Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water / Produção de anticorpos policlonais anti-Cryptosporidium e padronização da imunofluorescência direta para a detecção de oocistos na água

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20 percent of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20 por cento das amostras; não houve reação cruzada com cistos de Giardia duodenalis.

Immunoperoxidase technique using an anti-Leishmania (L. ) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis / Técnica da imunoperoxidase utilizando um soro hiperimune anti-Leishmania (L. ) chagasi no diagnóstico da leishmaniose tegumentar americana confirmada por cultura

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

O presente estudo relata a produção do soro policlonal de coelho anti-Leishmania (L.) chagasi, a padronização da técnica de imunohistoquímica (IHQ) e sua aplicação em lesões de leishmaniose cutânea (LC) diagnosticadas por isolamento de Leishmania sp. em cultura. Foram examinados 30 fragmentos de lesões ativas de LC e 10 fragmentos de lesões de etiologia fúngica, utilizados como grupo controle. A IHQ mostrou-se mais sensível na detecção de amastigotas que a coloração em hematoxilina-eosina (HE), sendo positiva em 24 fragmentos de LC (80%) e ao passo que a HE foi positiva em 16 (53%) (p = 0,028). A IHQ também marcou diferentes espécies de fungos causadoras de micoses cutâneas. Adicionalmente, verificou-se positividade no citoplasma de células mononucleares e células endoteliais. Entretanto, esse achado esteve presente no grupo controle. Conclui-se que o método de IHQ apresentou boa sensibilidade na detecção de formas amastigotas.

Alpha 2 macroglobulin activity in rats infected with Typanosoma lewisi and treated with cyclophosphamide and its effect on the malignancy of the disease.

BACKGROUND & OBJECTIVES: Trypanosoma lewisi is a common, flagellated parasite of the rat. Our previous study showed that rabbits injected with serum collected from rats infected with Trypanosoma lewisi and treated with cyclophosphamide (CyI) produced high levels of antibodies against a new protein in the CyI rat serum. RESULTS: In the present study, this protein was characterised as alpha2 macroglobulin (alpha2M) and the kinetics of its production and its influence on the malignancy of the disease were determined. In rats infected with T. lewisi, alpha2M was first demonstrated and peaked on the second day post-infection (972 microg/ml) and then reduced gradually, reaching a level of 32 microg/ml on the eighth day post-infection. However, in the CyI rats the level of alpha2M was gradually increased as the disease progressed, reaching a level of 890 microg/ml on the eighth day post-infection. Injection of both crude and purified alpha2M into rats infected with T. lewisi led to increased parasitaemia. INTERPRETATION & CONCLUSION: The present study suggests that increased levels of alpha2M in the CyI rats contribute to the malignancy of the disease.

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Epítopes de antígenos compartidos entre Plasmodium falciparum y Anopheles albimanus fueron identificados. Diferentes grupos de conejos fueron inmunizados con: extracto crudo de mosquito hembra de An. albimanus (EAaH), glóbulos rojos infectados con P. falciparum (EPfs) y la vacuna antimalárica sintética SPf66. Los anticuerpos policlonales producidos en conejos fueron evaluados por ELISA, inmunoensayo simultáneo de múltiples antígenos (MABA) e Immunoblotting. Todos los extractos resultaron inmunogénicos cuando se evaluaron por ELISA, MABA e Immunoblotting. Diez moléculas fueron identificadas en los mosquitos hembras y diez en los antígenos de P. falciparum por los sueros autólogos. El patrón electroforético por SDS-EGPA fue diferente para los tres antígenos evaluados. La reactividad cruzada de moléculas entre An. albimanus y P. falciparum fue demostrada por ELISA, MABA e Immunoblotting. Anticuerpos anti-P. falciparum y anti-SPf66 reconocieron diez y cinco componentes respectivamente en el extracto crudo de anofelinos (EAaH). Asimismo, sueros inmunes contra An. albimanus hembra identificaron cuatro moléculas en el extracto del antígeno de P. falciparum. Hasta el presente, este es el primer estudio en el que se demuestra la presencia de antígenos compartidos entre anofelinos y los parásitos de malaria. Este hallazgo podría ser de relevancia para el diagnóstico, vacunas e interpretación de la fisiopatología de la respuesta inmunitaria en malaria.

