Au vu des travaux de Certad et de ses collaborateurs, nous savons que Cryptosporidium parvum est également capable d’induire des adénomes avec des néoplasies digestives intraépithéliales de bas grade et de haut grade, ainsi que des adénocarcinomes in situ, chez des souris SCID (Severe Combined Immunodeficiency) déficientes en lymphocytes T et B, traitées par la dexaméthasone (SCID-D). De plus, nous savons aussi que Cryptosporidium muris induit une infection chronique mais pas de lésions néoplasiques. C’est pour faire suite à ces observations que nous avons entrepris dans un premier temps de déterminer la dose minimale de Cryptosporidium parvum (souche IOWA d’origine animale) pouvant infecter ce modèle et induire des néoplasies digestives. Nous avons montré qu’une dose très faible de parasites (théoriquement 1 oocyste) est capable d’induire non seulement une infection chronique chez les souris (SCID-D) mais également l’apparition de lésions néoplasiques aussi bien dans la région antropylorique de l’estomac qu’au niveau de la région iléo-caecale et cela dès 45 jours post infection. Nous avons également suivi la progression de ces lésions à la suite d’infection des souris par plusieurs doses de C. parvum (souche IOWA) (théoriquement : 1, 10, 100 et 105 oocystes). Ce travail a également été réalisé après inoculation d’une souche de C. parvum d’origine humaine (isolée à partir d’un patient immunodeprimée soufrant d’une cryptosporidiose grave à la suite d’une noyade). Pour cela, nous avons réalisé un suivi prolongé des animaux (au delà de 84 jours) et des analyses histopathologiques associées à la détection immuno-histochimique de la cytokeratine et de l’alpha actine lisse. Il a été observé, avec toutes les doses et pour les deux souches, aussi bien dans la région antropylorique qu’iléo-caecale des animaux, la présence d’adénomes contenant un grand nombre de parasites. Nous avons noté pour la première fois au niveau de ces lésions néoplasiques la présence d’une desmoplasie et de bourgeons de cellules tumorales envahissant le chorion de la muqueuse. En plus de ces éléments histologiques caractéristiques des adénocarcinomes invasifs, les différentes colorations et marquages ont mis en évidence d’autres signes d’invasion: une membrane basale discontinue, la présence de cellules épithéliales au niveau du stroma et enfin une interruption de la muscularis mucosa, voire une invasion de la musculeuse. Dans un deuxième temps, nous nous sommes intéressés aux voies de cancérogénèse impliquées dans le processus d’induction des lésions néoplasiques par C. parvum (IOWA) au niveau de la région iléocæcale. Afin d’initier nos travaux dans cette perspective, nous avons choisi quatre marqueurs impliqués dans les principales voies altérées dans les cancers colorectaux: APC, Beta-catenine, P53 et K-ras. Des études immunohistochimiques ont été réalisées et nous ont permis de noter qu’il y avait une localisation anormale aussi bien de l’APC, que de la Beta-catenine ou de la P53. La Beta-catenine (normalement localisée au niveau de la membrane cellulaire) et la P53 (normalement localisée dans le noyau) s’accumulent dans le cytoplasme alors que le marquage de l’APC dans les cellules néoplasiques diminue, voire même disparait. Le marquage de K-ras, quant à lui, est toujours membranaire comme dans les cellules normales. Tout cela semble indiquer l’implication des voies P53 et Wnt dans le phénomène de cancérogénèse chez notre modèle de souris (SCID-D). Des études visant à rechercher d’autres marqueurs et d’éventuelles mutations des gènes codant ces protéines sont en cours. / Certad and col, showed recently that Cryptosporidium parvum is also capable of inducing gastrointestinal adenomas with intraepithelial neoplasia of low and high grade, and adenocarcinomas in situ in SCID mice (Severe Combined immunodeficiency), treated with dexamethasone (SCID-D). In addition, we also know that Cryptosporidium muris induces a chronic infection but no neoplastic lesions.This is why we decided first to determine the minimum dose of C. parvum (IOWA) which can infect and cause digestive neoplasia in this model. This work allowed us to conclude that one oocyst is able to induce in SCID-D mice not only a chronic infection but also the development of neoplastic lesions in both the antropyloric region of the stomach and the ileocecal region at 45 days post infection. We also followed up the progression of these lesions after infection with several doses of C. parvum strain IOWA (theoretically: 1, 10, 100 and 105 oocysts). This work was also performed after inoculation of another strain of different origin isolated from an immunosupressed patient suffering from a severe cryptosporidiosis after a near-drowning). To do this we have achieved: an extended follow-up of animals (over 84 days) and an histopathological analysis based on immunohistochemical detection of cytokeratin and alpha smooth actin. For the first time it was noted with all doses and for the two strains, in both the