RESUMO
Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites, are suitable features that suggest their potential use for developing an epitope-based vaccine for fasciolosis control.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Fasciola hepatica/imunologia , Animais , Variação Antigênica/fisiologia , Epitopos/química , Epitopos/metabolismo , Fasciola hepatica/efeitos dos fármacos , Imuno-Histoquímica , Mercaptoetanol/farmacologia , Camundongos , Oxirredução , Ácido Periódico/farmacologia , Pronase/metabolismo , TemperaturaRESUMO
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.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/imunologia , Fasciolíase/veterinária , Animais , Epitopos , Fasciola hepatica/crescimento & desenvolvimento , Fasciolíase/parasitologia , Fasciolíase/prevenção & controle , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Coelhos , Caramujos/parasitologia , Vacinas/imunologiaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/análise , Portador Sadio/parasitologia , Peptídeo Hidrolases/análise , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/patogenicidade , Fatores de Virulência/análise , Portador Sadio/patologia , Feminino , Humanos , Proteínas de Protozoários/análise , Vaginite por Trichomonas/patologia , Vagina/parasitologia , VirulênciaRESUMO
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.
Assuntos
Variação Genética , Peptídeo Hidrolases/genética , Proteínas de Protozoários/genética , Trichomonas vaginalis/genética , Adolescente , DNA de Protozoário/genética , Feminino , Humanos , Filogenia , Polimorfismo de Fragmento de Restrição , Tricomoníase/parasitologia , Trichomonas vaginalis/enzimologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Fasciola hepatica/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Ascite/imunologia , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida , Imunoglobulina M/imunologia , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Fasciola hepatica/imunologia , Fasciolíase/transmissão , Lymnaea/parasitologia , Animais , Anticorpos Monoclonais , Cuba/epidemiologia , Fasciola hepatica/genética , Fasciola hepatica/isolamento & purificação , Fasciolíase/epidemiologia , Fasciolíase/parasitologia , Humanos , Técnicas Imunoenzimáticas/veterinária , Gado , Sensibilidade e Especificidade , Especificidade da Espécie , Inquéritos e QuestionáriosRESUMO
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.
Assuntos
Peptídeo Hidrolases/imunologia , Vacinas Protozoárias/imunologia , Vaginite por Trichomonas/prevenção & controle , Trichomonas vaginalis/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Toxina da Cólera/imunologia , Feminino , Imunização/métodos , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/imunologia , Vaginite por Trichomonas/imunologia , Vagina/imunologiaRESUMO
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.
Assuntos
Fasciola hepatica/fisiologia , Reação em Cadeia da Polimerase/métodos , Caramujos/parasitologia , Animais , DNA de Helmintos/genética , Fasciola hepatica/isolamento & purificação , Interações Hospedeiro-Parasita , RNA Ribossômico 18S/genéticaRESUMO
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.
Assuntos
Anticorpos Anti-Helmínticos , Anticorpos Monoclonais , Antígenos de Helmintos/análise , Vetores de Doenças , Fasciola hepatica/isolamento & purificação , Caramujos/parasitologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Monitoramento Epidemiológico , Fasciola hepatica/imunologia , Feminino , Camundongos Endogâmicos BALB C , Sensibilidade e EspecificidadeRESUMO
Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.
Assuntos
Cisteína Proteases/fisiologia , Proteínas de Protozoários/fisiologia , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/enzimologia , Vacinas Bacterianas , Líquidos Corporais/parasitologia , Adesão Celular , Membrana Celular/parasitologia , Feminino , Hemólise , Interações Hospedeiro-Parasita , Humanos , Evasão da Resposta Imune , Ferro/metabolismo , Lisossomos/parasitologia , Terapia de Alvo Molecular , Mucosa/parasitologia , Mucosa/patologia , Vaginite por Trichomonas/patologia , Trichomonas vaginalis/imunologia , Trichomonas vaginalis/patogenicidade , Vagina/parasitologia , Vagina/patologia , VirulênciaRESUMO
This study describes the use of bromo-deoxiuridine, a non-radioactive analogue of thymidine, to determine the adhesion of 40 T. vaginalis isolates, as compared with the clinical manifestations found in patients. Parasite-labeling was optimized and label detection by immunoassay was carried out. Bromo-deoxiuridine at 10 microM for 16 h produced the highest sensitivity. Once optimized, the assay was able to detect between 3.12 x 10(3) and 4 x 10(5) parasites, with a detection limit of 9.14 x 10(2), which is lower than with the use of [3H]-thymidine. The variation coefficients were 2.65% for repeatability and 3.8% for reproducibility. A correlation coefficient of 0.97 was found between the pattern curves obtained by both labeling procedures. The level of adhesion to HeLa cells was directly proportional to the severity of the clinical manifestations (P < 0.05).
Assuntos
Adesão Celular/fisiologia , Trichomonas vaginalis/citologia , Trichomonas vaginalis/patogenicidade , Adolescente , Animais , Bioensaio , Bromodesoxiuridina/metabolismo , Feminino , Células HeLa , Humanos , Imunoensaio , Técnicas In Vitro , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trichomonas vaginalis/metabolismo , VirulênciaRESUMO
Recently, we reported a partial characterization of the epitope recognized by the ES-78 monoclonal antibody (MoAb). This monoclonal antibody was obtained from spleen lymphocytes of a mouse immunized with excretory-secretory antigens of Fasciola hepatica adult worms. In the present study, we report the results obtained in experiments of passive protection using this MoAb in BALB/c mice infected with 15 Fasciola hepatica metacercariae. The monoclonal antibody was able to reduce the parasite burden when administered 24 h before challenge but not when delivered 7 days after challenge. The antibody recognition of digestive tract structures in 3-week-old parasites was demonstrated by immune histochemical techniques. The antigens purified by affinity chromatography using this antibody had molecular weights of 14-20, 25-29 and 36-45 kDa and demonstrated proteinase activity similar to cathepsin L. These results suggest that the antigens carrying the epitope recognized by the ES-78 MoAb may be used as target in the protection against fasciolosis.