MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii / Caracterização molecular e análise filogenética de sequências do antígeno de superfície 3 (SAG3) em isolados indianos (CHENNAI E IZATNAGAR) de Toxoplasma gondii

Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites. Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI. Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences. Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them. Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny. .

Contexto e objetivo. A caracterização molecular de isolados indianos de Toxoplasma gondii é importante para a investigação de variações genéticas existentes entre cepas do parasito em diferentes locos gênicos. Delineamento e disposição. A presente comunicação realizou a clonagem e o sequenciamento dos 1158 pares de base correspondendo à totalidade do quadro de leitura do antígeno de superfície 3 (SAG3) de Toxoplasma gondii em dois isolados indianos (Chennai e Izatnagar) mantidos em um biorrepositório localizado em IVRI. Método. As sequências do SAG3 dos dois isolados indianos foram clonadas, sequenciadas e posteriormente comparadas com sequências SAG3 de Toxoplasma gondii disponíveis em publicações. Resultados. A comparação das sequências revelou 99,9% de homologia com a cepa RH padrão; 99,3% de homologia com as cepas P-Br e CEP; e 98,4% de homologia com a cepa PRU. Os dois isolados indianos eram 100% idênticos no que diz respeito à sequência SAG3. Conclusão. Concluiu-se que os isolados indianos são filogeneticamente mais próximos da cepa RH em relação à cepa brasileira P-Br, ou às cepas CEP e PRU (USA). No entanto, a análise de outros genes de Toxoplasma gondii destes dois isolados indianos mostrou diferenças na composição de nucleotídeos, ao contrário do que foi encontrado para o locus SAG3. Estes resultados poderiam ser atribuídos ao fato do locus SAG3 ser altamente conservado, necessitando de estudos adicionais para determinar se SAG3 poderia ser utilizado no diagnóstico da toxoplasmose. No entanto, estes resultados são importantes do ponto de vista da filogenia molecular. .

Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

OBJECTIVE@#To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models.@*METHODS@#A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates.@*RESULTS@#Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons.@*CONCLUSIONS@#The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

Objective: To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods: A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions: The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

Vaccination against Murine Toxoplasmosis Using Recombinant Toxoplasma gondii SAG3 Antigen Alone or in Combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.

Vaccination against Murine Toxoplasmosis Using Recombinant Toxoplasma gondii SAG3 Antigen Alone or in Combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.