Seroepidemiological survey of trypanozoon infection in horses in the suspected dourine-infected Bale highlands of the Oromia region, Ethiopia

This paper presents the results of a seroepidemiological survey of trypanozoon infection in horses carried out between September 2007 and June 2008. The survey was conducted to determine the seroprevalence of anti-trypanozoon antibodies in 880 serum samples collected randomly from selected horse-breeding districts of the Bale highlands of Ethiopia. The seroprevalence of trypanozoon infection was found to be 173 (19.66%) and 140 (15.91%) for the CATT/T. evansi and LATEX/T. evansi tests, respectively. The high seroprevalence of trypanozoon infection strongly indicates that the infection is endemic. Neither test can differentiate between anti-trypanozoon antibodies caused by infection with T. equiperdum (the causative agent of dourine) and those of T. evansi (the causative agent of surra). The findings of the present study suggest that field-applicable screening serological tests such as the CATT/T. evansi and LATEX/T. evansi could be useful for epidemiological studies and the control of trypanozoon infection.     

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.     

Detection of Chagas infections using Trypanosoma evansi crude antigen demonstrates high cross-reactions with Trypanosoma cruzi.

Antigenic similarities between salivarian trypanosomes are known for a long time, but similarities between salivarian and stercorarian trypanosomes have been very little investigated. Phylogenetically, these genus and species appear to be far. However, in a preliminary work we had shown strong reactions of chagasic human sera using T. evansi antigens in Western-blotting and ELISA. In the current work an ELISA test using T. evansi crude antigens was probed with one hundred and two sera of chagasic Bolivian patients previously diagnosed which presented different pathologies. The sensitivity of the ELISA T. evansi was 92.6% similar to that of ELISA T. cruzi. The specificity evaluated using 20 sera of patients infected by Leishmania sp. reaches a comparable value of that obtained with the T. cruzi immunofluorescent assay. Finally, the sensitivity and the specificity of the ELISA T. evansi were not really different from conventional serology of Chagas. In spite of their taxonomic position in various sections and their old divergence, these observations prove a strong antigenic community between T. cruzi and T. evansi. Consequently, the common antigens which remain to be characterized, could be an alternative source of antigen for the detection of antibodies against T. cruzi. Given that T. evansi seems to have strong antigenic communities with the majority of the pathogenic current trypanosomoses of mammals, it is very attractive to identify and characterize these highly conserved antigens which could be suitable targets to develop tools for diagnosis, prophylaxy and chemotherapy against several human and animal trypanosomoses.     

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.     

Antibody capture radioimmunoassay for anti-rubella IgM.

An M-antibody capture radioimmunoassay (MACRIA) for anti-rubella IgM was developed. Under optimum conditions positive serum specimens bound up to 20 times as much radioactivity as negative specimens. Positive reactions were expressed in arbitrary units/ml by comparison with a calibration curve derived from results obtained with dilutions of a standard serum. The specificity of the assay was confirmed by testing IgM and IgG rich fractions of positive sera. One hundred and forty specimens from blood donors, patients whose sera contained rheumatoid factor and patients with acute, non-rubella, virus infections were tested by MACRIA. No significant non-specific reactions were detected. Paired sera from acute rubella (25 patients) and individual sera from suspected rubella (69 patients) were tested for anti-rubella IgM by MACRIA and by haemagglutination inhibition following sucrose-density-gradient fractionation. There was close agreement between the two methods. The capture assay was more sensitive and could be used to detect the weak IgM response in women given RA 27/3 vaccine. After the natural infection, the MACRIA was strongly positive for two months and remained weakly so for a further two months. Repeat testing of sera demonstrated good reproducibility of the assay. MACRIA proved a simple, sensitive and specific test for anti-rubella IgM and compared favourably with currently used techniques.     

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.     

Genetic diversity of Trypanosoma evansi in beef cattle based on internal transcribed spacer region.

This study was focused on genetic diversity of Trypanosoma evansi which is a widely distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer. The genetic diversity of T. evansi of beef cattle LAM19 was accomplished by using phylogenetic analysis based on internal transcribed spacer region (ITS). Blood sample was collected from a naturally infected beef cattle LAM 19 and parasitemia was raised by mouse inoculation. The parasites were collected and isolated by using DE 52 DEAE cellulose anion exchange column prior to DNA extraction. Upon PCR amplification of ITS region, the product of 1300bp in size was obtained. The ITS nucleotide sequences were analyzed and revealed that it could demonstrate the genetic diversity of T. evansi of beef cattle LAM19. Based on the ITS tree, beef cattle LAM 19 T. evansi were categorized into two main groups where the genetic diversity occurred within Group 1. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

The 'Phadiatop Paediatric': a useful in-vitro test on an atopic immune response in infants and young children

The aim of this study was to evaluate a new test for IgE antibody screening in children--the Phadiatop Paediatric. The mixture of allergens in this test contains the food and inhalant allergens for infants and children most relevant. 180 sera were selected from samples submitted for routine diagnostic IgE antibody assay. 91% of these sera were from children less than 5 year old. Half the sera were positive in RASTs that were initially requested by the physicians and the highest RAST scores for each serum were equally distributed over the range RAST class 1-4. 20 control sera were from children 1-4 years old. Results of the Phadiatop Paediatric were compared with results of the combination 'Phadiatop + mixed-food-RAST'. 22% of the sera that were negative in all RASTs that were initially requested by the physicians were positive in both tests. This suggests that an IgE antibody response to food components or inhalants was underdiagnosed in these cases. Whereas 135 sera were found positive in the Phadiatop Paediatric, only 116 sera had a positive Phadiatop result and/or a result of the mixed-food-RAST that was RAST class 1 (greater than or equal to 0.35 PRU/ml) or higher. In 11 of 16 sera with RAST class 0/1 (0.18-0.35 PRU/ml) in the mixed-food-RAST specific IgE against individual food allergens with RAST class 1 or higher could be demonstrated. Results of Phadiatop and Phadiatop Paediatric were not only expressed as 'positive' or 'negative', but also as a serum:reference ratio. Clinical documentation was collected retrospectively from the written case reports of 171 children.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of serological methods for the detection of B. abortus antibodies in sera from vaccinated and non-vaccinated cattle.

A total of 4551 sera from 863 Strain 19 vaccinated and non-vaccinated adult cattle, independent of disease status, were tested by five serological methods to detect the presence of antibodies to B. abortus. Results from Standard Agglutination Tube (SAT), Buffered Brucella Antigen or card (CT), Complement Fixation (CF), Enzyme Linked Immunosorbent Assay (ELISA) and Rivanol (Riv) methods were compared. There was a 95% probability for agreement among CT negative sera, between serological methods, for all groups of vaccinated and non-vaccinated cattle. The agreement between tests with Riv Positive sera, excluding the calfhood and adult vaccinated group tested by the CF method, was 91-100%. The probability of a serum which was serologically negative by other methods being Riv negative was 98%. The usefulness of serological results from Riv (greater than or equal to 1/50) tests for classifying the reactor status of cattle are of doubtful supplemental value to confirm card test positive results. Vaccination history is an important consideration when evaluating serological data on cattle sera particularly from SAT and CF methods.     

Serological studies on leptospirosis in livestock and chickens from Grenada and Trinidad

Sera from 1,206 livestock animals and chickens on Grenada and Trinidad were tested for leptospiral antibodies by the microscopic agglutination test. 376 of the sera were positive (25% of those tested on Grenada and 44% on Trinidad). The positive sera were obtained from 25% of 324 cattle, 35% of 130 pigs, 35% of 146 sheep, 25% of 44 goats and 11% of 175 chickens on Grenada; and 92% of 26 cattle, 53% of 122 pigs, 76% of 87 horses and donkeys and 11% of 144 chickens on Trinidad. Eight sera from ducks and geese on Trinidad were tested and found to be negative. The serogroups most commonly found to react with the sera of the Grenadian animals were Autumnalis, Icterohaemorrhagiae, Hebdomadis and the related serogroups Sejroe and Mini, and Pyrogenes; in the Trinidadian animals they were Icterohaemorrhagiae, Autumnalis, Hebdomadis and its related serogroups, and Panama. Strains of serogroup Pomona do not appear to have become established as livestock pathogens on the islands.     

False positive results of HIV virus tests in patients undergoing chronic hemodialysis

The sera of 173 haemodialysis patients treated in two dialysis centers in Hungary were tested for the presence of HIV (HTLV III/LAV) antibodies. Four different commercial enzyme immunoassay (EIA) kits and two types (CEM/LAV, and H9/HTLV III) of indirect immunofluorescence assay (IFA) were used. The Western blot technique was applied as confirmatory test in the study. No confirmed positive results were found in any of the cases. However, in 15 patients (8.7%) false positive (not confirmable by the Western blot assay) results were obtained in at least one but mostly in all of the three type 1 EIA kits (ORGANON, ELECTRONUCLEONICS, SORIN) applied. In 4 patients, the IFA assay also gave false positive results which could be repeated in sequential samples taken from the same patients. Increased reactivity in the control plate (coated with a concentrate of cellular material shed by uninfected H9 cell line) of the SORIN kit was found only in a few false positive samples and no fluorescence with the uninfected H9 or CEM cells was observed in any of the sera showing a false positive IFA. These results indicate that the false positive anti-HIV results frequently observable in haemodialysis patients are not simply the consequence of the presence of antibodies reacting with the uninfected H9 and/or CEM cells but they are most probably due to antibodies against antigens expressed on these cells only after infection with the human immunodeficiency virus.     

The current challenges of dourine: difficulties in differentiating Trypanosoma equiperdum within the subgenus Trypanozoon

During its 20th annual meeting in Paris in May 1999, the OIE (World organisation for animal health) Ad Hoc Group on Non-Tsetse Transmitted Animal Trypanosomoses expressed the following concerns about dourine: the discrepancies in some of the results of the complement fixation test (CFT), which is the only international diagnostic test officially recognised by the International Organisation for the Transportation of Equidae; the persistence of suspected cases of dourine in some Asian, European and African countries; the impossibility of differentiating Trypanosoma equiperdum from Trypanosoma evansi and of isolating new strains of T. equiperdum from clinical cases that have appeared in various parts of the world since 1982. In the light of these concerns, it was decided, in agreement with the Directorate of the Federal Veterinary Services of Russia in Moscow, to perform comparative trials on the value of CFT/dourine at the OIE Reference Laboratory for dourine in Moscow (The All-Russian Research Institute of Experimental Veterinary Medicine) using reagents (antigens and sera) from seven countries with extensive experience in the field of dourine diagnosis, namely, South Africa, France, Italy, Germany, Russia, the United States of America and the People's Republic of China. It is thanks to the successful co-operation of these countries that the trials were made possible. Results showed an overall concordance and were submitted for consideration to the OIE Biological Standards Commission, the commission which is in charge of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. These trials serve as a starting point for further study, particularly in the following areas: the isolation of new strains of T. equiperdum from clinical dourine cases; the identification of specific markers for T. equiperdum which would make it possible to differentiate it from among the other species within the subgenus Trypanozoon; the experimental infection of horses with newly isolated T. equiperdum strains to compare their pathogenicity with those currently used in national diagnostic laboratories and with that of T. evansi; phylogenetic studies; the proposal and validation of new, internationally recognised diagnostic test(s) for dourine.     

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.     

巴楚县2001年新疆出血热疫情的血清学证实

目的：以血清流行病学方法调查新闻出血热（XHF）病人，易感人群和主要宿主动物中疾病的感染情况。方法：分别收集2001年4－6月新疆巴县临床诊断为XHF的病人血清，易感人群血清和主要宿主动物的血清，用研制的诊断试剂以酶联免疫吸附试验（ELISA）检测XHF特异性IgG和IgM抗体；用抗原捕获ELISA检测XHF病毒抗原。结果：病人血清IgG抗体阳性率为39．62％（21／53）。IgM抗体阳性率为20．75％（11／53），抗原获ELISA有1份血清为XHF抗原阳性；易感人群血清IgG抗体阳性主为21．05％（4／19），IgM抗体检测和抗原捕获ELISA全部为阴性；羊血清IgG抗体阳性率为70％（56／80）。结论：血清流行病学研究证实该次疫情确系XHF、流行地区人畜均有较高水平的隐性感染。     

Search for epizootic-like Venezuelan encephalitis virus at enzootic habitats in Guatemala during 1969-1971.

Seventy-four strains of Venezuelan encephalitis (VE) virus recovered from sentinel hamsters or mosquitoes at enzootic habitats in Guatemala in the two years following the 1969 epidemic-equine epizootic were examined for ability to produce small plaques in Vero African green monkey kidney cell cultures, like isolates obtained during the epizootic. (a) One strain recovered from a sentinel hamster in late October 1969 at an enzootic habitat near the epicenter of the hemagglutination-inhibition (HI) and equine-virulence properties like epizootic virus; this strain retained its small plaque characteristic after inoculation and recovery from bloods of three horses. (b) None of the other 73 strains produced uniformly small plaques, but 31 formed a few small plaques among large ones. Virions from small plaques of five strains were cloned twice in Vero cell cultures. Four clones produced uniformly small plaques after one more passage in Vero cells; three had hemagglutination-pH properties compatible with epizootic virus or intermediate between epizootic and enzootic virus, but HI tests with these three hemagglutinins or with antibody to the fourth cloned strain showed them to be like Central American enzootic virus. One of three cloned strains tested in horses produced encephalitis and death in one of four horses; another strain produced encephalitis with recovery in one of two horses. (c) Thus these small Vero plaque clones resembled Central American enzootic strains of VE virus in HI and equine-virulence tests, and the small Vero plaque characteristic was not a satisfactory marker for consistently isolating equine-virulent, epizootic VE virions. Nevertheless, this technic led to recognition of one epizootic strain isolated at an enzootic habitat in Guatemala at the end of 1969 outbreak. Whether this strain was there before the outbreak or subsequently penetrated the habitat is uncertain. During the next two years, this strain did not become dominant in that enzootic focus.     

Rubella antibody measured by radial haemolysis. Characteristics and performance of a simple screening method for use in diagnostic laboratories.

A simple method for preparing radial haemolysis gels for rubella antibody screening is described. In use it gave clear zones of haemolysis when a standard serum was tested at dilutions down to 5.6 i.u./ml rubella antibody. In five laboratories 8404 sera were screened by the method and the results were read by comparing zones of haemolysis with that of a standard serum diluted to contain 15 i.u/ml antibody. A zone greater than or equal to 15 i.u./ml, indicating immunity, was given by 7433 (88.4%) of the sera. No zone indicating susceptibility was seen with 748 (8.9%) sera. Small zones, less than 15 i.u./ml standard, were given by 189 (2.2%) sera, and in only 34 cases (0.4%) did non-specific haemolysis interfere with the test readings. Further testing of the radial haemolysis interfere with the test readings. Further testing of the radial haemolysis negative and low positive sera by the haemagglutination inhibition test gave rise to some discrepant results which are discussed.     

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).     

WHO collaborative studies on enterovirus reference antisera. Third report

This paper smmarizes the results of the third part of co-operative studies undertaken by the WHO International Reference Centre for Enteroviruses and a number of WHO Regional Virus Reference Centres and WHO Virus Collaborating Laboratories and other laboratories in a comprehensive testing programme of enterovirus equine antisera prepared for long-term use as reference antisera. The studies were designed to appraise the specificity of the immune serum of horses inoculated with prototype enteroviruses (coxsackie-viruses A1, A5, A6, A12 and A22 and echoviruses 5, 6, 13-16, 18-20, 22-26, 29 and 32). Tests for neutralizing antibody were performed not only against the homologous viruses but also against regional homotypic strains. Tests for heterotypic antibody were made against the entire group of enteroviruses (except enterovirus 68), reoviruses 1-3, and adenoviruses 1-11, 13-17 and 19-22. Final vials of the dried serum were provided for the tests. Each serum sample represented a pool of the individual bleedings taken from a group of horses before and after immunization with each virus antigen. The results showed that the homologous geometric mean titres of 9 of 16 echovirus immune sera were 10 000 or above while the other 7 ranged from 2000 to 8000. The homologous antibody titre of one coxsackievirus type A serum was about 2000 while the titres of the other 4 type A sera ranged from about 5000 to 15 000. All corresponding pre-inoculation sera were negative. The results of the homotypic tests, though limited in number, showed the usefulness of the sera. Data on heterotypic antibody titres, if any, are recorded for gidance in the use of the sera. Co-operative testing of 19 additional enterovirus equine sera is now in progress.     

Characterization of Rickettsia spp. circulating in a silent peri-urban focus for Brazilian spotted fever in Caratinga, Minas Gerais, Brazil

The present study was intended to characterize Rickettsia spp. circulating in arthropod vectors in Caratinga, Minas Gerais, Brazil, by PCR and to investigate the presence of antibodies against the spotted fever Rickettsiae group (SFRG) in dogs and horses. 2,610 arthropods were collected and taxonomically identified. DNA samples obtained from these vectors were submitted to PCR and cycle-sequenced. Ctenocephalides and Amblyomma cajennense showed sequences presenting 100.0% homology with R. felis. A sequence obtained from Rhipicephalus sanguineus showed 99.0% homology with R. felis, and a sequence from A. cajennense showed 97.0% homology with R. honei and R. rickettsii. Canine (73) and equine (18) serum samples were tested by indirect fluorescent antibody (IFA) using R. rickettsii antigen. Only three of the equine sera tested (17.0%) had positive antibody titers. Molecular detection of rickettsiae species potentially pathogenic to humans in arthropod vectors and the presence of seroreactivity to SFRG in horses show the risk of transmission of rickettsiosis in this area and the need to maintain continuous epidemiological surveillance for rickettsial diseases.     

华枝睾吸虫病人治疗前后血清抗体的动态观察

用ＥＬＩＳＡ检测华支睾吸虫病人治疗前后血清特异性抗体ＩｇＧ，并进行动态观察，结果表明，ＥＬＩＳＡ的阳性符合率为９７％，抗体滴度在治疗后３、６和１２个月时不断下降。轻感组约６个月抗体降至正常；中感组１年左右；而重感组需１年半至２年。ＥＬＩＳＡ的阴转率随时间而增高。３、６和１２个月时间的阴转率，轻感组分别为３５％、８４．６％、９３．６％；中感组为１８％、５６．７％、８９．７％；重感组为０％、２５％５６