RESUMO
Intestinal epithelial cells possess the requisite molecular machinery to initiate cell-intrinsic defensive responses against intracellular pathogens, including intracellular parasites. Interferons(IFNs) have been identified as cornerstones of epithelial cell-intrinsic defense against such pathogens in the gastrointestinal tract. Long non-coding RNAs (lncRNAs) are RNA transcripts (>200 nt) not translated into protein and represent a critical regulatory component of mucosal defense. We report here that lncRNA Nostrill facilitates IFN-γ-stimulated intestinal epithelial cell-intrinsic defense against infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Nostrill promotes transcription of a panel of genes controlled by IFN-γ through facilitating Stat1 chromatin recruitment and thus, enhances expression of several genes associated with cell-intrinsic defense in intestinal epithelial cells in response to IFN-γ stimulation, including Igtp, iNos, and Gadd45g. Induction of Nostrill enhances IFN-γ-stimulated intestinal epithelial defense against Cryptosporidium infection, which is associated with an enhanced autophagy in intestinal epithelial cells. Our findings reveal that Nostrill enhances the transcription of a set of genes regulated by IFN-γ in intestinal epithelial cells. Moreover, induction of Nostrill facilitates the IFN-γ-mediated epithelial cell-intrinsic defense against cryptosporidial infections.
Assuntos
Criptosporidiose , Interferon gama , Mucosa Intestinal , RNA Longo não Codificante , Interferon gama/metabolismo , RNA Longo não Codificante/genética , Criptosporidiose/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/metabolismo , Animais , Humanos , Transcrição Gênica , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Camundongos , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Cryptosporidium/genética , Cryptosporidium/imunologia , Regulação da Expressão Gênica , Autofagia/imunologiaRESUMO
Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.
Assuntos
Linfócitos T CD4-Positivos , Criptosporidiose , Cryptosporidium , Células Dendríticas , Camundongos Endogâmicos C57BL , Animais , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Camundongos , Cryptosporidium/imunologia , Cryptosporidium/fisiologia , Intestinos/imunologia , Intestinos/parasitologia , Interleucina-12/metabolismo , Interleucina-12/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Células Th1/imunologia , Linfonodos/imunologia , Linfonodos/parasitologiaRESUMO
Infectious disease cryptosporidiosis is caused by the cryptosporidium parasite, a type of parasitic organism. It is spread through the ingestion of contaminated water, food, or fecal matter from infected animals or humans. The control becomes difficult because the parasite may remain in the environment for a long period. In this work, we constructed an epidemic model for the infection of cryptosporidiosis in a fractional framework with strong and weak immunity concepts. In our analysis, we utilize the well-known next-generation matrix technique to evaluate the reproduction number of the recommended model, indicated by [Formula: see text]. As [Formula: see text], our results show that the disease-free steady-state is locally asymptotically stable; in other cases, it becomes unstable. Our emphasis is on the dynamical behavior and the qualitative analysis of cryptosporidiosis. Moreover, the fixed point theorem of Schaefer and Banach has been utilized to investigate the existence and uniqueness of the solution. We identify suitable conditions for the Ulam-Hyers stability of the proposed model of the parasitic infection. The impact of the determinants on the sickness caused by cryptosporidiosis is highlighted by the examination of the solution pathways using a novel numerical technique. Numerical investigation is conducted on the solution pathways of the system while varying various input factors. Policymakers and health officials are informed of the crucial factors pertaining to the infection system to aid in its control.
Assuntos
Criptosporidiose , Criptosporidiose/transmissão , Criptosporidiose/imunologia , Criptosporidiose/epidemiologia , Humanos , Animais , Cryptosporidium/imunologiaRESUMO
Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells, type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium.
Assuntos
Linfócitos T CD8-Positivos , Criptosporidiose , Cryptosporidium , Células Dendríticas , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Animais , Criptosporidiose/imunologia , Camundongos , Cryptosporidium/imunologia , Interferon gama/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/imunologia , Humanos , Camundongos Transgênicos , Ativação Linfocitária/imunologia , Epitopos de Linfócito T/imunologia , Camundongos Endogâmicos C57BL , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Camundongos KnockoutRESUMO
Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.