Cytokine and antibody responses of reactivated murine toxoplasmosis upon administration of dexamathasone

Toxoplasma gondii has been shown to result in life-threatening encephalitis in immunocompromised patients after reactivation of dormant parasites. In order to obtain information on immune responses related to this phenomenon, BALB/c mice were infected with 25 cysts of the 76K strain of T. gondii, then, treated orally with dexamethasone (Toxo/Dexa-treated group) in order to reactivate the chronic toxoplasmosis. None of the T. gondii-infected mice died during the experimental periods, whereas the Toxo/Dexa-treated mice evidenced a significant attenuation of survival periods. Toxoplasma-specific IgG2a, IgA and IgM titers in sera were significantly depressed in the Toxo/Dexa-treated mice; however, the IgG1 sera titers were similar to those seen in the Toxoplasma-infected mice. The percentages of CD4+ and CD8 alpha + T cells in the Toxo/Dexa-treated mice were significantly reduced 2 weeks after dexamethasone treatment. IFN-gamma and IL-10 production levels in the Toxo/Dexa-treated mice were depressed significantly, whereas IL-4 production was increased temporarily. The expression levels of the Toxoplasma-specific P30 and B1 genes were found to have been increased in the Toxo/Dexa-treated mice in comparison with the Toxoplasmainfected mice. Collectively, the findings of this study demonstrate that reactivation of murine toxoplasmosis as the result of dexamethasone treatment induced a depression in Th1 immune responses, whereas Th2 immune responses were not significantly influenced.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani.

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.

Respuesta humoral específica en ratones Balb/c inoculados con una librería genómica de expresión de Trypanosoma cruzi / Specific humoral response in Balb/c mice inoculated with a genome library of expression of Trypanosoma cruzi

A genomic expression library of Trypanosoma cruzi (T. cruzi) was made using plasmid pcDNA3 as a vector, with which male mice from the Balb/c isogenic line were intramuscullary inoculated. It was used a positive control group that was administered soluble antigens of T. cruzi. Other 2 groups received genomic and plasmid DNA, respectively. One group was not immunized. Weekly blood samples were obtained from all the animals until the fourth week and 2 weeks after reimmunization to study the response of specific antibodies against the microorganism antigens by an indirect immunoenzymatic assay (ELISA). It was observed a significant increase of specific antibodies in the animals reimmunized with 50 micrograms of the library, as well as in the group immunized with soluble antigens of T. cruzi.

Serological evaluation of malaria patients in Thailand: antibody response against electrophoresed antigenic polypeptides of Plasmodium falciparum.

It was reported that a 47kDa antigenic polypeptide of Plasmodium falciparum had been strongly presented by the sera from 1) imported Japanese malaria patients with severe symptoms and 2) symptomatic and parasitemic inhabitants in endemic areas in the Sudan, Malaysia and the Philippines. In the present study, we observed the reactivity of the sera from falciparum malaria patients who had been hospitalized in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, and compared the antibody response against the 47kDa antigenic polypeptide according to the severity of the patients. It was observed that antibodies to this molecule were more commonly shared in sera from severer patients, although the IFAT titers against the whole P. falciparum parasite antigen were lower in the group, which suggested that this antibody against the 47kDa molecule was playing a specific role at a severe stage of the infection. Determination of the immunological features of the antigenic molecules of parasites by this type of sero-epidemiological study will provide a new assay system for evaluation of immune status of individuals in different severity and suggest a way of vaccine development.

