Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes / Avaliação da proteína recombinante GP60 de Cryptosporidium hominis e da IgY policlonal anti-GP60 recombinante para uso em pesquisa e diagnóstico

Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

Resumo Neste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.

Recombinant Cryptosporidium parvum p23 as a candidate vaccine for cryptosporidiosis

Cryptosporidium parvum is a ubiquitous protozoan, which develops within the microvillous membrane of enterocytes in a wide variety of vertebrates, including man. Cryptosporidiosis is an important parasite causing severe diseases in the immunodeficient people especially AIDS patients. Cryptosporidiosis has been also reported as a com-mon serious primary cause of outbreaks of diarrhea in newborn calves. The aim of this study was to confirm that P23 was an immunogenic antigen in domestic isolates of C. parvum. We isolated cryptosporidial oocysts from the naturally infected calves. The oocysts were then purified and characterized as C. parvum by nested PCR. To obtain the recombinant P23 protein, we isolated the mRNA from oocyst of C. parvum, and synthesized the cDNA. The cDNA was then amplified using specific primers for P23 gene. Sequencing of PCR product showed 100% homology to the known P23 sequences in GenBank. The double strand P23-cDNA was then cloned in pGEX-5X-2 expression vector and P23-recombinant protein was prepared. West-ern blot analysis of recombinant P23 showed that it could be recognized by the positive C. parvum serum. Furthermore, serum from immunized goat with the recombinant P23 protein also recognized a protein band with approximately 23 kDa in lysates prepared from the oocytes. Since P23 is an immunodominant surface glycoprotein expressed in the early phase of infection and the immunogenic epitopes are found in its residual chain of amino acid sequence, the recombinant P23 could be recom-mended as a favorable candidate for vaccination against C. parvum infection

Seasonal profile and level of CD4+ lymphocytes in the occurrence of cryptosporidiosis and cystoisosporidiosis in HIV/AIDS patients in the Triângulo Mineiro region, Brazil

Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7 percent of positivity for coccidian (8.6 percent with Cryptosporidium sp, 10.3 percent with Cystoisospora belli and 0.8 percent with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < 200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5 percent of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli.

Pacientes com AIDS são particularmente susceptíveis a infecção por coccídios intestinais e nesse estudo foi avaliada a freqüência de Cryptosporidium sp. e Cystoisospora belli entre pacientes HIV/AIDS na região do Triângulo Mineiro, Brasil. No período de julho de 1993 a junho de 2003, amostras de fezes de 359 pacientes foram submetidas à coloração pelo método de Ziehl-Neelsen modificado, sendo detectada a presença de coccídios em 19,7 por cento destas (8,6 por cento de Cryptosporidium sp, 10,3 por cento de Cystoisospora belli e 0,8 por cento de ambos coccídios). Pacientes com diarréia e níveis de linfócitos T CD4+ < 200 células/mm3 apresentaram maior frequência destes protozoários, demonstrando o perfil oportunista destas infecções e a relação com o status imunológico do indivíduo. Não foi possível determinar a influência da HARRT, pois apenas 8,5 por cento dos pacientes positivos para coccídios fazriam uso regular desta terapia. Parasitismo por Cryptosporidium sp foi mais freqüente no período compreendido de dezembro a fevereiro caracterizando padrão sazonal desta infecção, fato não observado com Cystoisospora belli.

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyer's patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.

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.

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn't show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heatkilled oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.

Infection by Cryptosporidium sp. in immunocompromised haematological patients

Determinou-se a freqüência de infecção intestinal por Cryptosporidium sp. em 60 pacientes atendidos no Serviço de Hematologia e Hemoterapia da Santa Casa de Misericórdia de São Paulo que apresentavam processos linfoproliferativos (Grupo 1). Como grupo controle (Grupo 2) examinaram-se 59 indivíduos sadios, que habitavam as mesmas localidades e pertenciam a faixa etária semelhante a dos pacientes do Grupo 1. Os exames parasitológicos de fezes revelaram freqüência de infecção por Cryptosporidium sp. de 10 por cento no Grupo 1, enquanto nos controles (Grupo 2) não se evidenciou nenhum caso de infecção por esse coccídeo. Entre os pacientes que eliminavam oocistos de Cryptosporidium sp. apenas um apresentava fezes diarrêicas.

