RESUMEN
INTRODUCCTION: Trichomonas vaginalis is a highly prevalent parasitic that causes the sexually transmitted disease trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, epidemiology of the infection and drug susceptibility. For this end, we conducted analysis of a fragment (23 kDa) of the p60 of T. vaginalis gene. MATERIAL AND METHODS: The restriction fragment length polymorphism (RFLP) methods was used. RESULT AND DISCUSSION: RFLP analysis showed the difference between T. vaginalis isolates from symptomatic and asymptomatic patients, suggesting a relation between the genetic identity of the isolates and their clinical manifestations.
Asunto(s)
Variación Genética , Péptido Hidrolasas/genética , Proteínas Protozoarias/genética , Trichomonas vaginalis/genética , Adolescente , ADN Protozoario/genética , Femenino , Humanos , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Tricomoniasis/parasitología , Trichomonas vaginalis/enzimologíaRESUMEN
Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía de Afinidad/métodos , Fasciola hepatica/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Ascitis/inmunología , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida , Inmunoglobulina M/inmunología , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Fasciolosis is one of the food-borne neglected trematodioses that has reemerged as a human disease while its effects on domestic animal health remains of significant economic consideration. Being snail-borne disease, the accurate and time-saving epidemiological surveillance of the transmission foci where infected lymnaeid snails occur could be essential to effectively focus or redirect control strategies. For this purpose, the first monoclonal antibody-based immunoenzymatic assay to detect Fasciola hepatica-infected snails (FasciMol-ELISA) was recently developed and showed a high sensitivity and specificity when tested in an experimental F. hepatica - Galba cubensis system. METHODS: Here, we surveyed populations of G. cubensis occurring in western Cuba for the assessment of the FasciMol-ELISA in determining natural F. hepatica infection in this intermediate host. A multiplex PCR, previously developed to detect F. hepatica in G. cubensis, was used for sample classification. Snail dissection method was also employed as screening technique. A Χ(2) test and a Kappa index were calculated to evaluate the positivity and the level of agreement between the FasciMol-ELISA and the snail dissection methods with the multiplex PCR, respectively. RESULTS: Galba cubensis was found in nine out of 12 sampled localities of which four were positive for F. hepatica infection as detected by both immunoenzymatic and PCR-based assays. The overall prevalence was higher than the natural infection rates previously reported for Cuban G. cubensis (range from 4.1 to 7.42% depending on the screening method). No significant differences were found between FasciMol-ELISA and multiplex PCR when determining parasite positivity (Χ(2) = 6.283; P = 0.0981) whereas an excellent agreement was also noted (Kappa = 0.8224). CONCLUSIONS: Our results demonstrate the importance of malacological surveys in assessing parasite transmission risk and constitute an alert on the need of accurate measures to control fasciolosis in western Cuba. The sensitivity and specificity of the FasciMol-ELISA as well as its time-saving capacity and the easy of performing the determination of a large number of samples, point at this assay as a novel tool suitable for large-scale monitoring of natural snails populations. To our knowledge, this is the first study that explores natural infection by F. hepatica in field-occurring lymnaeid snails using an immunoenzymatic assay.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/veterinaria , Fasciola hepatica/inmunología , Fascioliasis/transmisión , Lymnaea/parasitología , Animales , Anticuerpos Monoclonales , Cuba/epidemiología , Fasciola hepatica/genética , Fasciola hepatica/aislamiento & purificación , Fascioliasis/epidemiología , Fascioliasis/parasitología , Humanos , Técnicas para Inmunoenzimas/veterinaria , Ganado , Sensibilidad y Especificidad , Especificidad de la Especie , Encuestas y CuestionariosRESUMEN
Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.
Asunto(s)
Fasciola hepatica/fisiología , Reacción en Cadena de la Polimerasa/métodos , Caracoles/parasitología , Animales , ADN de Helmintos/genética , Fasciola hepatica/aislamiento & purificación , Interacciones Huésped-Parásitos , ARN Ribosómico 18S/genéticaRESUMEN
Fasciolosis is a globally distributed snail-borne disease which requires economic consideration due to its enormous impact on veterinary medicine. During recent decades, this parasitosis has also shown increasing prevalence in human populations worldwide. The dissemination and successful transmission of fasciolosis ultimately depends on the existence of susceptible snails that act as intermediate hosts. Therefore, to accomplish effective control of this disease, surveillance and detection of the infected intermediate host would be essential. The screening of trematodes within snails using classical parasitological examination of the larvae can be unreliable (sensitivity and specificity vary depending on the time of infection and the experience of the observer) and relatively costly when using molecular biological methods during large-scale monitoring. Here we propose a novel monoclonal antibody-based immunoenzymatic assay to detect ongoing Fasciola hepatica infection in lymnaeid snails. Anti-F. hepatica rediae mouse monoclonal antibodies were generated and used to develop a double monoclonal antibody-based ELISA for parasite detection. Fasciola hepatica-infected and uninfected laboratory-reared Galba cubensis and Pseudosuccinea columella were used for assessment of the developed ELISA. Experimentally infected snails were dissected and examined for parasite larvae as the "gold standard" method. Sensitivity results were 100% for both snail species, while specificity was 98% for G. cubensis and 100% for P. columella. No cross-reactivity was detected in lymnaeids infected with Trichobilharzia sp. or Cotylophoron sp. The ELISA enabled detection of the infection from day 8 p.i. in G. cubensis while in P. columella it was noted as early as day 4. To our knowledge no previous immunoassays have been reported to detect helminth-infected snails and the developed sandwich ELISA method is therefore suggested for infection status validation in natural populations of lymnaeid snails.
