG DNA as a Th1-promoting adjuvant in immunization against Trypanosoma cruzi

Th1-type immune response plays a critical role in resistance to Trypanosoma cruzi infection. We asked whether a synthetic oligodeoxynucleotide that contains immunostimulatory CpG motifs (CpG ODN), known to promote a Th1 response, could act as an adjuvant in immunization with parasite antigens. Mice immunized with a whole homogenate (WH) of T. cruzi antigens co-administered with CpG ODN presented high titers of T. cruzi antibodies (IgG2a isotype), strong delayed type hypersensitivity and a Th1-dominated (IFN-gamma and IL-12) cytokine profile. Furthermore, WH plus CpG ODN protected mice from challenge with an otherwise lethal dose of bloodstream trypomastigotes. As reported for leishmaniasis and malaria, CpG ODN holds considerable promise as an adjuvant for future vaccines against T. cruzi.     

Detection of serum antibodies in human Hymenolepis infection by enzyme immunoassay.

Although hymenolepiasis is the commonest cestode infection of man, there are no data available on the human immune response to this parasite. Thus, in order to determine if infection induces antibodies against Hymenolepis nana antigens, sera from 52 infected children were initially studied on Ouchterlony plates and then by enzyme-linked immunosorbent assay (ELISA), using a crude antigenic extract prepared from scolex and neck regions of adult worms. In addition, sera from persons with cysticercosis, taeniasis and other parasitoses, and normal human sera, were studied. Only one serum from the Hymenolepis group showed precipitin antibodies against H. nana antigen, while several were positive by ELISA. The sensitivity of the ELISA was 84.62% and its specificity was 100%. Very high cross-reactivity rates were obtained with taeniasis (70.6%) and cysticercosis (75%) sera. These results show that Hymenolepis infection in man induces a low but detectable humoral immune response. Although not useful for diagnostic purposes, this may be relevant to the serodiagnosis of other tissue cestode infections of man, since antibodies detected in serological tests used for the diagnosis of cysticercosis, and probably hydatidosis, could be induced by H. nana instead of Taenia solium or Echinococcus larvae.     

Cryostat sections with tissue amastigotes of Trypanosoma cruzi as a substrate for Chagas' disease serology

Sera from 30 chronic chagasic patients together with 52 control samples (34 with other pathological conditions and 18 from normal individuals) were titrated by the indirect immunofluorescent technique (IFA) on Trypanosoma cruzi amastigotes. Acetone-fixed cryostat sections of skeletal muscle of Rockland mice 10 days post-infection with the RA isolate of T. cruzi were used as substrate. Results were compared with titres obtained by conventional IFA on epimastigotes. All 52 control sera had amastigote titres less than or equal to 2 double dilutions (dd) as compared with epimastigote values. Out of the 30 chagasic samples, differences were greater than or equal to 4 dd (less than or equal to 1 log) for 22, 3 dd for 5 and less than or equal to 2 dd for the remaining 3, when comparing amastigote and epimastigote titres. These results show that the use of amastigotes in cryostat sections of infected tissue for performing Chagas' serology in a simple, adequate and sensitive method.     

双抗原夹心法ELISA在肾综合征出血热诊断中的应用

目的建立一种敏感、特异的检测肾综合征出血热(HFRS)血清总抗体的双抗原夹心法ELISA。方法应用重组汉坦病毒(HV)NP抗原e1.3S作为包被抗原和e6-119-HRP作为酶标抗原建立双抗原夹心法ELISA,检测血清总抗体,并与间接免疫荧光法(IFA)相比较。结果共检测188份人血清和378份鼠血清标本,2种方法的总符合率为97.70%。以IFA为参照标准,双抗原夹心法ELISA的敏感性为97.54%,特异性为97.87%。结论双抗原夹心法ELISA检测HFRS血清总抗体具有很高的敏感性和特异性,适用于大规模的流行病学调查。     

Mass screening for Trypanosoma cruzi infections using the immunofluorescence, ELISA and haemagglutination tests on serum samples and on blood eluates from filter-paper.

Methods used to diagnose Trypanosoma cruzi infection differ in their ability to discriminate between sera from infected and uninfected individuals. We compared the results of an immunofluorescence (IF) test, a haemagglutination (HA) test, and an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of T. cruzi infections in a large population-based survey in central Brazil using blood eluates from filter-paper and venous blood samples. The sensitivities of the tests on eluates, compared with results on serum samples, were low: ELISA (78.1%), IF (69.2%) and HA (64.6%). The level of agreement between the tests on eluates was very poor, with the best co-positivity for IF and ELISA. Both the positive and negative predictive values of the three tests on eluates were similar (around 96%) to those for sera. Higher co-positivity values were obtained for the three tests on sera. The implications of these results are discussed in relation to blood screening, routine medical practice, sero-epidemiological surveys, and the follow-up of patients admitted to therapeutic trials.     