antropyloric and ileocecal region of animals, a patern characteristic of invasif adenocarcinoma: desmoplasia and buds of tumor cells invading the lamina propria. In addition to these histological features, a discontinuous basement membrane, the presence of epithelial cells in the stroma, an interruption of the muscularis mucosa and an invasion of the muscularis were also detected. In the case of the strain of C. parvum of human origin, the adenocarcinoma also invaded the serosa and epithelial cells were observed inside blood vessels (vascular tumor emboli). Lesion’s progression was so fast that after only 60 days post-infection we observed at least, the invasion of the submucosa at ileocecal region. Furthermore, the results of this study showed for the first time the ability of an isolate of C. parvum of human origin to cause chologiocarcinoma in an experimental model. Finally, using an immunohistochemical approach, we explored metabolic pathways involved in the development of C. parvum-induced neoplastic lesions at the ileocecal region. Four markers involved in major pathways altered in colorectal cancer were chosen: APC, beta-catenin, p53 and K-ras. The assesment of tumor marker expression in the ileocaecal area showed an abnormal localization of APC, beta-catenin and p53. Beta-catenin and p53 accumulated in the cytoplasm, while APC labelling decreased or even disappeared. Meanwhile, K-ras was still at membrane level as in normal cells. these results suggest the involvement of p53 and Wnt pathway in the phenomenon of carcinogenesis in our mouse model (SCID-D). Studies to search the implication of other markers and possible mutations of the genes encoding these proteins are underway. In conclusion,, these findings show that different strains of C. parvum including a strain of human origin induce digestif invasive adenocarcinomas whatever the inoculum size administered to SCID-D mice. These results confirm the role of C. parvum in the induction of digestive cancer in immunocompromised hosts. In addition, the pathways involved in the process of carcinogenesis in mice (SCID-D) appeared to be the same as those altered in humans. Moreover, the Wnt signaling pathway in which the actin polymerization and rearrangement of the cytoskeleton are involved is a major event during Cryptosporidium infection and appears to play a role in the carcinogenic process induced by the parasite.

Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains 202 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 9.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-18Bitstream added on 2014-06-13T18:55:52Z : No. of bitstreams: 1 silva_dc_me_araca.pdf: 457686 bytes, checksum: 955900a36656c74b3d4e888ce706efe1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Devido à carência de informações relacionadas à epidemiologia da infecção por Cryptosporidium spp. em passeriformes, neste trabalho objetivou-se determinar a periodicidade da eliminação fecal de oocistos de Cryptosporidium, os sinais clínicos, a presença de mortalidade, e a caracterização molecular desse coccídio. Foram colhidas 480 amostras de fezes, provenientes de 40 aves, sendo 372 amostras de 31 aves adultas e 108 amostras de nove filhotes até 12 semanas de vida, com periodicidade mensal, no período de setembro de 2007 a setembro de 2008, com exceção do mês de abril. As aves estavam alojadas em cinco criatórios, com criação de bicudo (Oryzoborus maximiliani), curió (Oryzoborus angolensis), azulão (Passerina brissonii) e coleira do brejo (Sporophila collaris). As amostras foram conservadas em bicromato de potássio 2,5%, a 4ºC, até o processamento. Os oocistos foram purificados por centrífugo-flutuação em solução de Sheather, seguindo-se a extração do DNA genômico dos oocistos e a classificação molecular, por meio da reação em cadeia de polimerase-nested, para amplificação de fragmentos da subunidade 18S do gene do RNA ribossômico. Eliminação fecal intermitente de Cryptosporidium spp. foi observada em 91 (24,5%) amostras de aves adultas, com maior ocorrência nos períodos que se aproximam dos períodos de muda de penas e de reprodução das aves e em 14 amostras (13%) de aves jovens. O sequenciamento dos fragmentos de DNA amplificados possibilitou a identificação de somente Cryptosporidium galli. Embora em todos os criatórios houvesse aves positivas para C. galli, a presença de morbidade ou mortalidade foi observada em aves de somente um criatório, e estava associada à infecção concomitante com Escherichia coli e Isospora spp..Este é o primeiro relato de infecção por C. galli em P. brissonii, O. maximiliani e S. collaris / Due to the lack of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the frequency of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, the clinical signs and the presence of mortality, and accomplish its molecular characterization. Four hundred and eighty fecal samples were collected from 40 birds, 372 samples from 31 adult birds and 108 samples from young birds (up to 12 