Estudo comparativo entre métodos morfológico, imunológico e molecular aplicados ao diagnóstico da criptosporidiose em amostras fecais de pacientes portadores do vírus hiv e com aids / Cryptosporidiosis: comparative study of morfological, immunological and molecular diagnosis methods in fecal samples from patients with hiv and aids

Cryptosporidium parvum, protozoário da sub classe Coccidia, é um agente causador de diarréia aguda no homem e outros animais. A criptosporidiose é auto-limitada em indíviduos sadios, mas se apresenta como uma doença grave e crônica nos pacientes imuno-comprometidos, principalmente com AIDS. O diagnóstico laboratorial é feito geralmente pela detecção do parasita por métodos morfológicos, mas técnicas imunológicas ou moleculares podem também ser usadas. Neste estudo, utilizaram-se 100 amostras fecais, provenientes de pacientes portadores de HIV e com AIDS, que já haviam sido analisadas previamente por métodos parasitológicos. Estas amostras foram submetidas a diferentes métodos de detecção para Cryptosporidium sp, o método morfológico (visualização através de coloração por Kinyoun) e o teste imunoenzimático (ELISA) para pesquisa de coproantígenos. Destas, foram selecionadas 29 amostras, sendo 15 positivas e 14 negativas para Cryptosporidium pela coloração de Kinyoun, as quais foram submetidas à metodologia molecular de “Nested” PCR (N-PCR). Entre as 15 amostras positivas para Cryptosporidium por método morfológico, 13 foram positivas por ELISA e apenas 11 por N-PCR. Duas amostras positivas por método morfológico e negativas por ELISA, apresentaram-se negativas por N-PCR. Das 16 amostras positivas por N-PCR, 11 foram positivas e cinco negativas por métodos morfológico e ELISA. O método de coloração pelo Kinyoun demonstrou ser ainda um bom método para ser utilizado na rotina diagnóstica da criptosporidiose. O teste de ELISA apresentou resultados compatíveis com os resultados obtidos na coloração de Kinyoun e, devido à capacidade de automação, pode ser empregado em estudos epidemiológicos, otimizando exames em grande demanda. O método molecular (PCR e N-PCR), para ser introduzido em nosso meio como método de rotina diagnóstica da criptosporidiose, necessita de aprimoramentos nas técnicas de extração de DNA, entretanto já é conhecida sua sensibilidade e especificidade, constituindo-se em importante ferramenta para estudos epidemiológicos com a possibilidade de caracterizar diferentes espécies e genótipos de Cryptosporidium

Antibody profile, interferon-y and nutritional status in cryptosporidial infection among school children

This work studied the antibody profile, interferon-gamma [IFN-gamma] serum level and nutritional status among infected schoolchildren aged 11 years in a rural school in Abis 8 Village. Fecal examination showed that only 19 cases infected with Cryptosporidium and 10 children were free from parasites [controls]. The IgA, IgG and IgM serum levels in infected cases were within normal level, without significant differences as compared to controls. The IgE serum level was significantly high in infected children. The cytokine IFN-gamma was significantly lower in the infected cases as compared to controls. Malnutrition was diagnostic among infected children. The study showed that malnutrition is the important finding related to Cryptosporidium infection. Malnutrition decreased IFN- gamma, the recognized factor in protection from infection, and elevated serum IgE level

effect of breast feeding on amoebiasis, giardiasis, and cryptosporidiosis in children below the age of two years

