RESUMEN
Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Lenguado/parasitología , Enfermedades Transmitidas por los Alimentos/prevención & control , Gastroenteritis/prevención & control , Myxozoa/inmunología , Esporas Protozoarias/inmunología , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/genética , Secuencia de Bases , Enfermedades de los Peces/parasitología , Enfermedades Transmitidas por los Alimentos/parasitología , Gastroenteritis/parasitología , Japón , Músculos/parasitología , Myxozoa/genética , Myxozoa/aislamiento & purificación , Esporas Protozoarias/aislamiento & purificaciónRESUMEN
Myxobolus honghuensis infects the pharynx of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) and can cause high mortality. Only morphology-based diagnostic methods are currently available for clinical samples, but these methods are laborious and have low efficiency of detection. To overcome this problem, we designed a more sensitive diagnostic method. Two monoclonal antibodies (MAbs 1C7 and 3B7) were prepared by immunizing mice with soluble protein from sonicated M. honghuensis spores. Immunofluorescence analysis revealed that MAb 1C7 specifically reacts with polar filaments from spores, whereas MAb 3B7 identified protein localized on the spore valves. The isotypes of MAb 1C7 and MAb 3B7 were IgM and IgG1, respectively. Results of Western blot analysis revealed that MAb 1C7 recognized 2 prominent protein bands with molecular weights of 130 and 180 kDa, while MAb 3B7 recognized a protein band of 28 kDa. Thus, in this study we have developed 2 MAbs that have the potential for efficient detection of M. honghuensis. Moreover, identification of MAb 1C7 and MAb 3B7 allows for further studies of the functions and biochemical composition of polar filament and spore surface antigens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Myxobolus/inmunología , Esporas Protozoarias/metabolismo , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Myxobolus/metabolismo , Esporas Protozoarias/inmunologíaRESUMEN
BACKGROUND: The circumsporozoite protein (CS protein) on the malaria parasites in mosquitoes plays an important role in sporogony in mosquitoes. The RTS,S/AS01 malaria vaccine candidate, which has shown significant efficacy against clinical malaria in a large Phase 3 trial, targets the Plasmodium falciparum CS protein, but the ability of serum from vaccinated individuals to inhibit sporogony in mosquitoes has not been evaluated. METHODS: Previously a double-blind, randomized trial of RTS,S/AS01 vaccine, as compared with rabies vaccine, in five- to 17-month old children in Tanzania was conducted. In this study, polyclonal human antibodies were purified from the pools of sera taken one month after the third vaccination. IgGs were purified from four pools of sera from 25 RTS,S/AS01 vaccinated children each, and two pools of sera from 25 children vaccinated with rabies vaccine each. The ability of antibodies to inhibit P. falciparum oocyst formation and/or sporogony in the mosquito host was evaluated by a standard membrane-feeding assay. The test antibodies were fed on day 0 (at the same time as the gametocyte feed), or on days 3 or 6 (serial-feed experiments). The oocyst and sporozoite counts were performed on days 8 and 16, respectively. In addition, two human anti-CS monoclonal antibodies (mAb) and a control mAb were also evaluated. RESULTS: Polyclonal anti-CS IgG preparations from RTS,S-vaccinated children tested at concentrations of 149-210 ELISA units (EU)/ml did not show significant inhibition in oocyst and sporozoite formation when the antibodies were fed with gametocytes at the same time, or later (serial-feed experiments). Similarly, anti-CS mAbs tested at 6,421 or 7,122 EU/ml did not show reduction in oocyst and sporozoite formation. CONCLUSIONS: This study does not support the concept that anti-CS antibodies induced by the RTS,S/AS01 vaccines in humans noticeably reduce malaria transmission by blocking P. falciparum sporozoite development or salivary gland invasion in mosquitoes when taken up during feeding.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Culicidae/parasitología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/inmunología , Esporas Protozoarias/crecimiento & desarrollo , Esporas Protozoarias/inmunología , Vacunas Sintéticas/inmunología , Animales , Sangre/inmunología , Culicidae/efectos de los fármacos , Femenino , Humanos , Inmunoglobulina G/inmunología , Lactante , Masculino , Carga de Parásitos , Plasmodium falciparum/efectos de los fármacos , Ensayos Clínicos Controlados Aleatorios como Asunto , Esporas Protozoarias/efectos de los fármacos , TanzaníaRESUMEN
BACKGROUND: Myxomycete spores are present in the outdoor air but have not been studied for allergenicity. OBJECTIVE: To determine whether patients with seasonal allergic rhinitis (SAR) symptoms are sensitized to myxomycete spores. METHODS: Myxomycete specimens were collected in the field. Nine species of myxomycetes were collected and identified: Arcyria cinerea, Ceratiomyxa fruticulosa, Fuligo septica, Hemitrichia clavata, Lycogala epidendrum, Metatrichia vesparium, Stemonitis nigrescens, Tubifera ferruginosa, and Trichea favoginea. Allergen extracts were made for each species. Protein content of each extract was measured by bicinchoninic acid assay. Protein electrophoresis was performed. Subjects with a history of SAR symptoms were enrolled, and allergy skin prick testing was performed with each extract. RESULTS: Protein content of the extracts ranged from 1.05 to 5.8 mg/mL. Protein bands were seen at 10 to 250 kD. Allergy prick testing was performed in 69 subjects; 42% of subjects had positive prick test results for at least 1 myxomycete extract, with 9% to 22% reacting to each extract. Five of the 12 subjects who tested negative for all allergens on the standard aeroallergen panel had positive prick test results for myxomycetes. CONCLUSION: Forty-two percent of subjects with SAR were sensitized to myxomycete spores. A significant subset of subjects who had SAR symptoms and otherwise negative skin test results showed sensitization to myxomycetes. These spores are present in the outdoor air during the summer and autumn and might be significant aeroallergens.
Asunto(s)
Antígenos de Protozoos/inmunología , Mixomicetos/inmunología , Proteínas Protozoarias/inmunología , Rinitis Alérgica Estacional/inmunología , Esporas Protozoarias/inmunología , Adolescente , Adulto , Contaminantes Atmosféricos/inmunología , Alérgenos/inmunología , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Rinitis Alérgica Estacional/diagnóstico , Pruebas Cutáneas , Adulto JovenRESUMEN
Giardia is one of the most common human enteric parasites that continue to be a major cause of diarrheal disease globally. Wastewater is an important source of Giardia transmission, and control of the pathogen by appropriate treatment of wastewater would limit its transmission. In this study the occurrence of Giardia cysts at various stages of the wastewater treatment plants was monitored for a period of 18 mo. Using immunomagnetic separation and immunofluorescence with monoclonal antibodies, cysts were detected in all samples throughout the sampling period at a concentration ranging from 50 to 7548 cysts/L. The overall removal efficiency of the cysts in the treatment plants was 78%. Seasonal analyses of results revealed that the pathogens (cysts) were most prevalent in influents and effluents during autumn and winter.
Asunto(s)
Giardia/aislamiento & purificación , Esporas Protozoarias/aislamiento & purificación , Aguas Residuales/parasitología , Microbiología del Agua , Purificación del Agua , Animales , Monitoreo del Ambiente , Técnica del Anticuerpo Fluorescente , Alemania , Giardia/fisiología , Esporas Protozoarias/inmunologíaRESUMEN
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes opportunistic disease, particularly in immunocompromised individuals. Central to its transmission and pathogenesis is the ability of the proliferative stage (tachyzoite) to convert into latent tissue cysts (bradyzoites). Encystment allows Toxoplasma to persist in the host and affords the parasite a unique opportunity to spread to new hosts without proceeding through its sexual stage, which is restricted to felids. Bradyzoite tissue cysts can cause reactivated toxoplasmosis if host immunity becomes impaired. A greater understanding of the molecular mechanisms orchestrating bradyzoite development is needed to better manage the disease. Here, we will review key studies that have contributed to our knowledge about this persistent form of the parasite and how to study it, with a focus on how cellular stress can signal for the reprogramming of gene expression needed during bradyzoite development.
Asunto(s)
Interacciones Huésped-Parásitos , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Toxoplasmosis/parasitología , Animales , Humanos , Esporas Protozoarias/crecimiento & desarrollo , Esporas Protozoarias/inmunología , Esporas Protozoarias/patogenicidad , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis Animal/inmunologíaRESUMEN
BACKGROUND: Malaria is a significant health problem causing morbidity and mortality worldwide. Vaccine development has been an imperative for decades. However, the intricacy of the parasite's lifecycle coupled with the lack of evidence for robust infection-induced immunity has made vaccine development exceptionally difficult. OBJECTIVE: To review some of the key advances in the field and discuss potential ways forward for a whole-organism vaccine. METHODS: The authors searched PubMed using the words 'malaria and vaccine'. We searched for manuscripts detailing antigen characterisation and vaccine strategies with emphasis on subunit versus whole-parasite approaches. Abstracts were selected and relevant articles are discussed. The searches were not restricted by language or date. CONCLUSIONS: The early cloning of malaria antigens has fuelled rapid development of subunit vaccines. However, the disappointing results of clinical trials have resulted in reappraisal of current strategies. Whole-parasite approaches have re-emerged as an alternative strategy. Immunization using radiation or genetically attenuated sporozoites has been shown to result in sterile immunity and immunization with blood-stage parasites curtailed by antimalarials has demonstrated delayed parasitemia in rodent models as well as in human malaria.
Asunto(s)
Antígenos de Protozoos , Vacunas contra la Malaria , Plasmodium/inmunología , Animales , Antígenos de Protozoos/genética , Diseño de Fármacos , Humanos , Esporas Protozoarias/inmunología , Vacunas de SubunidadRESUMEN
In determining the effective vaccine spore dose of a low-virulence strain of Loma salmonae to limit microsporidial gill disease in trout, we found that fish receiving 10(3) to 10(5) killed spores had the best protection against experimental infection, with 85% fewer xenomas in their gills than in the controls. Intraperitoneal delivery of the vaccine was effective, and the addition of adjuvant did not improve vaccine performance against this disease-causing microsporidian.
Asunto(s)
Enfermedades de los Peces/inmunología , Loma/inmunología , Microsporidiosis/veterinaria , Oncorhynchus mykiss/inmunología , Vacunas/inmunología , Administración Oral , Animales , Enfermedades de los Peces/parasitología , Enfermedades de los Peces/prevención & control , Explotaciones Pesqueras , Branquias/parasitología , Microsporidiosis/inmunología , Microsporidiosis/parasitología , Oncorhynchus mykiss/parasitología , Organismos Libres de Patógenos Específicos , Esporas Protozoarias/inmunología , Factores de Tiempo , Vacunas/administración & dosificaciónRESUMEN
A monoclonal antibody (mAb) 3C2, against an exospore protein of the microsporidium Nosema bombycis (N. bombycis) was prepared, and its effects on microsporidial germination and reproduction in vitro were studied. MAb 3C2 was effective in inhibiting the germination and subsequent infection of Bombyx mori cells compared to the control mAb. The antigen was isolated by 2-dimensional gel electrophoresis. Immunoblotting revealed it to be an 84 kDa protein corresponding to pI (7.2) on the 2-D gel. The present results suggest that the antibodies can be used for diagnostic purposes and for developing new therapeutic strategies in controlling microsporidian diseases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Nosema/efectos de los fármacos , Nosema/fisiología , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos BALB C , Nosema/crecimiento & desarrollo , Reproducción/efectos de los fármacos , Reproducción/fisiología , Esporas Protozoarias/efectos de los fármacos , Esporas Protozoarias/inmunología , Esporas Protozoarias/fisiología , Factores de TiempoRESUMEN
Species of the phylum Myxozoa are common parasites of fish and can cause severe losses in cultured species. Although a number of myxozoan life-cycles have now been elucidated, little is known about the biology of these organisms in the fish host. Monoclonal antibody B4 raised to the myxozoan Tetracapsuloides bryosalmonae has been previously noted to react with a number of species infecting fish kidney. We present the results of a survey of 55 myxosporean species that determined that this antibody detects an antigen on the spore surface of 33 of these species in the genera Myxobolus, Sphaerospora and Thelohanellus. However, there appears to be no clear relationship between those spores that contain the MAb B4 reactive antigen and the host or organ in which they are detected. The antigen appears to be synthesized in the plasmodial cytoplasm and is intimately associated with the surface of the spore capsules and, where present, the mucus envelope. The nature of this envelope is further discussed in relation to its formation and distinctive properties.
Asunto(s)
Antígenos de Superficie/inmunología , Eucariontes/inmunología , Enfermedades de los Peces/parasitología , Estadios del Ciclo de Vida/inmunología , Animales , Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Superficie/metabolismo , Encéfalo/parasitología , Cartílago/parasitología , Eucariontes/clasificación , Eucariontes/ultraestructura , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/patología , Peces , Branquias/parasitología , Riñón/parasitología , Microscopía Electrónica/veterinaria , Microscopía Electrónica de Rastreo/veterinaria , Esporas Protozoarias/inmunología , Esporas Protozoarias/ultraestructuraRESUMEN
Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.
Asunto(s)
Enfermedades de los Peces/inmunología , Loma/inmunología , Microsporidiosis/veterinaria , Oncorhynchus mykiss/inmunología , Vacunas/inmunología , Administración Oral , Animales , Enfermedades de los Peces/parasitología , Enfermedades de los Peces/prevención & control , Explotaciones Pesqueras , Branquias/parasitología , Microsporidiosis/inmunología , Microsporidiosis/prevención & control , Oncorhynchus mykiss/parasitología , Organismos Libres de Patógenos Específicos , Esporas Protozoarias/inmunología , Factores de Tiempo , Vacunas/administración & dosificaciónRESUMEN
Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 x 10(5) individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M. rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M. rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Eucariontes/genética , Enfermedades de los Peces/parasitología , Carpa Dorada/parasitología , Infecciones Protozoarias en Animales/parasitología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/genética , Afinidad de Anticuerpos , Antígenos de Protozoos/inmunología , China , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Eucariontes/inmunología , Femenino , Enfermedades de los Peces/inmunología , Vectores Genéticos/análisis , Vectores Genéticos/genética , Hibridomas , Ratones , Ratones Endogámicos BALB C , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Infecciones Protozoarias en Animales/inmunología , Sensibilidad y Especificidad , Esporas Protozoarias/genética , Esporas Protozoarias/inmunología , Esporas Protozoarias/aislamiento & purificaciónRESUMEN
Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Enterocytozoon/inmunología , Microsporidiosis/diagnóstico , Microsporidiosis/inmunología , Animales , Especificidad de Anticuerpos , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/aislamiento & purificación , Reacciones Cruzadas , Diarrea/diagnóstico , Diarrea/inmunología , Diarrea/parasitología , Enterocytozoon/crecimiento & desarrollo , Heces/parasitología , Femenino , Humanos , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos BALB C , Esporas Protozoarias/inmunologíaRESUMEN
The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble. The potential role of the surface components in initiating infection was also discussed.
Asunto(s)
Formación de Anticuerpos , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Microsporida , Microsporidiosis/veterinaria , Perciformes , Esporas Protozoarias/aislamiento & purificación , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Microsporidiosis/inmunología , Esporas Protozoarias/inmunologíaRESUMEN
We examined the effects of both fish age and size on the development of resistance to whirling disease in Erwin strain rainbow trout. Previously, we demonstrated that juvenile rainbow trout became resistant to development of the disease when first exposed to triactinomyxons of the parasite Myxobolus cerebralis at about 9 wk post-hatch when raised at 12 degrees C, but ages and sizes of fish used in that experiment were confounded (Ryce EKN, Zale AV, MacConnell E [2004] Dis Aquat Org 59:225-233). In this study, rainbow trout of the same age and different sizes, and the same size and different ages, were exposed to the parasite to distinguish the influences of age and size. Fish were reared at 3 different water temperatures prior to exposure to produce groups with different growth rates and were exposed to the parasite at 7 or 9 wk post-hatch. Disease severity was affected by both age and size at first exposure, but the effects were not independent. An increase in fork length from 36 to 40 mm among fish exposed at 7 wk post-hatch did not confer increased resistance, but the same increase in size at 9 wk post-hatch did. Similarly, an increase in age from 7 to 9 wk post-hatch among fish exposed at 36 mm fork length did not confer increased resistance, but the same increase in age at 40 mm did. Rainbow trout must be both 9 wk post-hatch or older and at least 40 mm in fork length at time of exposure to exhibit enhanced resistance to whirling disease. Resistance to disease was not associated with the level of skeletal ossification.
Asunto(s)
Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Inmunidad Innata/inmunología , Oncorhynchus mykiss , Infecciones Protozoarias en Animales/inmunología , Factores de Edad , Animales , Tamaño Corporal , Huesos/patología , Enfermedades de los Peces/mortalidad , Infecciones Protozoarias en Animales/mortalidad , Esporas Protozoarias/inmunología , Coloración y Etiquetado , Natación/fisiología , TemperaturaRESUMEN
Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 microm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Especificidad de Anticuerpos , Enterocytozoon/aislamiento & purificación , Enterocytozoon/fisiología , Heces/parasitología , Esporas Protozoarias/aislamiento & purificación , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Centrifugación por Gradiente de Densidad/métodos , Enterocytozoon/inmunología , Humanos , Ratones , Microsporidiosis/parasitología , Conejos , Esporas Protozoarias/inmunologíaRESUMEN