months old), housed in five herds, monthly, from September 2007 to September 2008, with the exception of the April. The birds were originated from flocks were the following species were herd: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Passerina brissonii) and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate 2.5% at 4°C, until processing. The oocysts were purified by centrifugal flotation in Sheather solution, followed by genomic DNA extraction from oocysts and molecular characterization using the nested polymerase chain reaction for amplification of fragments of the 18S subunit ribosomal RNA gene. Intermittent fecal shedding of Cryptosporidium spp. was observed in 91 (24.5%) samples from adult birds, with more frequent in periods approaching the periods of moulting and reproduction of birds and 14 samples (13%) of young birds.The sequencing of the amplified fragments allowed the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in birds from only one aviary, and was associated to concomitant infection with Escherichia coli and Isospora sp. This is the first report of infection by C. galli in Oryzoborus maximiliani, Passerina brissonii, and Sporophila collaris

Orientador: Marcelo Vasconcelos Meireles / Banca: Valéria Marçal Félix de Lima / Banca: Rodrigo Martins Soares / Resumo: Devido à carência de informações relacionadas à epidemiologia da infecção por Cryptosporidium spp. em passeriformes, neste trabalho objetivou-se determinar a periodicidade da eliminação fecal de oocistos de Cryptosporidium, os sinais clínicos, a presença de mortalidade, e a caracterização molecular desse coccídio. Foram colhidas 480 amostras de fezes, provenientes de 40 aves, sendo 372 amostras de 31 aves adultas e 108 amostras de nove filhotes até 12 semanas de vida, com periodicidade mensal, no período de setembro de 2007 a setembro de 2008, com exceção do mês de abril. As aves estavam alojadas em cinco criatórios, com criação de bicudo (Oryzoborus maximiliani), curió (Oryzoborus angolensis), azulão (Passerina brissonii) e coleira do brejo (Sporophila collaris). As amostras foram conservadas em bicromato de potássio 2,5%, a 4ºC, até o processamento. Os oocistos foram purificados por centrífugo-flutuação em solução de Sheather, seguindo-se a extração do DNA genômico dos oocistos e a classificação molecular, por meio da reação em cadeia de polimerase-nested, para amplificação de fragmentos da subunidade 18S do gene do RNA ribossômico. Eliminação fecal intermitente de Cryptosporidium spp. foi observada em 91 (24,5%) amostras de aves adultas, com maior ocorrência nos períodos que se aproximam dos períodos de muda de penas e de reprodução das aves e em 14 amostras (13%) de aves jovens. O sequenciamento dos fragmentos de DNA amplificados possibilitou a identificação de somente Cryptosporidium galli. Embora em todos os criatórios houvesse aves positivas para C. galli, a presença de morbidade ou mortalidade foi observada em aves de somente um criatório, e estava associada à infecção concomitante com Escherichia coli e Isospora spp..Este é o primeiro relato de infecção por C. galli em P. brissonii, O. maximiliani e S. collaris / Abstract: Due to the lack of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the frequency of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, the clinical signs and the presence of mortality, and accomplish its molecular characterization. Four hundred and eighty fecal samples were collected from 40 birds, 372 samples from 31 adult birds and 108 samples from young birds (up to 12 months old), housed in five herds, monthly, from September 2007 to September 2008, with the exception of the April. The birds were originated from flocks were the following species were herd: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Passerina brissonii) and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate 2.5% at 4°C, until processing. The oocysts were purified by centrifugal flotation in Sheather solution, followed by genomic DNA extraction from oocysts and molecular characterization using the nested polymerase chain reaction for amplification of fragments of the 18S subunit ribosomal RNA gene. Intermittent fecal shedding of Cryptosporidium spp. was observed in 91 (24.5%) samples from adult birds, with more frequent in periods approaching the periods of moulting and reproduction of birds and 14 samples (13%) of young birds.The sequencing of the amplified fragments allowed the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in birds from only one aviary, and was associated to concomitant infection with Escherichia coli and Isospora sp. This is the first report of infection by C. galli in Oryzoborus maximiliani, Passerina brissonii, and Sporophila collaris / Mestre

