Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas'' disease patients.

Approximately 70% of the cDNA clones identified by immunoscreening Leishmania donovani expression libraries with serum from a patient with visceral leishmaniasis (kala-azar) were found to encode the highly conserved Hsp90 and Hsp70 members of the heat shock protein family. Recombinant fusion proteins containing the C-terminal portions of L. donovani Hsp90 and Hsp70 were used as target antigens in enzyme-linked immunosorbent assays of various sera. Sera from four patients with visceral leishmaniasis recognized recombinant Leishmania Hsp90 and Hsp70, while sera from seven patients with Chagas' disease did not, despite the fact that Trypanosoma cruzi Hsp90 and Hsp70 share more than 80% amino acid identity with their counterparts in Leishmania spp. Thus, Leishmania Hsp90 and Hsp70 elicit strong humoral responses and are potential candidates for specific serodiagnostic assays capable of distinguishing between L. donovani and T. cruzi infections.     

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.     

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis.

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral leishmaniasis.     

Mapping of the antigenic determinants of the T. cruzi kinetoplastid membrane protein-11. Identification of a linear epitope specifically recognized by human Chagasic sera

The high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino- and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease.     

Use of full-length recombinant calflagin and its c fragment for improvement of diagnosis of Trypanosoma cruzi infection

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.     

Sera from chronic Chagasic patients and rodents infected with Trypanosoma cruzi inhibit trans-sialidase by recognizing its amino-terminal and catalytic domain.

We investigated whether sera from chronic Chagasic patients and animals infected with Trypanosoma cruzi inhibit the removal of sialic acid from human erythrocytes and the transfer of sialic acid from sialyllactose to [14C]lactose in the reactions catalyzed by the parasite trans-sialidase. Sera from Swiss mice and Calomys callosus animals infected with three different T. cruzi strains inhibit both reactions. Inhibition increases during the infection, reaching maximal levels when the parasitemia decreases. Among 44 sera of untreated chronic Chagasic patients, 40 inhibit both reactions. Inhibition is observed with total, defatted sera or with purified immunoglobulins. Whereas most of the inhibitory antibodies from Chagasic patients react with the papain fragment of trans-sialidase in immunoblots, a few patients have noninhibitory antibodies that react only with the entire trans-sialidase. These findings may be relevant for the pathology of Chagas' disease.     

Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.     

Cross-reactive studies in visceral leishmaniasis: comparison of antigens of some Leishmania species in Kenya

Little is known about the common antigens of the various strains of Leishmania in Kenya, and their possible role in immunity to disease. Kenyan isolates of Leishmania donovani, L. Major and L. tropica were cultured, crude antigens prepared and electrophoresced in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots by pooled sera from cutaneous and visceral leishmaniasis patients revealed common antigens within the 110-116kD, 70kD and the 63kD ranges. Only the 112-116kD antigens from L. major strains were reacted to by both cutaneous and visceral leishmaniasis patient sera. Sera from normal individuals living in an endemic area for leishmaniasis also showed reactivity to the 116kD antigen. We suggest that these common antigens may be of value in future vaccine studies     

Enzyme-linked immunoassay using recombinant trans-sialidase of Trypanosoma cruzi can be employed for monitoring of patients with Chagas' disease after drug treatment

trans-Sialidase is an enzyme present on the surface of Trypanosoma cruzi and is an important antigen recognized by sera from patients with Chagas' disease. In the present study we investigated whether the benznidazole treatment of patients with Chagas' disease induced changes in the reactivity of serum toward a recombinant form of trans-sialidase in order to develop an assay for monitoring of patients after treatment for Chagas' disease, which is needed at Chagas' disease control centers. By using an enzyme-linked immunosorbent assay containing a recombinant protein corresponding to the catalytic domain of trans-sialidase, we found that the antigen had a high specificity for sera from untreated patients with Chagas' disease. Sera from healthy individuals or patients with active visceral leishmaniasis minimally cross-reacted with the antigen. Anti-trans-sialidase immunoglobulin was detected in 98% of 151 untreated patients with Chagas' disease. Of these, 124 patients were treated for 60 days with benznidazole (5 mg/kg of body weight/day), and their sera were assayed for reactivity with the recombinant trans-sialidase. By using this methodology, three groups of patients could be established. The first group (60 patients), which was considered to have been successfully treated, showed no reactivity after treatment. The second group (46 patients) still showed signs of infection, and after treatment their sera recognized trans-sialidase, but with reduced titers. The third group (18 patients) was considered to be resistant to drug treatment, and their sera presented identical reactivities before and after treatment. These results suggest that determination of the absence of antibodies to recombinant trans-sialidase in treated patients by the present assay is indicative of treatment success, while the presence of antibodies may indicate the persistence of infection. Therefore, this method may be useful for the diagnosis and monitoring of patients undergoing benznidazole treatment.     

