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Con el fin de investigar los niveles de anticuerpos y antígenos en muestras de lavado broncoalveolar (LBA) y suero, ratas hembras inmunosuprimidas o no inmunosuprimidas e infectadas experimentalmente con Strongyloides venezuelensisy sus crías fueron utilizadas. Para ello, el ensayo inmunoabsorbente ligado a enzimas (ELISA) se realizó usando extractos alcalinos de larvas y anti-L3 anticuerpo policlonal producido en conejos. Los resultados mostraron que las crías de las hembras inmunosuprimidas fueron significativamente mayores para la detección de IgG en el suero y LBA. Por otra parte, la detección de antígeno en muestras de suero también fue más evidente en las crias de ratas inmunosuprimidas. Así pues, aquí reforzamos la ruta transmamaria de la infección por Strongyloides en el modelo experimental de infección.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A estrongiloidíase humana é uma parasitose intestinal causada pelo nematódeo Strongyloides stercoralis com distribuição mundial e predominância em áreas tropicais e subtropicais. O diagnóstico parasitológico apresenta baixa sensibilidade devido à intermitente eliminação de larvas nas fezes e por aproximadamente 50% dos indivíduos acometidos serem assintomáticos. O diagnóstico sorológico, apesar de mais sensível, apresenta reatividade cruzada com outras parasitoses. A busca por antígenos que apresentem bons padrões diagnósticos, se faz necessário, e o uso de modelos experimentais podem auxiliar na obtenção de antígenos heterólogos aplicáveis ao diagnóstico. Strongyloides venezuelensis é frequentemente utilizado para avaliar a interação parasito-hospedeiro, os aspectos moleculares durante a infecção, a eficácia de novas terapias e outras características imunológicas da estrongiloidíase. O uso de produtos de excreção/secreção (E/S) de larvas filarioides de S. venezuelensis no diagnóstico da estrongiloidiase, apresenta-se como uma ferramenta promissora. O objetivo da pesquisa foi obter produtos de E/S de larvas filarioides de S. venezuelensis, aplicá-los no diagnóstico da estrongiloidiase e identificar e caracterizar parcialmente as proteínas imunorreativas. Testes ELISA para detecção de IgG anti-Strongyloides foram realizados, com amostras de soro de ratos experimentalmente infectados com S. venezuelensis ou de soro humano, utilizando extrato total salino (TS) e produtos E/S (RPMI E/S e PBS E/S). Immunoblotting foi realizado para detectar as frações proteicas imunorreativas, e posteriormente foram analisadas por espectrometria de massas e bioinformática. Todas as preparações detectaram IgG anti-Strongyloides com destaque para o RPMI E/S (Se = 83,3% e Es = 100%), na análise com soro de rato e PBS E/S (Se = 81,7% e Es = 88,9%) em amostras de soro humano. No immunoblotting foram identificadas bandas proteicas específicas com aproximadamente 17 kDa nas três preparações. Na análise de bioinformática, foram identificadas proteínas contendo domínio CAP pertencente a superfamília CRISP (A0A0K0FJA1) nas três preparações, no TS uma proteína ligante de cálcio, RPMI E/S duas proteínas contendo domínio CAP e PBS E/S uma proteína não caracterizada da família ADF/Cofilin. Em sua maioria as proteínas identificadas são produtos de E/S, apresentando sequências preditas como epítopos de célula B. Conclui-se que os produtos de E/S apresentaram desempenho diagnostico superior ao TS, tanto na estrongiloidiase experimental quanto humana. Bandas proteicas de 17 kDA especificas para Strongyloides, apresentaram em sua composição, proteínas contendo domínio CAP. A construção dos modelos tridimensionais das proteínas e suas validações foram satisfatórias e permitiram entender como essas proteínas apresentam sua conformação. A predição de epítopos de célula B, pelo programa Bepipred 2.0, identificou sequências como epítopos de célula B, podendo serem utilizadas como possíveis ferramentas diagnosticas, como também em testes vacinais. Strongyloides venezuelensis EXCRETORY/SECRETORY PRODUCTS: APPLICATION TO STRONGYLOIDIASIS DIAGNOSIS AND CHARACTERIZATION OF IMMUNOREACTIVE PROTEINS. The Human strongyloidiasis is an intestinal parasitosis caused by the nematode Strongyloides stercoralis with worldwide distribution and predominance in tropical and subtropical areas. Parasitological diagnosis presents low sensitivity due to intermittent elimination of larvae in the faeces and approximately 50% of the affected individuals are asymptomatic. In the serological diagnosis, although more sensitive, cross reactivity still occurs with other parasitoses. The search for antigens that present greater specificity, if necessary, and the use of experimental models may aid in the production of heterologous antigens. Strongyloides venezuelensis is often used to evaluate host-parasite interaction, molecular aspects during infection, efficacy of new therapies, and other immunological characteristics of strongyloidiasis. Thus, use of excretory/secretory products (E/S) of filaroid larvae of S. venezuelensis in the diagnosis of strongyloidiasis presents itself as a promising tool. The objective of the research was to obtain E/S products of filaroid larvae of S. venezuelensis, apply them in the diagnosis of strongyloidiasis and to identify and partially characterize the immunoreactive proteins. ELISA tests for anti-Strongyloid IgG were carried out using samples of rat serum experimentally infected with S. venezuelensis or human serum using total saline extract (TS) and E/S products (RPMI E/S and PBS E/S). Immunoblotting was performed to detect the immunoreactive protein fractions and were later analyzed by mass spectrometry and bioinformatics. All preparations detected anti-Strongyloides IgG with emphasis on RPMI E/S (Se = 83.3% and Sp = 100%) on analysis with mouse serum and PBS E/S (Se = 81.7% and Sp = 88.9%) in human serum samples. In immunoblotting, specific protein bands with approximately 17 kDa were identified in the three preparations. In the bioinformatics analysis, proteins containing CAP domain belonging to the superfamily CRISP (A0A0K0FJA1) were identified in the three preparations, in TS a calcium binding protein, RPMI E/S two proteins containing CAP domain and PBS E/S an uncharacterized protein of the family ADF/Cofilin. Most of the identified proteins are E/S products, presenting predicted sequences as B cell epitopes. The E/S products presented superior diagnostic performance to TS, both in experimental and human strongyloidiasis. Protein bands of 17 kDa specific for Strongyloides, presented in their composition, proteins containing CAP domain. The construction of the three-dimensional models of the proteins and their validations were satisfactory and allowed to understand how these proteins present their conformation. Prediction of B-cell epitopes by the Bepipred 2.0 program identified sequences as B-cell epitopes and could be used as possible diagnostic tools as well as in vaccine trials. Tese (Doutorado)

Strongyloides venezuelensis is a model to study human strongyloidiasis, which infects wild rodents and shares common antigenic epitopes with Strongyloides stercoralis. This study aimed to evaluate parasitological and immunological parameters of prednisolone immunosuppression protocols in rats (Rattus novergicus) infected with S. venezuelensis. Rats were divided into six groups (n = 36): untreated and uninfected (-) or infected (+); oral treatment and uninfected (o-) or infected (o+); subcutaneous treatment and uninfected (sc-) or infected (sc+). For oral immunosuppression, 5 mg/mL of water diluted prednisolone were given five days before infection, and in the days 8 and 21 (for 5 days). For subcutaneous immunosuppression, 10 mg/kg of prednisolone were given daily. The infection was established by the subcutaneous injection of approximately 3,000 S. venezuelensis filarioid larvae per animal. All animals from the (+) and (o+) groups survived, while four rats from the (sc+) died prior to necropsy date. Parasitological analysis showed higher egg elimination in (o+) in comparison to (+) and (sc+) on 7, 13 and 26 days post infection (d.p.i.).The recovery of parasitic females at day 30 was significantly higher in (o+), compared to (+). The (+) and (o+) groups showed a clear increase in anti-S. venezuelensis IgG, IgG1 and IgG2 from 13th d.p.i. Oral immunosuppression led to a higher number of adult females and increased egg output while maintaining IgG and subclasses antibody levels comparable to the positive control.

Strongyloidosis is a neglected disease that affects millions of people around the world. The cases that particularly deserve attention are those related to hyperinfection, mainly in immunocompromised patients. In this sense, there is a need to improve the serological diagnosis of this helminth. The objective of this study was therefore to produce and characterize excretory/secretory (E/S) antigens of Strongyloides venezuelensis infective larvae (L3) for use as a heterologous antigen in the diagnosis of human strongyloidosis and other parasitic infection groups. Soluble antigenic preparations were produced as total saline extract (SE), E/S in Roswell Park Memorial Institute 1640 (RPMI) and E/S in phosphate buffered saline (PBS). The three antigenic preparations showed similar protein bands. An ELISA showed that the E/S antigens were profitable, easy to use, and more sensitive and specific than SE, eliminating cross-reactivity with other parasites in serum samples. The detection of anti-Strongyloides stercoralis in the sera of patients with strongyloidosis and those with immunosuppressive conditions using S. venezuelensis L3 larvae E/S antigens was satisfactory. RPMI and PBS E/S antigens were also superior in terms of specificity than SE.

One of the problems frequently faced in laboratory facilities is the possibility of the natural parasitic infection of lab animals, which can interfere with biomedical research results. The present study aimed to evaluate cross-reactivity among serum samples from Wistar rats (Rattus norvegicus) naturally infected with Syphacia muris and experimentally infected with Strongyloides venezuelensis. Forty rats were divided into four groups of ten animals each. Parasite load was evaluated by quantifying the adult worms from both helminthes species recovered from the intestines and the S. venezuelensis eggs eliminated in feces. Serological cross-reactivity by parasite-specific IgG detection was tested via enzyme linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT) and immunoblotting. The results demonstrated that the quantity of S. venezuelensis eliminated eggs and parthenogenetic females decreased significantly in cases of co-infection with S. muris. ELISA revealed 100% cross-reactivity of serum samples from both species against the opposing antigen. IgG cross-reactivity was confirmed by IFAT using tissue sections of S. venezuelensis larvae and adult S. muris. Immunoblotting showed that IgG antibodies from the sera of animals infected with S. muris recognized eight antigenic bands from S. venezuelensis saline extract and that IgG antibodies from the sera of animals infected with S. venezuelensis recognized seven bands from S. muris saline extract. These results demonstrate the serological cross-reactivity between S. muris and S. venezuelensis in infected rats.

In the present study, antigens from parthenogenetic females and eggs ofStrongyloides venezuelensis, or anti-parthenogenetic-female and anti-egg antigens were used to detect specific IgG and immune complex responses, respectively. Serum samples from experimentally infected immunocompetent and immunosuppressed rats were analysed on days 5, 8, 13 and 21 post-infection (dpi). An enzyme-linked immunosorbent assay (ELISA) was performed using alkaline parasite extract for specific IgG detection, and anti-parthenogenetic-female or anti-egg antigens for immune complex detection. The data were analysed using analysis of variance (ANOVA), followed by a Bonferroni test. When parthenogenetic female or egg extracts were used as antigens, specific IgGs were not detected in either immunocompetent or immunosuppressed rats. When anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used, immune complexes were detected for the duration of the infection in immunosuppressed animals and were only detected between 5 and 13 dpi in immunocompetent animals. The duration of infection was not significantly different between the immunocompetent and immunosuppressed groups when anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used. Parthenogenetic female extracts yielded significant differences between antibody and immune complex responses in immunocompetent rats from 5 to 13 dpi, but only on day 5 dpi in immunosuppressed rats. Exposure toS. venezuelensisegg extract yielded significant differences in both antibody and immune complex detection between immunocompetent and immunosuppressed rats for the duration of the infection. In conclusion, ELISA using alternative antigens may be a successful strategy for identifying immune complexes in serum samples and diagnosing active strongyloidiasis, particularly under conditions of immunosuppression.

This study aimed to compare three qualitative parasitological methods for the diagnosis ofSyphacia murisinfection in 30 Wistar rats (Rattus norvegicus) infected naturally. Methods of spontaneous sedimentation (Hoffman, Pons and Janer, or HPJ) and spontaneous flotation (Willis) for faecal samples and a method of taping (Graham) were performed and compared. The Graham and Willis methods were more sensitive than the HPJ method (PS. muriseggs in 100% of the samples. Eggs were detected in 83% and 60% of the samples using the Willis and HPJ methods, respectively. Method choice is important for screening for parasites of rats kept under laboratory conditions, as accurate diagnosis helps prevent future environmental contamination and infection. We concluded that the Graham method was the most efficient of those tested in this study for detection ofS. murisinfection in rats. This method is also rapid, inexpensive and practical, and should be implemented as a necessary measure for infection control.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais The nematode Strongyloides stercoralis responsible for strongyloidiasis diagnosed by detection of larvae in feces. However, the elimination of low larvae hampers the detection of disease particularly in cases of immunosuppression. Immunodiagnostic tests have been developed, but the difficulty of obtaining larvae of S. stercoralis for the production of homologous antigenic extract, thereby Strongyloides venezuelensis and their different evolutionary forms for the production of antigen extracts. The aim of this study was to evaluate the detection of antibodies anti S. venezuelensis against alkaline extracts of L3 larvae, parthenogenetic females or eggs in experimental strongyloidiasis associated with immunosuppression. Male rats were divided into two groups: non-immunosuppressed and immunosuppressed experimentally infected and serum from all animals were obtained at 0, 5th, 8th, 13th and 21th days post infection (d.p.i.). The test enzyme-linked immunosorbent assay (ELISA) was performed to compare the reactive potential of alkaline extracts of L3 larvae, parthenogenetic females and eggs of S. venezuelensis in rats non-immunosuppressed and immunosuppressed. Immunoblotting was also evaluated for detection of IgG anti S. venezuelensis in both groups front to alkaline of the three developmental forms of the parasite extracts. In all comparative ELISA extracts were skilled in detecting IgG S. venezuelensis. The extract of larvae showed reactivity from the 5th dpi ceasing at 21th d.p.i in the non-immunosuppressed group while the group immunosuppressed this reactivity remained at 21th d.p.i. immunoblotting showed the reactivity profile with immunoreactive fractions in immunosuppressed group from the 5th d.p.i. while the non-immunosuppressed group reactivity from 8th d.p.i. The alkali extracts of parthenogenetic females and eggs showed a similar profile of reactivity in both ELISA and immunoblotting. Immunoreactive protein fractions of 26 and 17 kDa present in the alkaline extract of larvae presented as important markers of infection in immunosuppressed animals. It is concluded that all alkali extracts have diagnostic potential in experimental strongyloidiasis especially the larval extract in cases of immunosuppression, and immunoblotting an important complementary diagnostic tool. O nematódeo Strongyloides stercoralis é responsável pela estrongiloidíase cujo diagnóstico é realizado pela detecção de larvas nas fezes. Porém a baixa eliminação de larvas dificulta a detecção da doença em especial em casos de imunossupressão. Testes imunodiagnósticos têm sido desenvolvidos, porém há dificuldade de se obter larvas de S. stercoralis para a produção de extrato antigênico homólogo assim o parasito Strongyloides venezuelensis e suas diferentes formas evolutivas são utilizados para produção de extratos antigênicos. O objetivo do presente estudo foi avaliar a detecção de anticorpos anti S. venezuelensis a partir de extratos alcalinos de larvas L3, fêmeas partenogenéticas ou ovos na estrongiloidíase experimental associada à imunossupressão. Ratos machos foram divididos em dois grupos: não-imunossuprimidos e imunossuprimidos experimentalmente infectados e o soro de todos os animais foram obtidos no 0, 5º, 8º, 13º e 21º dias pós-infecção (d.p.i.). O teste enzyme-linked immunosorbent assay (ELISA) foi realizado para comparar o potencial reativo dos extratos alcalinos de larva L3, fêmeas partenogenéticas e ovos de S. venezuelensis em ratos não-imunossuprimidos e imunossuprimido. Immunoblotting foi avaliado quanto sua detecção de IgG anti S. venezuelensis em ambos os grupos frente aos extratos alcalinos das três formas evolutivas do parasito. No ELISA comparativo todos extratos foram efetivos na detecção de anti-IgG de S. venezuelensis. O extrato de larvas apresentou reatividade a partir do 5º d.p.i cessando no 21º d.p.i. no grupo não-imunossuprimido enquanto o grupo imunossuprimido essa reatividade permaneceu no 21º d.p.i.. O immunoblotting apresentou perfil de reatividade com frações imunorreativas no grupo imunossuprimido a partir do 5º d.p.i. enquanto o grupo não-imunossuprimido a reatividade a partir do 8º d.p.i. Os extratos alcalinos de fêmeas partenogenéticas e de ovos apresentaram similar perfil de reatividade tanto no ELISA comparativo quanto no immunoblotting. Frações proteicas imunorreativas de 26 e 17 kDa presentes no extrato alcalino de larvas apresentaram como importantes marcadores de infecção em animais imunossuprimidos. Conclui-se que todos os extratos alcalinos possuem potencial diagnóstico na estrongiloidíase experimental com destaque para o extrato de larvas em casos de imunossupressão, sendo o immunoblotting uma importante ferramenta diagnóstica complementar. Mestre em Imunologia e Parasitologia Aplicadas

The nematode Strongyloides stercoralis is responsible for strongyloidiasis in humans. Diagnosis of infection occurs through detection of larvae in feces, but low elimination of larvae often hampers the detection of disease, particularly in cases of patient immunosuppression. Immunodiagnostic tests have been developed; however obtaining S. stercoralis larvae for the production of homologous antigen extract is technically difficult. Thus, the use different developmental forms of Strongyloides venezuelensis has become an alternative method for the production of antigen extracts. The aim of this study was to evaluate immunoblotting using alkaline extracts from S. venezuelensis L3 larvae, parthenogenetic females or eggs to test detection of experimental strongyloidiasis associated with immunosuppression. Immunocompetent and immunosuppressed male rats were experimentally infected, and serum sample from all animals were obtained at 0, 5, 8 13, and 21 days post infection (d.p.i.). Immunoblotting was evaluated for use in detection of anti-S. venezuelensis IgG in both experimental rat groups. The larval extract immunoblotting profile had the most immunoreactive fractions in the immunosuppressed group beginning at 5 d.p.i., while the immunocompetent group reactivity began on 8 d.p.i. Immunoreactive protein fractions of 17 kDa present in larval alkaline extract presented as possible markers of infection in immunosuppressed rats. It is concluded that all extracts using immunoblotting have diagnostic potential in experimental strongyloidiasis, particularly larval extract in immunosuppressed individuals.