To determine the effect of breast feeding on amoebiasis, giardiasis and cryptosporidiosis in infants in our region. Stool samples were collected from 180 children below 2 years of age who suffered from acute diarrhea. There were 81 breast fed, 49 bottle fed, and 50 mixed bottle and breast fed children. The direct smear method and then formalin-ether sedimentation were carried out to detect intestinal parasites. Fecal smears were prepared from the sediment and stained by the modified Ziehl-Neelsen method for the recovery of acid-fast oocysts of Cryptosporidium. Intestinal amoebiasis, giardiasis and cryptosporidiosis were found at the highest rates [53%, 55% and 64% respectively] among bottle fed children. Collectively, 16.3% of the bottle fed children with diarrhea tested positive for parasitic infections. Breast feeding appears to reduce the risk of these parasitic infections and should be encouraged

Immunofluorescent detection of both giardia lamblia and cryptosporidium parvum using anti-cryptosporidium oocyst antibodies

In the present work, a polyspecific anti-cryptosporidium oocyst antibodies was used for simultaneous detection of both parasites in human stool. Known positive formalinized human stool specimens of Giardia sp. [n = 10], Cryptosporidium sp. [n=7] mixed infection [n=3] and negative specimens [n = 20] were tested using direct fluorescent technique against the developed antibodies. All positive stool samples for Cryptosporidium and 9 out of 10 Giardia samples or each alone showed fluorescence with variable intensities, while no negative sample harbored other parasites had fluorescence. This newly used polyspecific antibodies offer the advantages of screening of a large number of patients, particularly in outbreaks. Additionally, it represents a cheaper alternative for the most sophisticated and costly immunoassay kits using the monoclonal antibodies with more or less the same diagnostic potentials

Role of B-Cells in the Development of Immunity to Cryptosporidium Infection

Studies have shown that symptomatic improvement of cryptosporidiosis in immunocompromised patients is possible following treatment with" hyperimmune" colostrum containing anti-sporozoite antibodies from cattle immunized with Cryptosporidium parvum antigens. The contribution to immunity of antibodies developed during Cryptosporidium infection is unclear, however. Previously, we showed that SCID [severe combined immunodeficiency] mice acquired immunity to C. muris infection if injected with immunocompetent splenocytes. To investigate the role of the splenic donor B-cells in this immunity, groups of CB-17 SCID mice received BALB/c splenocytes, or splenocytes depleted of B-cells by immunomagnetic separation before infection. Both groups recovered from infection at similar times and displayed little difference in patterns of oocyst excretion. Subsequently, B-cells and serum anti-parasite antibodies were found only in mice reconstituted with unfractionated spleen cells. These results suggested that B-cells may not normally be required in immunity against Cryptosporidium

Prevalencia de parasitismo intestinal en niños supuestamente sanos. Valoración de su inmunidad humoral / Prevalence of intestinal parasitism in child

Se estudió la prevalencia de parasitismo intestinal en un total de 132 niños, clasificados como supuestamente sanos desde el punto de vista gastroentérico, los cuales forman parte de la matrícula de un círculo infantil ubicado en el municipio Habana Vieja. Se recolectaron 3 muestras de heces en cada caso, y se pesquisó, además de otros protozoarios, el Cryptosporidium. También se realizó la cuantificación de inmunoglobulinas G; A; M y E a los casos con Giardia lamblia. La prevalencia obtenida fue de 47,7 por ciento , con predominio de la giardiasis en el 96,8 por ciento de éstos. El 40 por ciento de los niños valorados inmunológicamente presentó algún tipo de déficit, fundamentalmente el de la inmunoglobulina A

Serological and histopathological studies on experimental cryptosporidiosis

120 albino rats were orally infected with Cryptosporidium oocyts isolated from diarrheal malnourished children in order to study the effect of the duration of infection on the pathology, extent of infection in the intestine and the antibody response in the sera. Small segments from different levels of the gut were processed for histological examination and cryostat sectioning. The sera of these rats were subjected to serological study using cryostat sections as antigen in the indirect immunofluorescence [IIF] test. The study declared that the duration of infection has no effect on the extent of infection. But the severity of the pathological changes and the grades of seropositivity in the sera varied with the duration of infection being maximum on the 15th day of infection. The possible role of the humoral immunity in clearance and protection from infection was discussed