Assuntos
Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium/imunologia , Células Dendríticas/imunologia , Interações Hospedeiro-Parasita/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Células Th1/imunologia , Animais , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Homeostase , Mucosa Intestinal/metabolismo , Camundongos , Microbiota , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/metabolismoRESUMO
Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.
Assuntos
Subunidade alfa de Receptor de Interleucina-21/deficiência , Subunidade alfa de Receptor de Interleucina-21/genética , Adolescente , Linfócitos B/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Criptosporidiose/genética , Criptosporidiose/imunologia , Cryptosporidium/imunologia , Feminino , Genômica/métodos , Humanos , Imunidade Humoral/genética , Imunidade Humoral/imunologia , Lactente , Subunidade alfa de Receptor de Interleucina-21/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Células B de Memória/imunologia , Infecção Persistente/genética , Infecção Persistente/imunologia , Fenótipo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Adulto JovemRESUMO
Cryptosporidium is a widely distributed enteric parasite that has an increasingly appreciated pathogenic role, particularly in pediatric diarrhea. While cryptosporidiosis has likely affected humanity for millennia, its recent "emergence" is largely the result of discoveries made through major epidemiologic studies in the past decade. There is no vaccine, and the only approved medicine, nitazoxanide, has been shown to have efficacy limitations in several patient groups known to be at elevated risk of disease. In order to help frontline health workers, policymakers, and other stakeholders translate our current understanding of cryptosporidiosis into actionable guidance to address the disease, we sought to assess salient issues relating to clinical management of cryptosporidiosis drawing from a review of the literature and our own field-based practice. This exercise is meant to help inform health system strategies for improving access to current treatments, to highlight recent achievements and outstanding knowledge and clinical practice gaps, and to help guide research activities for new anti-Cryptosporidium therapies.
Assuntos
Antiparasitários/uso terapêutico , Criptosporidiose/tratamento farmacológico , Criptosporidiose/epidemiologia , Cryptosporidium/efeitos dos fármacos , Nitrocompostos/uso terapêutico , Tiazóis/uso terapêutico , Pré-Escolar , Cryptosporidium/imunologia , Diarreia/parasitologia , Surtos de Doenças , Hidratação , Humanos , Hospedeiro Imunocomprometido/imunologia , Lactente , Recém-NascidoAssuntos
Coinfecção/diagnóstico , Coinfecção/parasitologia , Criptosporidiose/diagnóstico , Estrongiloidíase/diagnóstico , Trombocitopenia/diagnóstico , Trombocitopenia/imunologia , Idoso , Animais , Coinfecção/imunologia , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium/imunologia , Cryptosporidium/patogenicidade , Feminino , Humanos , Hospedeiro Imunocomprometido , Púrpura Trombocitopênica Idiopática/complicações , Strongyloides stercoralis/imunologia , Strongyloides stercoralis/patogenicidade , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologia , Trombocitopenia/parasitologiaRESUMO
The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.
Assuntos
Criptosporidiose/imunologia , Cryptosporidium/imunologia , Interferon Tipo I/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais , Animais , Animais Recém-Nascidos , Criptosporidiose/parasitologia , Diarreia/imunologia , Diarreia/parasitologia , Células Epiteliais/parasitologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/parasitologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Intestinos/parasitologia , Camundongos , Transcrição Gênica , Regulação para CimaRESUMO
Cryptosporidiosis is common in early childhood, and both diarrheal and subclinical infections are associated with adverse developmental outcomes. Improved therapeutic medications may help reduce the burden of cryptosporidial diarrhea; however, an effective vaccine would be better able to prevent the detrimental impact of both diarrheal and subclinical disease. A more complete understanding of naturally occurring immunity may further inform strategies to develop an effective vaccine. In this prospective cohort study of Bangladeshi children, greater fecal IgA at 12 months, but not plasma IgG, directed against two sporozoite-expressed, immunodominant and vaccine candidate antigens was associated with delayed time to subsequent cryptosporidiosis to 3 years of life. These findings extend prior work and further support the role of mucosal antibody responses in naturally developing protective immunity to Cryptosporidium.
Assuntos
Anticorpos Antiprotozoários/química , Antígenos de Protozoários/imunologia , Cryptosporidium/imunologia , Fezes/parasitologia , Imunoglobulina A/imunologia , Anticorpos Antiprotozoários/sangue , Bangladesh/epidemiologia , Pré-Escolar , Criptosporidiose , Cryptosporidium/metabolismo , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , MasculinoRESUMO
AIMS: Immunocompromised mice are extensively used in the screening of vaccines and drugs for Cryptosporidium, but this study model does not reflect the real status of infection in immunocompetent animals. This study aimed to provide an optimized animal model for future studies of Cryptosporidium vaccine. METHODS AND RESULTS: Three mouse strains (ICR, BALB/c and KM) with or without immunosuppression were compared after challenge with Cryptosporidium tyzzeri (C tyzzeri). The results indicated that ICR mice shed a greater number of faecal oocysts (20 346 ± 203 oocysts/g) compared with BALB/c (2077 ± 142 oocysts/g) and KM mice (3207 ± 431 oocysts/g) after experimental infection with C tyzzeri (P < .001). However, ICR mouse model is uniquely effective for C tyzzeri, not for other Cryptosporidium spp. such as C parvum. ICR mice were then used to determine the immunoreactions and immunoprotection of P23-DNA vaccine (pVAX1-P23) to C tyzzeri experimental infection. The results showed that a significant increase in anti-P23 antibody levels was induced by the pVAX1-P23 vaccine. Compared to pVAX1, TB and blank control mice, pVAX1-P23 immunized mice produced specific spleen cell proliferation as well as enhanced IL-5, IL-12p70 and IFN-γ production in sera. After challenge with 5 × 106 C tyzzeri oocysts, the oocyst shedding of the pVAX1-P23 immunized group was reduced by 69.94% comparing to the infection control. CONCLUSION: These results provide an optimized animal model for the study of prophylactic vaccines and this model might be applied to other candidates against Cryptosporidium, not only for pVAX1-P23.
Assuntos
Criptosporidiose/prevenção & controle , Cryptosporidium/imunologia , Vacinas Protozoárias/imunologia , Vacinas de DNA/imunologia , Animais , Formação de Anticorpos/imunologia , Criptosporidiose/imunologia , Modelos Animais de Doenças , Fezes/parasitologia , Interferon gama/sangue , Subunidade p35 da Interleucina-12/sangue , Interleucina-5/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Oocistos/imunologia , VacinaçãoRESUMO
The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0-9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child's house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1-9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia's Amhara region may reduce exposure to these enteropathogens in young children.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Antiprotozoários/sangue , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/imunologia , Criptosporidiose/sangue , Cryptosporidium/imunologia , Entamebíase/sangue , Giardíase/sangue , Criança , Pré-Escolar , Infecções por Chlamydia/sangue , Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/genética , Estudos Transversais , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/genética , Entamoeba histolytica/genética , Entamoeba histolytica/imunologia , Entamebíase/epidemiologia , Entamebíase/parasitologia , Etiópia/epidemiologia , Feminino , Água Doce/parasitologia , Giardia lamblia/genética , Giardia lamblia/imunologia , Giardíase/epidemiologia , Giardíase/parasitologia , Humanos , Masculino , Estudos SoroepidemiológicosRESUMO
OBJECTIVE: Cryptosporidium is an enteric protozoan parasite that affects human and many animal species in worldwide. Staining methods or stool antigen detecting methods are using for detection of Cryptosporidium in faeces. It is known that the ELISA method has high sensitivity and specificity in practice. The aim of this study is to demonstrate the frequency of this parasite by ELISA in samples sent to our laboratory between 2010 January and 2018 September. METHODS: The study was conducted on a total of 723 patients, 431 men and 292 women, who were referred to the Parasitology Laboratory from various outpatient clinics due to digestive system complaints. The presence of Cryptosporidium spp. antigen was investigated by ELISA method. RESULTS: Cryptosporidium spp. was not found in any patient with Nativ-lugol method, whereas Cryptosporidium spp. antigen positivity was detected in 2.8% of 723 patients. In the study, 2.5% of the males and 3.1% of the females were found positive in terms of having the parasite, and there was no significant difference in gender between the parasite frequencies. The highest rate of parasite positivity (4.5%) was found in the 0-6 age group. CONCLUSION: The high rate of cryptosporidiosis detected in our study is thought to be related to factors such as widespread animal husbandry in our region, poor hygiene rules and low socio-economic level. As a result of considering the findings of our study, evaluation of patients with intestinal complaints in terms of Cryptosporidium will be useful for accurate diagnosis, regardless of whether they have diarrhea and/or they are immunocompromised.
Assuntos
Antígenos de Protozoários/análise , Criptosporidiose/diagnóstico , Cryptosporidium/imunologia , Fezes/parasitologia , Adolescente , Adulto , Distribuição por Idade , Criação de Animais Domésticos , Animais , Criança , Pré-Escolar , Criptosporidiose/epidemiologia , Cryptosporidium/isolamento & purificação , Diarreia/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Hospedeiro Imunocomprometido , Lactente , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Distribuição por Sexo , Fatores Socioeconômicos , Turquia/epidemiologia , Adulto JovemRESUMO
When considering methods of detecting Cryptosporidium in patient samples, clinical and public health laboratories have historically relied primarily on microscopy. However, microscopy is time intensive and requires trained personnel to accurately identify pathogens that are present. Even with skilled analysts, the parasitemia level has the potential to fall below the level of detection. In addition, public health laboratories do not always receive specimens in fixatives that are compatible with the desired microscopic method. Antigen-based and molecular methods have proven to be effective at identifying Cryptosporidium at low levels and require less training and hands-on time. Here, we have developed and validated a real-time polymerase chain reaction (RT-PCR) laboratory-developed test (LDT) that identifies Cryptosporidium hominis and Cryptosporidium parvum, and also includes detection at the genus level to identify additional species that occasionally cause disease in humans. Results of the molecular test were compared with those obtained from modified acid-fast microscopy, immunofluorescent microscopy, an antigen-based detection rapid test, and a commercial gastrointestinal panel (GI panel). Of 40 positive samples, microscopy and antigen-based methods were able to detect Cryptosporidium in only 20 and 21 samples, respectively. The GI panel detected 33 of the 40 positive samples, even though not all specimens were received in the recommended preservative. The LDT detected Cryptosporidium in all 40 positive samples. When comparing each method for the detection of Cryptosporidium, our results indicate the LDT is an accurate, reliable, and cost-effective method for a clinical public health reference laboratory.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Técnicas de Laboratório Clínico , Criptosporidiose/parasitologia , Cryptosporidium/imunologia , Cryptosporidium parvum/genética , Cryptosporidium parvum/imunologia , Fezes , Feminino , Humanos , Imunoensaio/métodos , Lactente , Masculino , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Coloração e Rotulagem , Adulto JovemRESUMO
A major obstacle to developing vaccines against cryptosporidiosis, a serious diarrheal disease of children in developing countries, is the lack of rodent models essential to identify and screen protective immunogens. Rodent models commonly used for drug discovery are unsuitable for vaccine development because they either are purposefully immunodeficient or immunosuppressed. Here, we describe the development and optimization of an immunocompetent intratracheal (IT) rat model susceptible to infections with sporozoites of Cryptosporidium parvum and Cryptosporidium hominis - the primary causes of human cryptosporidiosis. A model suitable for screening of parasite immunogens is a prerequisite for immunogen screening and vaccine development.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Cryptosporidium parvum/imunologia , Cryptosporidium/imunologia , Modelos Animais , Ratos Sprague-Dawley/imunologia , Animais , Antígenos de Protozoários , Criptosporidiose/prevenção & controle , Feminino , Imunidade Humoral , Imunocompetência , Ratos , Ratos Sprague-Dawley/parasitologia , Esporozoítos/imunologia , Traqueia/parasitologia , Vacinação/métodosRESUMO
Stained microscopy of fecal smears was the cornerstone of Cryptosporidium diagnosis for many years, and still provides a low-cost method for detecting oocysts. The development and commercialization of improved enzyme immunosorbent assays (EIA) for coproantigen detection provided an automatable method for mass testing, and rapid diagnostics when incorporated onto a cartridge format. Similarly, immunochromatographic lateral flow assays (ICLF) enable rapid diagnostics. Nevertheless, it is important that positive reactions by EIA or ICLF are confirmed. Here we describe microscopical methods using tinctorial stains for the diagnosis of acute cryptosporidiosis, and using immunofluorescent reagents for diagnosis or for confirmation of EIA or ICLF positive reactions.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/imunologia , Imunofluorescência/métodos , Técnicas Imunoenzimáticas/métodos , Microscopia de Fluorescência/métodos , Microscopia/métodos , Oocistos/imunologia , Animais , Corantes , Criptosporidiose/imunologia , Fezes/parasitologia , Humanos , Microscopia/instrumentação , Microscopia de Fluorescência/instrumentação , Oocistos/isolamento & purificação , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Fluxo de TrabalhoRESUMO