Asunto(s)
Anticuerpos Antihelmínticos , Anticuerpos Monoclonales , Antígenos Helmínticos/análisis , Vectores de Enfermedades , Fasciola hepatica/aislamiento & purificación , Caracoles/parasitología , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Monitoreo Epidemiológico , Fasciola hepatica/inmunología , Femenino , Ratones Endogámicos BALB C , Sensibilidad y EspecificidadRESUMEN
The control of fasciolosis, as that of other vector-borne diseases, must be related to the control of the lymnaeid snails, the intermediate hosts of the parasite. Thus, an accurate epidemiological surveillance of the transmission foci where the infected mollusks occur is essential. For this purpose, immunoassays could be a useful tool. However, information regarding specific proteins of intramolluscan larvae and previous studies concerning monoclonal antibody generation against asexual stages of trematodes are scarce. Therefore, we explored the antigenic features of intramolluscan rediae of Fasciola hepatica to evaluate three antigenic preparations in order to use the most promising one for developing specific monoclonal antibodies. Mouse antiserum was generated against each antigen for assessing the polyclonal antibody response against the crude extract of rediae and the cross-reactivity against lymnaeids. The specific C-terminal of F. hepatica cytochrome c oxidase subunit I (first antigen), selected by in silico analyses, might not be the appropriate target for immunoassay detection of infected snails, due to its low representation in the total extract of rediae. The majoritarian mixture of low-molecular-weight proteins (<30 kDa) from the rediae homogenate (second antigen) revealed a significant cross-reactivity with lymnaeids. Evidence of the existence of mimetic immunogenic epitopes in this fraction of F. hepatica rediae was achieved. High immunogenicity of the crude extract of rediae (third antigen), mainly related to parasite's specific epitopes, was regarded. Therefore, the rediae homogenate is stated as the most promising antigen from those evaluated, for monoclonal antibody development with potentialities for detecting F. hepatica-infected snails.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Helmínticos/inmunología , Fascioliasis/veterinaria , Animales , Epítopos , Fasciola hepatica/crecimiento & desarrollo , Fascioliasis/parasitología , Fascioliasis/prevención & control , Femenino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Proteica , Conejos , Caracoles/parasitología , Vacunas/inmunologíaRESUMEN
The aim of this study was to analyze the presence of 62 kDa proteinase and anti-62 kDa proteinase antibody in clinical samples of symptomatic and asymptomatic infected women. Proteinase was detected in all the swabs vaginal of infected women. Significantly, amounts of antigen (mean optical density (OD) values) were detected in swabs vaginal of symptomatic as compared to asymptomatic women. This protein was not detected in the group of patients with Trichomonas vaginalis-culture-negative results and in the groups of samples infected with other agents. Antibody to 62 kDa was detected in the swabs vaginal the only 66.6% of the symptomatic and 55.5% of the asymptomatic infected women. Antibody to 62 kDa was also detected in 7/30 of the swabs vaginal from uninfected women. No significant difference was observed in mean OD values of vaginal swabs of T. vaginalis-infected symptomatic as compared to asymptomatic women. The presence of proteinase in 100% of T. vaginalis-infected women suggested that 62 kDa proteinase could be a virulence factor.
Asunto(s)
Anticuerpos Antiprotozoarios/análisis , Portador Sano/parasitología , Péptido Hidrolasas/análisis , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/patogenicidad , Factores de Virulencia/análisis , Portador Sano/patología , Femenino , Humanos , Proteínas Protozoarias/análisis , Vaginitis por Trichomonas/patología , Vagina/parasitología , VirulenciaRESUMEN
Trichomonosis, caused by the protozoan parasite Trichomonas vaginalis, is one of the most frequent sexually transmitted diseases and is widely spread in all continents. Trichomonas vaginalis as well as other protozoan organisms have high levels of proteolitic activity mainly of the cysteine-proteinase type. This activity is necessary for recognition and adhesion of the parasite to the superficial epithelial cells of the host. In the present study, we show that intranasal immunisation with a 62 kDa cysteine-proteinase purified from T. vaginalis excretion-secretion products in combination with cholera toxin or with synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG-ODN) elicits 62kDa specific IgG and IgA in vaginal lavage fluid and specific IgG in serum. This immunisation protocol resulted in enhanced elimination of parasites following intravaginal challenge of BALB/c mice.