RESUMO
Cryptosporidium parvum is a protozoan parasite of the phylum Apicomplexa responsible for cryptosporidiosis in calves, a disease that causes significant diarrhea and impairs gain of body weight, generating important production losses. As to now, no effective drugs or vaccines are available for the treatment or prevention of bovine cryptosporidiosis. Several reports suggest that development of a vaccine to prevent cryptosporidiosis is feasible, but relatively few vaccine candidates have been characterized and tested. The most prominent C. parvum antigen is gp60, an O-glycosylated mucin-like protein tethered to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor. Gp60 has been shown to be involved in essential mechanisms for the survival of C. parvum, such as recognition, adhesion to, and invasion of host cells. This work was aimed at expressing gp60 in Tetrahymena thermophila, a ciliated protozoon with numerous advantages for the heterologous expression of eukaryotic proteins, as a first approach for the development of a recombinant vaccine for bovine cryptosporidiosis. T. thermophila-expressed gp60 localized to the protozoon cell surface and oral apparatus, and partitioned into the Triton X-114 detergent phase. This indicates that the protein entered the reticuloendothelial system of the ciliate, and suggests it contains a GPI-anchor. Homogenates of gp60-expressing T. thermophila cells were recognized by sera from calves naturally infected with C. parvum demonstrating their immunoreactivity. In summary, the heterologous expression of gp60, a C. parvum-encoded GPI-anchored protein, has been successfully demonstrated in the ciliate T. thermophila.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Tetrahymena thermophila/genética , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Criptosporidiose/imunologia , Criptosporidiose/prevenção & controle , Cryptosporidium parvum/genética , Vacinas Sintéticas/sangue , Vacinas Sintéticas/genéticaRESUMO
The apicomplexan protozoan Cryptosporidium parvum is an important causative agent of diarrhea of neonatal bovines. Vaccination has been proposed as an advantageous strategy against cryptosporidiosis of calves since besides protection against disease it has also the potential to prevent dissemination of infective oocysts into the environment. Antigens anchored to the parasite surface via glycosylphosphatidylinositol (GPI) are implicated in host cell attachment and invasion and represent promising vaccine candidates. A reverse vaccinology approach was employed to (i) identify the GPI-anchored proteome of C. parvum using available web-based bioinformatic tools and (ii) characterize previously unrecognized novel vaccine antigens. Altogether, 14 putative GPI-anchored proteins could be determined of which CpH1 and CpSUB2 as well as GP60 were further characterized. Sequencing and comparison of GP60, CpH1, and CpSUB1 alleles amplified from different geographic isolates showed a high degree of conservation. All three antigens were recombinant expressed and immunoblotted using sera of 12 Cryptosporidium-infected calves sampled at age periods 1-11 and 12-28 days after birth. Specific antibody reactions against the studied antigens were detected in all analyzed calves, demonstrating their immunreactivity and expression, and recognition in vivo at an early stage of host infection. Besides the acknowledged GP60 vaccinogen, the presented reverse vaccinology approach reveals the additional vaccine candidates CpH1 and CpSUB1 for inclusion into a subunit vaccine formulation.
Assuntos
Doenças dos Bovinos/prevenção & controle , Criptosporidiose/prevenção & controle , Vacinas Protozoárias/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Anti-Helmínticos/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium/imunologia , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologia , VacinologiaRESUMO
In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
Assuntos
Galinhas/imunologia , Criptosporidiose/diagnóstico , Cryptosporidium/química , Imunoglobulinas/imunologia , Animais , Criptosporidiose/imunologia , Escherichia coli/metabolismo , Feminino , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
Resumo Neste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.
Assuntos
Animais , Feminino , Imunoglobulinas/imunologia , Galinhas/imunologia , Criptosporidiose/diagnóstico , Cryptosporidium/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Criptosporidiose/imunologia , Escherichia coli/metabolismoRESUMO
In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.(AU)
Neste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.(AU)
Assuntos
Animais , Galinhas/imunologia , Criptosporidiose/diagnóstico , Criptosporidiose/imunologia , Criptosporidiose/prevenção & controle , Escherichia coli/metabolismo , Imunoglobulinas/imunologia , Proteínas Recombinantes , Técnica Indireta de Fluorescência para Anticorpo , Western BlottingRESUMO
MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, have been detected in a variety of organisms ranging from ancient unicellular eukaryotes to mammals. They have been associated with numerous molecular mechanisms involving developmental, physiological and pathological changes of cells and tissues. Despite the fact that miRNA-silencing mechanisms appear to be absent in some Apicomplexan species, an increasing number of studies have reported a role for miRNAs in host-parasite interactions. Host miRNA expression can change following parasite infection and the consequences can lead, for instance, to parasite clearance. In this context, the immune system signaling appears to have a crucial role.
Assuntos
Cryptosporidium/imunologia , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Parasita/genética , MicroRNAs/genética , Plasmodium/imunologia , Toxoplasma/imunologia , Criptosporidiose/imunologia , Criptosporidiose/microbiologia , Regulação da Expressão Gênica/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Malária/imunologia , Malária/microbiologia , Transdução de Sinais/imunologia , Toxoplasmose/imunologia , Toxoplasmose/microbiologiaRESUMO
Cryptosporidium parvum subtype IIaA21G1R1 oocysts were used to infect dexamethasone immunosuppressed N: NIH Swiss mice. This is the first Cryptosporidium mouse model in which the relationship between infection and apoptosis has been histologically studied at each portion of the gut in order to observe this dynamic in chronic cryptosporidiosis. Histology showed developmental stages in the duodenum, proximal and distal jejunum, ileum, cecum and colon, with the small intestine remaining infected until day 35 post infection. At proximal jejunum an inverse correlation between infection and apoptosis was observed at days 28 and 35 p.i. Data suggests that jejunum could be an interesting place to carry out further studies on the dynamics of Cryptosporidium infection and apoptosis. Based on these findings, this mouse model was useful to evaluate clinical, parasitological and histological aspects of C. parvum subtype IIaA21G1R1 infection, and it will be an appropriate tool to investigate different aspects of Cryptosporidium infection.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , Animais , Criptosporidiose/imunologia , Dexametasona/farmacologia , Hospedeiro Imunocomprometido , Imunossupressores/farmacologia , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/patologia , Intestinos/parasitologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7% of positivity for coccidian (8.6% with Cryptosporidium sp, 10.3% with Cystoisospora belli and 0.8% with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < or =200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5% of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Linfócitos T CD4-Positivos/imunologia , Criptosporidiose/epidemiologia , Diarreia/epidemiologia , Isosporíase/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Adolescente , Adulto , Idoso , Animais , Brasil/epidemiologia , Criança , Pré-Escolar , Criptosporidiose/diagnóstico , Criptosporidiose/imunologia , Diarreia/parasitologia , Fezes/parasitologia , Feminino , Humanos , Isosporíase/diagnóstico , Isosporíase/imunologia , Masculino , Pessoa de Meia-Idade , Prevalência , Estações do AnoRESUMO
Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7 percent of positivity for coccidian (8.6 percent with Cryptosporidium sp, 10.3 percent with Cystoisospora belli and 0.8 percent with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < 200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5 percent of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli.
Pacientes com AIDS são particularmente susceptíveis a infecção por coccídios intestinais e nesse estudo foi avaliada a freqüência de Cryptosporidium sp. e Cystoisospora belli entre pacientes HIV/AIDS na região do Triângulo Mineiro, Brasil. No período de julho de 1993 a junho de 2003, amostras de fezes de 359 pacientes foram submetidas à coloração pelo método de Ziehl-Neelsen modificado, sendo detectada a presença de coccídios em 19,7 por cento destas (8,6 por cento de Cryptosporidium sp, 10,3 por cento de Cystoisospora belli e 0,8 por cento de ambos coccídios). Pacientes com diarréia e níveis de linfócitos T CD4+ < 200 células/mm3 apresentaram maior frequência destes protozoários, demonstrando o perfil oportunista destas infecções e a relação com o status imunológico do indivíduo. Não foi possível determinar a influência da HARRT, pois apenas 8,5 por cento dos pacientes positivos para coccídios fazriam uso regular desta terapia. Parasitismo por Cryptosporidium sp foi mais freqüente no período compreendido de dezembro a fevereiro caracterizando padrão sazonal desta infecção, fato não observado com Cystoisospora belli.
Assuntos
Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , /imunologia , Criptosporidiose/epidemiologia , Diarreia/epidemiologia , Isosporíase/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Brasil/epidemiologia , Criptosporidiose/diagnóstico , Criptosporidiose/imunologia , Diarreia/parasitologia , Fezes/parasitologia , Isosporíase/diagnóstico , Isosporíase/imunologia , Prevalência , Estações do AnoRESUMO
OBJECTIVES: To study the prevalence of Cryptosporidium infection by measuring the levels of anti-Cryptosporidium IgG antibodies among people inhabiting three neighbourhoods of a periurban area of Salvador, Northeast of Brazil; and to investigate the effects of environmental sanitation measures, hygienic habits and household water supply, storage and handling on the frequency of these antibodies in sera of the studied population. METHODS: Cryptosporidium inter-household transmission was studied by comparing the frequency of anti-Cryptosporidium IgG antibodies among people inhabiting areas with or without different environmental sanitation measures and intra-household transmission by comparing the presence of these antibodies in families with or without cases of diarrhoea, associated with the presence of Cryptosporidium oocysts in their stools. Children or family members with diarrhoeal episodes were evaluated parasitologically for Cryptosporidium infection by testing stool specimens with the Ritchie-modified formol-ether concentration and the acid-fast staining methods. All groups were serologically evaluated for parasite exposure by an indirect enzyme-linked immunosorbent assay. RESULTS: A statistically significant difference was detected in the prevalence of Cryptosporidium infection between area 1 which had no environmental sanitation measures and area 3 which had improved environmental sanitation measures (P = 0.044). Most of the hygienic habits investigated did not correlate with the presence of anti-Cryptosporidium antibody in sera of the population studied. However, positive associations were found between both poor household water supply (OD = 0.17; 90% CI = 0.09-0.32; P = 0.0001) and drinking unboiled/unfiltered water (OD = 0.40; 90% CI = 0.24-0.67; P = 0.0002) with high levels of anti-Cryptosporidium antibodies in sera. CONCLUSIONS: These data suggest that although uncorrected household water supply, storage and handling play an important role on Cryptosporidium transmission in periurban areas of developing country cities, like Salvador, Brazil, inadequate environmental conditions may also contribute to the spread of this parasite.