Cryptosporidium spp são protozoários cosmopolita que acometem peixes, répteis, anfíbios, aves e mamíferos. Mais de 20 espécies são reconhecidas dentro deste gênero. Os roedores, grupos de organismos abundantes e ubíquos, têm sido considerados reservatórios de Cryptosporidium para humanos e animais de produção. As seqüências codificadoras da menor unidade ribossômica (18S rRNA) de Cryptosporidium spp caracterizam-se por intercalações entre regiões conservadas e polimórficas ao longo dos seus 1700 pares de bases. O objetivo deste estudo foi desenhar primers específicos para o gene 18S rRNA, potencialmente capazes de amplificar qualquer espécie ou genótipo de Cryptosporidium spp. e avaliar os atributos diagnósticos da nested-PCR baseadas em tais sondas. O desenho dos primers foi realizado de forma a amplificar um segmento de menor dimensão possível para se maximizar a sensibilidade do ensaio molecular e preservando o potencial discriminatório das seqüências amplificadas. A nestedPCR padronizada neste estudo (nPCR-SH) foi comparada com outro ensaio similar que vem sendo largamente utilizado para detecção e identificação de Cryptosporidium spp. no mundo todo (nPCR-XIAO). Também se objetivou caracterizar molecularmente amostras de Cryptosporidum spp. isoladas de roedores sinantrópicos, empregando-se estas sondas e sondas moleculares direcionadas. Foram capturados 45 roedores em áreas públicas da região urbana da cidade de Umuarama, Paraná. As amostras foram submetidas a três provas moleculares, sendo duas direcionadas ao gene18S rRNA (nPCR-SH e nPCR-XIAO) e outra, ao gene codificador da actina. A nPCR-SH foi testada com as amostras de Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, Cryptosporidum serpentis e todas foram positivas. Dezesseis amostras de roedores foram positivas para a nPCR-SH, seis pela nPCR-XIAO e cinco pela nPCR dirigida ao gene codificador da actina. O sequenciamento dos fragmentos amplificados possibilitou a identificação de Cryptosporidum muris em três amostras de Rattus rattus, e dois novos genótipos de Cryptosporidium, os genótipos rato II e III. Genótipo rato II foi encontrado em uma amostra de Mus musculus e o genótipo III em doze amostras, sendo cinco Rattus rattus e sete Mus musculus. Os resultados deste estudo demonstraram que os primers desenhados para detecção do Cryptosporidium spp em amostras de fezes foram mais eficientes em amplificar regiões que permitem a distinção entre as espécies do parasito do que aqueles usados na PCR-XIAO. Nas amostras estudadas não foram encontrados genótipos ou espécies de Cryptosporidium que podem ser transmitidos a outras espécies como os zoonóticos, o que sugere que a importância destes animais na transmissão zoonótica de criptosporidiose é pouco relevante. / Cryptosporidium spp are cosmopolitan protozoan that infect fish, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are groups of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for infection of humans and livestock. The coding sequences of the smallest unit ribosomal (18S rRNA) of Cryptosporidium spp are characterized by intercalations amongst polymorphic and conserved regions along its 1700 base pairs. The aim of this study was to design primers specific for the 18S rRNA gene, potentially capable of amplifying any species or genotype of Cryptosporidium spp., and evaluate the attributes of the nested-PCR diagnosis based on such probes. The design of primers was performed to amplify a smaller segment as possible to maximize the sensitivity of molecular testing and preserving the discriminatory potential of the sequences amplified. The nested-PCR standardized in this study (nPCR-SH) was compared with another similar assay that has been widely used for detection and identification of Cryptosporidium spp. worldwide (nPCR-XIAO). It also aimed to characterize molecularly Cryptosporidum spp. isolated from synanthropic rodents, using these probes and targeted molecular probes. Forty five rodents were captured in public areas of the urban area of Umuarama, Paraná. The samples were subjected to three molecular tests, two targeted to gene18S rRNA (nPCR-SH and nPCR-XIAO) and another targeted to the gene encoding actin. The nPCR-SH was tested with strains of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis Cryptosporidum canis, Cryptosporidum serpentis and all were positive. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR targeted to the gene encoding actin. The sequencing of the amplified fragments allowed the identification of Cryptosporidum Muris in three samples of Rattus rattus, and two new genotypes of Cryptosporidium rat genotype II and III. It was found rat genotype II in a sample of Mus musculus and genotype III in twelve samples, five from Rattus rattus and seven from Mus Musculus.The results showed that the primers designed for detection of Cryptosporidium spp in fecal samples were more efficient in amplifying regions that allow the distinction amongst the parasite species than those used in the PCR-XIAO. Cryptosporidium species or genotypes transmitted to other species such as zoonotic were not found in the studied samples, which suggest that the importance of these animals in the zoonotic transmission of cryptosporidiosis is very limited.