Optimal protection against malaria may require induction of high levels of protective antibody and CD8(+) and CD4(+) T cell responses. In humans, malaria DNA vaccines elicit CD8(+) cytotoxic T cells (CTL) and IFNgamma responses as measured by short-term (ex vivo) ELISPOT assays, and recombinant proteins elicit antibodies and excellent T cell responses, but no CD8(+) CTL or CD8(+) IFNgamma-producing cells as measured by ex vivo ELISPOT. Priming with DNA and boosting with recombinant pox virus elicits much better T cell responses than DNA alone, but not antibody responses. In an attempt to elicit antibodies and enhanced T cell responses, we administered RTS,S/AS02A, a partially protective Plasmodium falciparum recombinant circumsporozoite protein (CSP) vaccine in adjuvant, to volunteers previously immunized with a P. falciparum CSP DNA vaccine (VCL-2510) and to naïve volunteers. This vaccine regimen was well tolerated and safe. The volunteers who received RTS,S/AS02A alone had, as expected, antibody and CD4(+) T cell responses, but no CD8(+) T cell responses. Volunteers who received PfCSP DNA followed by RTS,S/AS02A had antibody and CD8(+) and CD4(+) T cell responses (Wang et al., submitted). Sequential immunization with DNA and recombinant protein, also called heterologous prime-boost, led to enhanced immune responses as compared to DNA or recombinant protein alone, suggesting that it might provide enhanced protective immunity.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Vacunas de ADN/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antiprotozoarios/análisis , Linfocitos B/inmunología , Femenino , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Esquemas de Inmunización , Vacunas contra la Malaria/efectos adversos , Masculino , Esporas Protozoarias/inmunología , Linfocitos T/inmunología , Vacunas de ADN/efectos adversos , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
Encephalitozoon cuniculi continues to pose a problem for immunocompromised patients. Previous studies from our laboratory have elucidated the importance of the CD8(+) T cell subset in the protection against systemic parasite infection. There have been no studies related to the mucosal immunity induced against this orally acquired pathogen. In the present study, the immune response generated in the gut after oral E. cuniculi infection was evaluated. An early and rapid increase of the intraepithelial lymphocyte (IEL) population of orally infected animals was observed. This increase in the IEL population started as early as day 3 and peaked at day 7 postinfection with persistent elevation thereafter. At day 7 postinfection, IELs expressed strong cytokine messages (IFN-gamma and IL-10) and were highly cytotoxic for parasite-infected syngeneic macrophages. At an E:T ratio of 80:1, these cells were able to cause >60% Ag-specific target cell lysis. A significant increase in the CD8alphaalpha subset of IEL in response to an oral E. cuniculi infection was observed. To the best of our knowledge, such an early expansion of an IEL population exhibiting strong ex vivo cytotoxicity has not been reported with infectious models. These data suggest that IELs act as important barriers for multiplication of this organism leading to the successful resolution of infection. The protective role of IELs may be due both to their inflammatory (IFN-gamma production and cytotoxic response) as well as immunoregulatory (IL-10 production) properties.
Asunto(s)
Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/inmunología , Encephalitozoon cuniculi/inmunología , Encefalitozoonosis/inmunología , Mucosa Intestinal/inmunología , Activación de Linfocitos/inmunología , Administración Oral , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , División Celular/inmunología , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Susceptibilidad a Enfermedades , Encefalitozoonosis/mortalidad , Encefalitozoonosis/prevención & control , Femenino , Interferón gamma/biosíntesis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/trasplante , Depleción Linfocítica , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Esporas Protozoarias/inmunologíaRESUMEN
Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 micro m and height of 14 microm, and possessed two soft valves surrounding two spherical polar capsules (2 microm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 microm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Sondas de ADN , Eucariontes/fisiología , Oncorhynchus mykiss/parasitología , Esporas Protozoarias/inmunología , Orina/parasitología , Animales , Briozoos/parasitología , Eucariontes/genética , Eucariontes/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Interacciones Huésped-Parásitos , Enfermedades Renales/inmunología , Enfermedades Renales/parasitología , Enfermedades Renales/veterinaria , Infecciones Protozoarias en Animales/inmunología , Infecciones Protozoarias en Animales/parasitología , Esporas Protozoarias/genética , Esporas Protozoarias/aislamiento & purificaciónRESUMEN
The possible role of humoral antibodies in the immune response to microsporidiosis was studied using a novel anti-exospore monoclonal antibody (MAb) P5/H1 which recognised Encephalitozoon cuniculi. The effect of the P5/H1 MAb on microsporidial growth in vitro resulted in a reduction of the numbers of E. cuniculi spores in a Vero E6 cell-line culture. This reduction in the number of infected cells and the decrease of intracellular spores in infected cells was found when MAb P5/H1 was present in cultures, compared to cultures with an irrelevant isotype control MAb. Moreover, the presence of P5/H1 MAb increased the number of phagocytosed spores in macrophage cultures, and increased the activation of macrophages measured by nitrite oxide production. These results suggest a possible partial role of specific humoral antibodies in the protection against E. cuniculi infection.