For more than 35 years, various assay formats have been used to detect Cryptosporidium-specific antibodies in human and animal sera. Cryptosporidium parvum 17- and 27-kDa antigens, identified from invasive sporozoites, have been used in serologic antibody assays to identify individuals infected in outbreaks of diarrheal disease caused by this protozoan parasite and to monitor exposures in communities. During infection, immunoglobulin (Ig) A, IgM, and IgG responses are elicited by these immunodominant antigens, and the parasite-specific Ig responses diminish following the resolution of infection. Using the recombinant forms of the 17- and 27-kDa C. parvum antigens and the relatively recently developed multiplex bead assay (MBA), data from serologic antibody responses can be economically and efficiently acquired, especially when the Cryptosporidium assays are integrated with assays for antibody responses to antigens from other pathogens monitored in community-wide or nation-wide serosurveys. Here we describe the coupling of the C. parvum recombinant antigens to carboxylated polystyrene beads, the data acquisition and analysis of IgG antibodies bound to the coupled beads, and the quality control methods required for data validation using the Luminex/MBA system.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Criptosporidiose/diagnóstico , Cryptosporidium parvum/imunologia , Cryptosporidium/imunologia , Imunoensaio/métodos , Antígenos de Protozoários/genética , Criptosporidiose/imunologia , Cryptosporidium/isolamento & purificação , Cryptosporidium parvum/isolamento & purificação , Humanos , Imunoensaio/instrumentação , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Técnicas de Imunoadsorção , Fluxo de TrabalhoRESUMO
Development of an effective vaccine against cryptosporidiosis is a medical and veterinary priority. However, many putative Cryptosporidium vaccine candidates such as surface and apical complex antigens are posttranslationally modified with O- and N-linked glycans. This presents a significant challenge to understanding the functions of these antigens and the immune responses to them. Isolation of large amounts of native antigen from Cryptosporidium oocysts is expensive and is only feasible for C. parvum antigens. Here, we describe a method of producing recombinant, functional Cryptosporidium glycoprotein antigens in Toxoplasma gondii. These functional recombinant proteins can be used to investigate the role of glycotopes in Cryptosporidium immune responses and parasite-host cell interactions.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Cryptosporidium parvum/metabolismo , Glicoproteínas/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Toxoplasma/metabolismo , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Linhagem Celular , Cromatografia de Afinidade/métodos , Cryptosporidium/crescimento & desenvolvimento , Cryptosporidium/imunologia , Cryptosporidium/metabolismo , Cryptosporidium parvum/crescimento & desenvolvimento , Cryptosporidium parvum/imunologia , Imunofluorescência/métodos , Expressão Gênica , Vetores Genéticos , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Oocistos/crescimento & desenvolvimento , Oocistos/isolamento & purificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , Transfecção/métodos , Fluxo de TrabalhoRESUMO
Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.
Assuntos
Arqueologia/métodos , Cryptosporidium/isolamento & purificação , Entamoeba/isolamento & purificação , Fezes/parasitologia , Giardia lamblia/isolamento & purificação , Técnicas Imunoenzimáticas/normas , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/genética , Cryptosporidium/genética , Cryptosporidium/imunologia , Entamoeba/genética , Entamoeba/imunologia , Entamoeba histolytica/genética , Entamoeba histolytica/imunologia , Entamoeba histolytica/isolamento & purificação , Giardia lamblia/genética , Giardia lamblia/imunologia , Humanos , Enteropatias Parasitárias/parasitologia , Roedores , Sensibilidade e Especificidade , América do SulRESUMO
Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium. Here, we derive an in vivo model of Cryptosporidium infection in immunocompetent C57BL/6 mice by isolating parasites from naturally infected wild mice. Similar to human cryptosporidiosis, this infection causes intestinal pathology, and interferon-γ controls early infection while T cells are critical for clearance. Importantly, mice that controlled a live infection were resistant to secondary challenge and vaccination with attenuated parasites provided protection equal to live infection. Both parasite and host are genetically tractable and this in vivo model will facilitate mechanistic investigation and rational vaccine design.