Cryptosporidium spp são protozoários cosmopolita que acometem peixes, répteis, anfíbios, aves e mamíferos. Mais de 20 espécies são reconhecidas dentro deste gênero. Os roedores, grupos de organismos abundantes e ubíquos, têm sido considerados reservatórios de Cryptosporidium para humanos e animais de produção. As seqüências codificadoras da menor unidade ribossômica (18S rRNA) de Cryptosporidium spp caracterizam-se por intercalações entre regiões conservadas e polimórficas ao longo dos seus 1700 pares de bases. O objetivo deste estudo foi desenhar primers específicos para o gene 18S rRNA, potencialmente capazes de amplificar qualquer espécie ou genótipo de Cryptosporidium spp. e avaliar os atributos diagnósticos da nested-PCR baseadas em tais sondas. O desenho dos primers foi realizado de forma a amplificar um segmento de menor dimensão possível para se maximizar a sensibilidade do ensaio molecular e preservando o potencial discriminatório das seqüências amplificadas. A nestedPCR padronizada neste estudo (nPCR-SH) foi comparada com outro ensaio similar que vem sendo largamente utilizado para detecção e identificação de Cryptosporidium spp. no mundo todo (nPCR-XIAO). Também se objetivou caracterizar molecularmente amostras de Cryptosporidum spp. isoladas de roedores sinantrópicos, empregando-se estas sondas e sondas moleculares direcionadas. Foram capturados 45 roedores em áreas públicas da região urbana da cidade de Umuarama, Paraná. As amostras foram submetidas a três provas moleculares, sendo duas direcionadas ao gene18S rRNA (nPCR-SH e nPCR-XIAO) e outra, ao gene codificador da actina. A nPCR-SH foi testada com as amostras de Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, Cryptosporidum serpentis e todas foram positivas. Dezesseis amostras de roedores foram positivas para a nPCR-SH, seis pela nPCR-XIAO e cinco pela nPCR dirigida ao gene codificador da actina. O sequenciamento dos fragmentos amplificados possibilitou a identificação de Cryptosporidum muris em três amostras de Rattus rattus, e dois novos genótipos de Cryptosporidium, os genótipos rato II e III. Genótipo rato II foi encontrado em uma amostra de Mus musculus e o genótipo III em doze amostras, sendo cinco Rattus rattus e sete Mus musculus. Os resultados deste estudo demonstraram que os primers desenhados para detecção do Cryptosporidium spp em amostras de fezes foram mais eficientes em amplificar regiões que permitem a distinção entre as espécies do parasito do que aqueles usados na PCR-XIAO. Nas amostras estudadas não foram encontrados genótipos ou espécies de Cryptosporidium que podem ser transmitidos a outras espécies como os zoonóticos, o que sugere que a importância destes animais na transmissão zoonótica de criptosporidiose é pouco relevante. / Cryptosporidium spp are cosmopolitan protozoan that infect fish, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are groups of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for infection of humans and livestock. The coding sequences of the smallest unit ribosomal (18S rRNA) of Cryptosporidium spp are characterized by intercalations amongst polymorphic and conserved regions along its 1700 base pairs. The aim of this study was to design primers specific for the 18S rRNA gene, potentially capable of amplifying any species or genotype of Cryptosporidium spp., and evaluate the attributes of the nested-PCR diagnosis based on such probes. The design of primers was performed to amplify a smaller segment as possible to maximize the sensitivity of molecular testing and preserving the discriminatory potential of the sequences amplified. The nested-PCR standardized in this study (nPCR-SH) was compared with another similar assay that has been widely used for detection and identification of Cryptosporidium spp. worldwide (nPCR-XIAO). It also aimed to characterize molecularly Cryptosporidum spp. isolated from synanthropic rodents, using these probes and targeted molecular probes. Forty five rodents were captured in public areas of the urban area of Umuarama, Paraná. The samples were subjected to three molecular tests, two targeted to gene18S rRNA (nPCR-SH and nPCR-XIAO) and another targeted to the gene encoding actin. The nPCR-SH was tested with strains of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis Cryptosporidum canis, Cryptosporidum serpentis and all were positive. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR targeted to the gene encoding actin. The sequencing of the amplified fragments allowed the identification of Cryptosporidum Muris in three samples of Rattus rattus, and two new genotypes of Cryptosporidium rat genotype II and III. It was found rat genotype II in a sample of Mus musculus and genotype III in twelve samples, five from Rattus rattus and seven from Mus Musculus.The results showed that the primers designed for detection of Cryptosporidium spp in fecal samples were more efficient in amplifying regions that allow the distinction amongst the parasite species than those used in the PCR-XIAO. Cryptosporidium species or genotypes transmitted to other species such as zoonotic were not found in the studied samples, which suggest that the importance of these animals in the zoonotic transmission of cryptosporidiosis is very limited.