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 > > IgG3 > IgG2 > IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.     

Antiactin and antitubulin antibodies in canine visceral leishmaniasis

Visceral leishmaniasis, a chronic and often fatal disease, is caused by the protozoan parasite Leishmania donovani. Both specific and nonspecific antibodies are produced in the course of the disease, and autoantibodies may be involved in pathogenesis. Tubulin and actin have been found to be associated with L. donovani. To learn whether antiactin and antitubulin antibodies are present in visceral leishmaniasis, we tested sera from 263 infected dogs by enzyme-linked immunosorbent assay for antibodies to the antigens L. donovani, actin, and tubulin. All samples reacted positively with L. donovani, and a high percentage reacted positively with all three antigens. Sera from 202 uninfected dogs were also tested, none reacted with L. donovani antigen, although positive reactions were observed for 8 of the samples with actin or tubulin. It was found that the antibody-antigen reaction occurred at the Fab portion of the immunoglobulin molecule. Competitive enzyme immunoassays showed that the reaction was inhibited if the positive serum was first incubated with L. donovani antigen, actin, or tubulin and then tested by enzyme-linked immunosorbent assay. These results suggest that antiactin and antitubulin antibodies are present in the sera of dogs infected with visceral leishmaniasis.     

Antigens shared by Leishmania species and Trypanosoma cruzi: immunological comparison of the acidic ribosomal P0 proteins.

Patients with visceral leishmaniasis produce high levels of immunoglobulin, but the specificities of antibodies produced are not well characterized. In an effort to identify leishmania antigens that are specific to Leishmania species or are cross-reactive with other parasitic protozoa, we have cloned and characterized full-length genomic and cDNA clones encoding a Leishmania chagasi acidic ribosomal antigen, LcP0, recognized during human infections. The protein is homologous to the Trypanosoma cruzi and human ribosomal proteins TcP0 and HuP0, respectively. Unlike most higher eukaryotes, but similar to TcP0, LcP0 has a C-terminal heptapeptide sequence resembling those of the archaebacterial acidic (P-like) proteins. The highly charged C-terminal acidic domain of LcP0 contains a serine residue typically found in most eukaryotes but lacking in all T. cruzi P proteins we have characterized thus far. L. chagasi-infected individuals as well as those with T. cruzi infections have antibodies cross-reactive with recombinant LcP0 and TcP0 as well as HuP0. However, the properties of anti-P0 antibodies in T. cruzi and L. chagasi infection sera are quite different. Through the use of synthetic peptides, we showed that while T. cruzi infection anti-TcP0 antibodies are exclusively directed against the C-terminal domain of TcP0, L. chagasi infection sera contain antibodies reactive with epitopes other than the C-terminal sequence of LcP0. Thus, anti-LcP0 antibodies in L. chagasi infection sera represent the first characterized deviation from the restricted immunodominant C-terminal epitope involved in the generation of anti-P0 antibodies following infection or autoimmune diseases.     

Rapid immunochromatographic test for serodiagnosis of canine leishmaniasis

An rK39 immunochromatographic test and immunofluorescent-antibody test (IFAT) for serodiagnosis of canine leishmaniasis were evaluated. The two tests showed correlation for all but one of the sera obtained from 68 dogs confirmed as leishmaniasis cases and 40 dogs (22 healthy dogs and 18 dogs with other diseases) from areas where the disease is not endemic. Specificity was 100% for both tests, while sensitivity was 97% for the rapid test and 99% for IFAT.     

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.     

Cross-reactivity studies and differential serodiagnosis of human infections caused by Trypanosoma cruzi and Leishmania spp; use of immunoblotting and ELISA with a purified antigen (Ag163B6).

Results of our studies on the reactivity of chagasic and leishmaniasic sera with the purified T. cruzi-specific antigen 163B6, as assessed by ELISA, and with complex antigenic mixtures from T. cruzi and Leishmania mexicana, by immunoblotting, are presented here. Our objective was to identify the antigens responsible for the exhibited cross-reactivity between trypanosomiasis and leishmaniasis, and to find a specific reactivity pattern corresponding to each parasitosis. In spite of the high cross-reactivity observed with the immunoblotting, the use of 7.5% A-B gels made it possible to identify a characteristic pattern for each parasitosis, that could be distinguished by the naked eye. The characteristic pattern corresponding to chagasic patients was ascribed to reactivity with T. cruzi bands of mol. wts 131, 125, 116, 111, 51-45 and 43 kD, that were not recognized by leishmaniasic sera. Trypanosoma cruzi antigens of mol. wts 85, 81, 70, 65-60, 37 and 32 kD were considered as crossing antigens, since they were recognized by leishmaniasis sera. With L. mexicana, most of the chagasic patients presented reaction with antigen of mol. wts 124, 107, 92, 59 and 32 kD, while bands of mol. wts 155, 140, 73, 56 and 48 kD were recognized only by leishmaniasic sera. In this study we found 12 out of 45 sera of patients with leishmaniasis, from a region endemic for both parasitoses, which exhibited a pattern of bands very similar to those corresponding to chagasic individuals, strongly suggesting a mixed infection. This hypothesis was verified by using a purified specific antigen of T. cruzi, Ag163B6, which would be the major cysteine proteinase of this specie (cruzipain). By ELISA, these 12 sera showed a positive reaction with this purified antigen, as those of chagasic patients, thus leading to the confirmation of the presence of a mixed infection.     

Rapid detection of Leishmania infantum infection in dogs: a comparative study using fast agglutination screening test (FAST) and direct agglutination test (DAT) in Iran

Canine visceral leishmaniasis (CVL) is not only a veterinary problem but has also a serious public health importance. Rapid detection of CVL is highly important for control of human visceral leishmaniasis in Iran. This study was aimed to compare the fast agglutination screening test (FAST) with direct agglutination test (DAT) as a standard serological test for the detection of anti-Leishmania antibodies on dog serum samples. DAT and FAST antigens were prepared in the School of Public Health, Tehran University of Medical Science. Altogether, 73 serum samples from Leishmania infantum infection dogs and 74 sera from healthy controls were collected from human VL/CVL endemic and non-endemic areas of Iran, respectively. All the sera were evaluated with both FAST and DAT techniques. A sensitivity of 98.60% (95% CI, 98.57–98.62) and specificity of 78.70% (95 CI%, 69.20–88.20) were found at a 1:160 -(cut-off) titer when DAT confirmed cases were compared with healthy control. A good degree of agreement was observed between FAST and DAT (86.8%) by kappa analysis (p < 0.01). In conclusion, this study showed that FAST is very practical and simple diagnostic tool for the sero-diagnosis of CVL in endemic areas of Iran.     

Visceral leishmaniasis : activation of human T-lymphocytes by two specific Leishmania antigens

T cell responses to a specific Leishmania antigens, 70kD and 116kD were investigated in individuals from an endemic area for leishmaniasis. Peripheral blood mononuclear cells were obtained from 38 individuals (24 test an d14 controls) and T cell responses to phytohaemaglutinin (PHA), Concanavalin A (ConA) and the specific Leishmania antigens were examined. PHA and Con A-induced responses were all positive in both the test and control groups. Eighteen out of 24 individuals with previous history of visceral leishmaniasis and 12 out of 14 normal individuals residing in the endemic areas responded to the 70kD antigen. In contrast, all the eight normal individuals all the eight normal individuals from a non-endemic area for leishmaniasis did not responded, indicating that cellular responses to the 70kD antigen are specific for Leishmanai spp. The 116kD antigen, on the other hand, exhibited poor specificity: about 30% of the individuals with a previous history of leishmaniasis did not respond to this antigen. A significant proportion of the population from an endemic area, but with no history of visceral leishmaniasis, responded to the 70kD antigen. This is an indicator of a prior exposure to Leishmania parasite and is consistent with previous studies which have shown that subclinical infections of visceral leishmaniasis do occur in such areas. Results from the present study suggest that the 70kD antigen is a strong candidate for vaccine development.     

Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera.

A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.     

Leishmania major-like antigen for specific and sensitive serodiagnosis of human and canine visceral leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.     

Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.