Giardia duodenalis: Analysis of humoral immune response in experimentally infected gerbils (Meriones unguiculatus)

In this work, we have analyzed the humoral immune response in Mongolian gergils infected with Giardia duodenalis trophozoites of strains P-1 ad WB. The course of infection in the animals was assessed by monitoring cyst shedding in feces, and serum samples were collected at weekly intervals to measure antibody levels by ELISA. Parallel studies were carried out to determine the patterns of total and surface antigens of the parasite recognized by antibodies using Western blot and radioimmunoprecipitation (RIP) assays with the use of homospecific enzyme conjugates. Typical patterns of cyst shedding were observed in the infected animals and cyst numbers per grams of feces were consistently higher in gerbils infected with WB strain. Antibody levels to G. duodenalis antigens were observed by week 2 post-infection and were still detectable 4 months after infection. G. duodenalis antigen showed a complex but quantitatively and qualitatively different recongnition pattern by infection-induced antibodies in Western blot assays which related to infecting strain. However, RIP assays showed a more restricted and common pattern of recognition of surface antigens from either strain. Taken together, the data obtained in this study provides further information regardin direct comparisons among infecting strain, pattern of infectivity, and host immune response toward G duodenalis antigens in the gerbil model

Dynamics of natural antibody responses to malaria parasite surface proteins in the intermediate rainfall zone of Sri Lanka.

Antibodies against repetitive epitopes on Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins and epitopes on the 45 kDa and 185-200 kDa P. falciparum merozoite surface proteins were measured by radioimmunoassay in a two year longitudinal study in Nikawehera village located in the intermediate rainfall zone of Sri Lanka. The prevalence and concentrations of specific antibodies were in many, but not all instances, greater in adults than in children who were aged 7-15 yr at the beginning of the study. The concentrations and prevalence of antibodies were associated with malaria transmission levels previously determined from entomological and hospital admission data in the area. Antibody responses to epitopes on different P. falciparum antigens, two different epitopes within the 185-200 kDa merozoite surface protein and between the P. falciparum and P. vivax CS repeats were significantly correlated. Antibody concentrations against a conserved epitope in the 185-200 kDa protein were significantly higher in P. falciparum infected individuals than in non-parasitaemic subjects. Antibody concentration and prevalences in Nikawehera were lower than at Weheragala, a site located 70 km away in the dry zone of Sri Lanka. It is postulated that lower levels of immunity in the population in areas such as Nikawehera, that are adjacent to more highly malaria endemic areas, may promote epidemics when conditions favour transmission.

Trypanocidal activity of 1,2,3,4-Tetrahydrocarbazoles

Uma nova série de tetrahidrocarbazois di-substituidos e sua atividade tripanomicida foi avaliada quanto à sua habilidade para lisar formas epimastigotas de T. cruzi marcadas com trítio. Os doze compostos examinados nestas séries mostraram-se ativos

A comparative study of different isolated fractions of Entamoeba histolytica in amoebic liver abscess.

Fifty six patients with amoebic liver ubscess were assessed for antibody production and lymphokine release using different antigenic fractions such as sephadex G-200 eluted fraction-I (F-I), shed of bile salt treated amoebae (SBSTA), amoebic membrane glycoprotein (AMG), amoebic RNA and whole amoebic lysate (WAL). Antibody production in response to WAL, F-I, SBSTA, AMG and RNA was assessed by indirect haemagglutination assay. Five and 53 fold increased titres of IHA was observed with F-I and SBSTA respectively compared to WAL. The difference between mean titres of F-I, SBSTA and WAL was found to be highly significant (P < 0.001). Amoebic RNA did not show pronounced antibody titres. Lymphokine release by T lymphocytes in response to F-I, SBSTA, AMG, RNA and WAL was assessed by leukocyte migration inhibition (LMI) test. A highly increased release of LMIF by RNA, AMG, F-I and SBSTA was observed compared to whole amoebic lysate. The difference between the means of the purified fractions and WAL, to release of LMIF was found to be highly significant (P 0.001). These findings suggest that SBSTA, F-I and AMG fractions might have an important role as a potent antigen in inducing humoral and cell mediated immunity in patients with amoebic liver abscess and these findings also confirm the multiple antigenicity of E. histolytica.

Correlation between antiamoebic IgG and autoreactive anti-IgG in amoebic liver abscess cases.

IgG isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography was used for detection of its own antibodies and correlated with the antiamoebic antibody titres in amoebic liver abscess cases. The mean titres with standard deviation of the self reactive antibodies to serum IgG both ALA cases and healthy controls show a highly significant difference, and antiamoebic antibody titres (IgG) are very much correlated with the autoreactive anti IgG titres in amoebic liver abscess cases. This correlation suggests that as antiamoebic IgG levels reach to its maximum, autoreactive anti IgG are produced to switch off antiamoebic anti IgG production in amoebic liver abscess cases.