The humoral response of humans, calves and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sporozoite antigens form sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The number of antigens recognized by immune sera from humans and animals increased with time post infection (P.I.). A 20 kDa antigen appeared to be a major sporozoite surface determinant since it was labelled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20 kDa band occurred in 3 week post infection (P.I.) sera from all species tested. Sera reactivity to the 20 kDa band diminished significantly within 5 months P.I. when infected humans had no further recurrence of cryptosporidial diarrhea. In contrast, 12 month P.I. sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3 week convalescent sera. Therefore, reactivity to the 20 kDa antigen appeared to be a good indicator of exposure to Cryptosporidium. Anti-sporozoite indirect immunofluorescent titers decrease in reactivity from convalescent to post convalescent sera which correlated with western blot results. Chromosomal DNA of five Cryptosporidium parvum isolates and one Cryptosporidium baileyi isolate were compared by field inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the five Cryptosporidium parvum isolates were indistinguishable. Distinct differences in chromosomal DNA were evident between the Cryptosporidium baileyi and Cryptosporidium parvum isolates, yet the overall pattern was similar. Five C. parvum isolates were also compared using two dimensional electrophoretic analyses. Silver stained patterns of sporozoite proteins showed a shift in a 106 kDa protein in three of the isolates. One isolate (Mexico) showed a complete absence of this protein (106 kDa) and the presence of an additional 40 kDa protein not found in any other isolate.

Memoria para optar al título Profesional de Médico Veterinario / Cryptosporidium spp. es un agente parasitario protozoario zoonótico capaz de infectar a múltiples especies animales en todo el mundo, definido como prioritario para monitoreo en fauna silvestre. Produce diarrea en individuos inmunocomprometidos, siendo transmitido por vía fecal-oral, donde el agua juega un rol epidemiológico importante. Este patógeno, ya ha sido detectado en aguas marinas y superficiales y en animales acuáticos, incluyendo pingüinos de la Antártica. Los Sphenisciformes se consideran centinelas oceánicos, ya que reflejan cambios en la productividad oceánica y cambios ambientales producidos por actividades humanas. Con el objetivo de detectar este patógeno en pingüinos de zonas templadas, en este estudio se tomaron muestras a partir de tórulas cloacales y heces frescas desde el ambiente provenientes de Pingüinos de Magallanes (Spheniscus magellanicus) pertenecientes a dos colonias reproductivas, Isla Magdalena y Seno Otway, en la Región de Magallanes y Antártica Chilena. En Isla Magdalena, un 3,5% de las muestras obtenidas directamente desde las aves fueron positivas al agente, mientras que en Seno Otway un 3,4% de ellas dieron positivas, no existiendo diferencias significativas en los niveles de infección entre las colonias. Por otro lado, en las muestras ambientales, tomadas solamente en Isla Magdalena, se encontró un 29,1% de positividad a la presencia este parásito, siendo esta la primera descripción de Cryptosporidium spp. en S. magellanicus, y a su vez en pingüinos de zonas templadas. / Proyecto FONDECYT 1110255

Thèse doctorat : Médecine. Parasitologie : Université de Nantes : 2003. / Bibliogr. f. 197-217.

No description available.