Spatio-temporal survey of small mammal-borne Trypanosoma lewisi in Cotonou,Benin, and the potential risk of human infection

Human trypanosomoses are the sleeping sickness in Africa and Chagas disease in Latin America. However, atypical human infections by animal trypanosomes have been described, but poorly investigated. Among them, the supposed rat-specific T. lewisi was shown to be responsible for a few severe cases. In Africa, the scarcity of data and the null awareness about the atypical human trypanosomoses suggest that the number of cases may be higher that currently thought. Furthermore, T. lewisi is resistant to normal human serum and therefore a potential human pathogen. In order to document T. lewisi distribution and ecology, a qPCR- and 16DNA sequencing-based survey was conducted in 369 rodents from three urban districts of Cotonou city, Benin, during three different periods of the same year. Our study demonstrated very high prevalence (57.2%) even when considering only individuals identified as positive through DNA sequencing (39.2%). Black rats represented the most dominant as well as the most T. lewisi-parasitized species. No difference was retrieved neither between seasons nor districts, suggesting a large infestation of rodents by trypanosomes throughout the year and the city. Our results suggest that conditions are gathered for rat to human transmission of T. lewisi in these socio-environmentally degraded urban areas, thus pointing towards the rapidly urbanizing Abidjan-Lagos corridor as a region at particular risk.     

Tenth international meeting on Trypanosoma evansi: report of the working group. Paris, 24 May 1989

Some epidemiological surveys have provided information on the incidence of Trypanosoma evansi infection among dromedaries in Mali, and among buffaloes in Java and Indonesia. The disease among camels has been reported again from Kazakhstan in the USSR, with the coexistence of T. evansi and Cephalopina titillator in animals which developed acute infection. The disease has been studied among horses in Venezuela and among buffaloes in Vietnam and Indonesia, and suspected among horses in Brazil. Diagnostic kits for rapid and reliable detection of T. evansi are being made available free of charge, upon request, by the institutes which have developed these new techniques, namely: detecting the parasite by agglutination-lysis; detecting antibody (by a modification of CATT); detecting antigen (by using monoclonal antibodies). Once these various diagnostic procedures developed by competent institutes have been evaluated and used widely, the next step will be to standardise the techniques and the antigens. Differential diagnosis of T. evansi and T. equiperdum is still difficult in the case of akinetoplastic strains. For improved evaluation of T. evansi isolates, a proposal has been made to form collections of complementary DNA (cDNA) with a view to exchanging these copies and the original strains. The advice of the International Commission on Zoological Nomenclature has been requested for definitive adoption of a binomial designation for the species T. evansi. With more extensive data on the pharmacology and pharmacokinetics of Cymelarsan and laboratory testing of a new trypanocide called "IMOL 881", research on trypanocides continues.     

Persistence of Trypanosoma cruzi antigens in the inflammatory lesions of chronically infected mice

Different tissues and organs of mice infected with Trypanosoma cruzi trypomastigotes have been examined for the presence of parasites and parasitic antigens during both the acute and the chronic phases of infection. Specimens of skeletal and cardiac muscles, spleen, liver, brain and sciatic nerves were studied by histological and immunological methods. During the acute phase of infection, the parasites were commonly observed in these tissues. In the chronic phase of the experimental infection, pseudocysts filled with amastigotes were seen in less than 1% of the tissue sections, while immunohistological methods showed that T. cruzi antigens were present in 11% of the inflammatory infiltrates. These findings suggest that antigenic stimulation persists throughout the chronic phase, even though the parasites are not morphologically detectable.     

kDNA gene signatures of Trypanosoma cruzi in blood and oesophageal mucosa from chronic chagasic patients

Trypanosoma cruzi presents a high degree of intraspecific variability, with possible implications for the pathogenesis of Chagas disease. The aim of this study was to evaluate T. cruzi kDNA minicircle gene signatures using the low-stringency single-specific-primer PCR technique in both peripheral blood and oesophageal mucosa from chronic chagasic patients, with or without megaesophagus, alone or in combination with cardiopathy and megacolon. It was not possible to identify a uniform pattern of shared bands between blood and oesophageal mucosa samples from individuals with the same clinical form or mixed forms, suggesting multiple T. cruzi infections with differential tissue tropism. Thus, the results indicate that there is an intense intraspecific variability in the hypervariable regions of T. cruzi kDNA, which has so far made it impossible to correlate the genetic profile of this structure with the clinical manifestations of Chagas disease.     

Cell surface glycoproteins of Trypanosoma cruzi: protective immunity in mice and antibody levels in human chagasic sera

Two cell surface glycoproteins from Trypanosoma cruzi have been compared for their ability to protect mice against an acute lethal infection. One of these, a 90,000 glycoprotein found on all stages of the parasite, protected against both bloodstream and metacyclic trypomastigote challenge. A 72,000 glycoprotein found only on insect-derived stages of T. cruzi protected only against a metacyclic trypomastigote challenge. Antibody against both of these glycoproteins was present in human chagasic sera.