Kinetics of specific IgM in patients of hepatic amoebiasis.

Entamoeba histolytica (EH) specific IgM was measured in 54 patients with diagnosed amoebic liver abscess (ALA), 13 with non-suppurative hepatic amoebiasis (NSHA) and 50 controls. The mean levels of EH specific IgM, estimated by ELISA were significantly raised in patients of invasive amoebiasis (both ALA and NSHA) compared to controls (P less than 0.05). EH specific IgG was also raised in both groups of patients. Follow up of patients with ALA showed a significant decline (P less than 0.05) in the specific IgM levels three months after treatment while the specific IgG antibodies persisted in high titres (1:160). Only four patients of NSHA could be followed up and all showed a decline in specific IgM levels. Raised specific serum IgM seems to be an indicator of active (invasive) amoebiasis.

Evolution of subpatent parasitaemia in Trypanosoma cruzi chronically infected mice with the help of a cyclophosphamide amplification transfer assay

Avaliamos o potencial do ensaio classico de subinoculacao, modificado pelo tratamento com ciclofosfamida dos animais receptores, na deteccao de parasitemias ocultas em camundongos com infeccao cronica pelo Trypanosoma cruzi. O ensaio, alem de simples, mostrou ter uma alta sensibilidade; assim, utilizando-se parasitas da fase aguda, o tratamento com ciclofosfamida revelou parasitemias em 53,8 por cento dos animais infectados com um tripanosoma da cepa y, e em 20 por cento dos animais infectados com um tripanosoma da cepa CL. O tratamento com ciclofosfamida aumentou a sensibilidade do ensaio de subinoculacao nas infeccoes pela cepa CL, e resultou em igual sensibilidade quando utilizada a cepa Y. Nos camundongos de fase cronica, obtidos a partir de diversos esquemas de imunoprofilaxia (BCG, soro de camundongo imune) ou quimioterapia, o ensaio revelou parasitemias ocultas em 99 por cento dos animais. Auxiliados pelo metodo da subinoculacao-ciclofosfamida estudamos no espaco de um ano a evolucao das parasitemias ocultas em um grupo de camundongos infectados que soberviveram a fase aguda pelo tratamento com Benzonidazol. O ensaio revelou parasitemias ocultas em 100 por cento dos animais. Entretanto, padroes continuos e descontinuos de positividade puderam ser detectados.

Characterization of surface associated antigens of axenic Giardia lamblia trophozoites & their recognition by human sera.

Two surface associated antigens (GLSA-82 and GLSA-56) of axenically grown G. lamblia trophozoites (PI strain) were affinity purified from its sonic extract. Both GLSA-82 and GLSA-56 were heat labile, sensitive to treatment with pronase, trypsin and were also sodium metaperiodate modifiable as assessed by micro ELISA. Lectin binding studies revealed that GLSA-82 specifically bound concanavalin A and pokeweed mitogen, and had alpha-methyl mannoside and n-acetyl-B-d-glucosamine sugar moieties. However, GLSA-56 selectively bound Ricinus communis agglutinin and phytohaemagglutinin, and had B-d-galactose and n-acetyl-B-d-galastosamine as sugar moieties. Human sera obtained during acute G. lamblia infection recognised GLSA-82 and GLSA-56 antigens. However, the antibody levels to GLSA-82 were significantly lower (P less than 0.05) during active giardiasis infection. Such surface associated antigens may be target of antiparasitic immune responses and thus, may modulate disease processes.

The immunodominant surface antigen of Plasmodium gallinaceum is present in both the salivary gland and oocyst sporozoites

1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midadgusts containing oocysts (OoS). 2. All of the 15 MAbs teted (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoiter precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) patten identical to that produced with serum from mice lyperimmunized with viable intacts sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecutlar weight 78 and 64KDa. 4. No difference in the WB pattern was observed when-or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similary was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel