Made available in DSpace on 2014-06-12T23:02:37Z (GMT). No. of bitstreams: 2 arquivo8569_1.pdf: 776272 bytes, checksum: afcf5071cf95d763da3e9f4ccb484cf5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A criptosporidiose tem se destacado como um problema de saúde pública e animal. A associação de casos da doença na população humana com a presença de Cryptosporidium spp. spp. em águas de consumo ou de recreação tem motivado a pesquisa do parasito no ambiente aquático. Este estudo objetivou a detecção de oocistos de Cryptosporidium spp. em águas superficiais na Região Metropolitana de Recife. A técnica de centrífugo- flutuação com solução saturada de cloreto de sódio foi usada para recuperação experimental de oocistos de Cryptosporidium spp.. Oocistos foram pesquisados em mananciais e no sistema de tratamento (água bruta e tratada) através da filtração das amostras em membrana, sendo identificados, sem prévia purificação, pelos métodos de coloração Kinyoun e imunofluorescência direta (IFD) associada ao 4 6 - Diamidino-2-Phenylindole (DAPI), analisando-se amostras durante 12 meses, nos períodos seco (Setembro à Fevereiro) e chuvoso (Março à Agosto). A qualidade da água foi avaliada através dos parâmetros microbiológicos (coliformes totais e fecais) e físicoquímicos (turbidez, pH). A técnica de PCR foi realizada para a pesquisa de oocistos de Cryptosporidium spp. de origem fecal a partir dos iniciadores gênero e espécie específicos, CPB-DIAGF/CPB-DIAGH e HB-1/HB-2, sendo o DNA extraído com proteinase K e solução de lise sob congelamento/descongelamento. Os resultados mostraram que a técnica de purificação garantiu um material mais limpo, porém o percentual de recuperação de oocistos variou de 39,0% a 98,4%. Na água tratada não se verificou a presença de oocistos de Cryptosporidium spp., enquanto nas amostras de água bruta foram encontradas estruturas álcool-ácido resistentes similares aos oocistos de Cryptosporidium spp. em 100% (05/05) dos locais e em 40% (24/60) das amostras analisadas pelo método Kinyoun, sendo a presença do parasito confirmada pela técnica IFD/DAPI em 40% (02/05) dos locais e em 5% (03/60) das amostras, com o número variando de 16 a 40 oocistos/l, e verificando sua ocorrência no período seco e chuvoso. Todas as amostras encontraram-se dentro dos limites microbiológicos e físico-químicos padrões, exceto no parâmetro turbidez. Os produtos de PCR foram obtidos apenas com o uso dos primers CPB-DIAGF/CPB-DIAGR, sendo o melhor perfil de amplificação observado quando 105 oocistos foram usados para a extração do DNA, e após o emprego dos protocolos de extração e de amplificação modificados. Conclui-se que a técnica de purificação de oocistos de Cryptosporidium spp. influencia os percentuais de recuperação. Os oocistos de Cryptosporidium spp. estão presentes em águas de rio na Região Metropolitana de Recife, sendo este o primeiro relato de Cryptosporidium spp. em mananciais de Pernambuco e do Nordeste. A técnica IFD/DAPI permite uma melhor identificação de oocistos de Cryptosporidium spp. em amostras de água, no entanto a coloração histoquímica demonstrou ser útil como uma técnica de triagem. As técnicas de extração e amplificação avaliadas neste estudo podem ser usadas para a pesquisa de Cryptosporidium spp. em amostras com elevado número de oocistos

Cryptosporidium spp. is an oocyst-forming apicomplexan protozoan, which infects humans and a large variety of animals. Several species and genotypes are potentially zoonotic and ruminats are considered as an important source of infection. Pre-weaned calves are major hosts for zoonotic C. parvum, and show higher rates of infection than post-weaned or adult animals. Cryptosporidium infection has been demonstrated in a wide variety of wild animals, which may co tribute to environmental contamination. In sub-Saharan Africa, where the HIV infection prevalence is the highest in the world, high incidence of severe and even fatal Cryptosporidium infection have been reported in humans. This study investigated the epidemiology of Cryptosporidium spp. simultaneously in wildlife, indigenous cattle and young children living at the wildlife, livestock and human interface on the western boundary of the Kruger National Park (KNP) in Mpumalanga Province, South Africa. Initially, a pilot study was carried out to assess the zoonotic or anthroponotic importance of Cryptosporidium in diarrhoeic children in South Africa, representing the human group most likely to be infected. This geographically broad study involved hospitals from four provinces in South Africa. Stool samples from hospitalized diarrhoeic children from 0-1 year of age were analysed by microscopy (modified Ziehl-Neelsen (MZN) acid-fast staining) and molecular techniques: polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing. An overall prevalence of Cryptosporidium infection of 12.2% was revealed, mainly involving species of anthroponotic origin, such as C. hominis (76%) and C. parvum of anthroponotic nature (20%). Only one species of uncertain zoonotic importance (C. meleagridis) was detected in this study. These findings were in accordance with reports from sub-Saharan Africa (including South Africa), where anthroponotic species of Cryptosporidium were responsible for most infections in humans. The study then concentrated on the public health importance of cryptosporidiosis at the wildlife/livestock/human interface of the KNP. Cryptosporidium oocysts were detected in elephant, impala and buffalo samples collected in three different study areas of the KNP; two located close to the boundaries of the KNP and a third one in the centre of the KNP. The MZN staining technique and an immunofluorescent antibody (IFA) test were applied to identify oocysts from faecal samples. The prevalence detected with MZN was higher than that detected with IFA, however both tests found a higher prevalence in elephants (25.8% and 4.2%, respectively) compared to the other species. The prevalence of Cryptosporidium in buffalo was 5.5% and 1.4% with MZN and IFA, respectively, and 4.2% and 1.8% respectively in impala. In the two study areas adjacent to the fence of KNP, the combined prevalence was significantly higher compared to the area in the centre of the KNP. The agreement between the MZN staining technique and the IFA test was assessed for each wildlife species; the estimates of kappa suggested moderate agreement in buffalo and impala and fair to poor agreement in elephant. The above results of were analysed further by the use of molecular techniques in order to reveal the species and genotypes of the parasite in wildlife and in addition faecal samples collected from post-weaned calves. A questionnaire was also conducted among farmers to investigate observed contacts between cattle and wildlife species in grazing areas outside and inside the KNP. Four of the 241 wildlife samples were PCR-positive (2.8% each in impala and buffalo and of 0.0% in elephant) and sequencing revealed the presence of C. ubiquitum in two impala and one buffalo and C. bovis in one buffalo. Cryptosporidium ubiquitum has been commonly found in a large number of animals, including humans. Among calf samples, 8% (4/51) were PCR-positive and were identified as C. andersoni (2/4) and C. bovis (2/4). The probability of contact between cattle and wildlife outside the KNP, observed by farmers, was higher for buffalo (Pr=0.6) and impala (Pr=0.46) than for elephant (Pr=0.04). This suggests that the detection of C. bovis in both cattle and buffalo might be due to direct or indirect contact between these two species. The detection of C. ubiquitum in wildlife, with its zoonotic potential, suggests that Cryptosporidium may be of public health concern for people living at the interface. We further investigated the prevalence of Cryptosporidium infection in cattle and humans, this time targeting younger (pre-weaned) calves and children. Children <5 years were sampled at six rural clinics within the same interface and stool samples were screened by the MZN staining technique. All MZN-positive and suspicious samples of children and samples of 36 calves within the age of 0-4 months were analysed by nested PCR. Eight of the 143 children (5.6%) were positive on PCR, and sequencing identified predominantly C. hominis, while one sample was identified as C. meleagridis. Eleven of the 36 calf samples (30.5%) were PCR-positive and were identified as C. bovis and C. ryanae. Due to limited resources, molecular analysis could not be performed on more samples. Variables such as source of drinking water, age and contact with animals for children, were analysed as potential risk factors for humans and cattle; however, none were statistically significant. In conclusion, the prevalence of Cryptosporidium detected in human and wildlife was low compared to that reported in other studies in Africa. The species and genotypes detected in humans were predominantly of anthroponotic nature; however, the isolation of C. Ubiquitum from buffalo and impala shows that at least one species of zoonotic importance is present at the wildlife/livestock/human interface. The prevalence of HIV/AIDS in our study area is one of the highest worldwide; therefore the potential public health importance of this parasite should be investigated further. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Production Animal Studies / unrestricted

Orientador: Katia Denise Saraiva Bresciani / Resumo: A criptosporidiose, uma das principais causas de diarreia infantil, é causada por parasitos do Filo Apicomplexa pertencentes ao gênero Cryptosporidium. A falta de medicamentos eficazes está motivando pesquisas para desenvolver tratamentos alternativos. Para este objetivo, o impacto dos probióticos no curso da criptosporidiose foi investigado. A microbiota intestinal nativa de camundongos imunossuprimidos livres de patógenos específicos foi inicialmente esgotada com antibióticos administrados por via oral. Um produto probiótico comercialmente disponível destinado ao consumo humano foi subsequentemente adicionado à água potável. Os camundongos foram infectados com oocistos de Cryptosporidium parvum. Em média, os camundongos tratados com probiótico desenvolveram uma infecção mais grave. Os probióticos alteraram significativamente a microbiota fecal, mas não foi observada associação direta entre a ingestão de bactérias probióticas e sua abundância na microbiota fecal. Esses resultados sugerem que os probióticos alteram indiretamente o microambiente intestinal ou o epitélio intestinal de maneira a favorecer a proliferação de C. parvum. / Abstract: Cryptosporidiosis, a leading cause of infant diarrhea, is caused by apicomplexan parasites classified in the genus Cryptosporidium. The lack of effective drugs is motivating research to develop alternative treatments. To this aim, the impact of probiotics on the course of cryptosporidiosis was investigated. The native intestinal microbiota of specific pathogen-free immunosuppressed mice was initially depleted with orally administered antibiotics. A commercially available probiotic product intended for human consumption was subsequently added to the drinking water. Mice were infected with Cryptosporidium parvum oocysts. On average, mice treated with probiotic developed a more severe infection. The probiotics significantly altered the fecal microbiota, but no direct association between ingestion of probiotic bacteria and their abundance in fecal microbiota was observed. These results suggest that probiotics indirectly altered the intestinal microenvironment or the intestinal epithelium in a way that favors proliferation of C. parvum. / Doutor

Evalúa la presencia de Cryptosporidium spp. en alpacas y su relación con las variables diarrea y relación madre/cría, durante la temporada de parición de alpacas del 2011. Estudia muestras de heces de 1650 alpacas, 825 madres y 825 crías de hasta 15 días de edad, de las localidades de Keyosenca, Colores, U.N.A. Chuquibambilla y Santa Rosa en la provincia de Melgar del departamento de Puno. Realiza la coloración de las muestras utilizando la tinción de Ziehl–Neelsen modificado y las examina en un microscopio para su diagnóstico. Los resultados se evalúan en tablas de contingencia, con intervalos de confianza del 95%. Los datos son analizados utilizando una regresión logística múltiple. En este análisis no se encuentra relación entre la presencia de diarrea (liquida y pastosa) con la presencia de Cryptosporidium spp. La prevalencia en las crías es de 5%, porcentaje que se puede considerar como relativamente bajo. En el caso de las madres la prevalencia es del 11%. Las crías y sus madres se muestrearon a los 15 días de la parición, tiempo después de la depresión inmune periparto lo cual indica que la presencia de Cryptosporidium en las madres no es un factor de riesgo para la presencia en las crías. La baja prevalencia de Cryptosporidium parvum puede deberse a buenas condiciones de manejo, como la rotación periódica de dormideros, la utilización de estercoleros o la ingestión de calostro después del parto. Al no encontrarse relación madre/cría con la presencia de Cryptosporidium se sugiere estudiar las prevalencia de Cryptosporidium en las alpacas madres y las crías durante la depresión inmune periparto, secuenciando las cepas para ver si existe relación entre los Cryptosporidium de las madres y sus crías. / Tesis

The human serine protease inhibitor (serpin) alpha-1-antitrypsin (AAT) was studied for potential interaction with components of the protozoan parasite Cryptosporidium parvum. A homogenate prepared from C. parvum oocysts was incubated with purified human AAT, and complexes formed between the serpin and components of the homogenate were detected using an enzyme-linked immunosorbent assay (ELISA). Serpin:parasite infections were effectively blocked by preincubating AAT with a cognate target enzyme, porcine pancreatic elastase, prior to performing the ELISA on the homogenate. Incubation of a mixture of C. parvum oocysts and sporozoites with AAT demonstrated preferential fluorescence labeling of the sporozoite surface membrane by indirect immunofluorescence assay. Localization of serpin complexes on sporozoites was confirmed by immunogold electron microscopy. AAT was evaluated for in vitro anticryptosporidial activity in a bovine fallopian tube epithelial (BFTE) cell culture system using both oocysts and filter purified sporozoites as inocula. Serial dilutions of AAT were mixed with oocysts (or sporozites) and used to inoculate BFTE cell monolayers. Inoculted cells were maintained at 37ºC/5% CO2 and collected at 24-,48-,72-, and 96-hr post-inoculation intervals. The addition of AAT and other select protease inhibitors (i.e.,antipain, aprotinin, leupeptin, soybean trypsin inhibitor, and phenylmethylsulfonyl floride) significantly inhibited parasite infection (P<0.01) in a concentration- and time-dependent manner when bleach-decontaminated oocysts were used in the inoculum. The anticryptosporidial activity of AAT is postulated to be linked to an antagonistic effect on oocyst excystation and, putatively, the forced expenditure of bioenergetic reserves prior to host cell invasion. This postulate was supported by the observations that serpin activity had no statistically significant effect on reducing established in vitro infections (i.e., 24 hr post-inoculation prior to addition of AAT) and did not inhibit infection of BFTE cells when inoculted with sporozoite preparations. The combined application of AAT and the aminoglycoside paromomycin demonstrated a synergistic anticryptosporidial effect on in vitro infection and suggested the basis for a multi-agent therapeutic protocol in preventing cryptosporidosis. These studies collectively demonstrated an inticryptosporidial potential for serine protease inhibitors, in particular for AAT, and suggest an alternative approach to conventional therapeutic strategies.

The purpose of this study was to quan ti tate the populations o f lymphocytes in the s pleens and intestines of normal and immunosuppressed adult C57BL/6N mice that were noninfected or infected with Cryptosporidi um parvum. This was accomplished by using the following methodologies: immunohistochemistry, ELISA-spot assay, and flow cytometry. Mice in groups 1 and 2 were immunosuppressed, but only group 2 was in fected. Mice in group 3 were only infected, whereas group 4 served as the normal control . Mice were immunosuppressed with dexamethasone (DEXI at a dosage of 125~g / mouse/day. Infected mice received 106 oocysts per os . The numbers o f lymphocytes were monitored from day 0 to day 18 postinfect i on. Flow cytometry using antibodies directed against CD4+ and CDS+ T cells (helper and cytotoxic, respectively) and B cells (expressing IgG, IgM, and IgA receptors) revealed that c. parvum did not evoke an alteration in the phenotypic profile of lymphocytes within spleens or Peyer's patches (PP) of mice in groups 2 and 3 that was statist i cally different from groups 1 and 4. Immunosuppressed mice (groups 1 and 2) had significantly fewer lymphocytes (bearing CD4+, IgG, IgM, and IgA receptors) within the spleen when compared with mice in groups 3 and 4 (P

Cryptosporidium parvum and Toxoplasma gondii are obligate intracellular parasites transmitted by ingestion of resilient walled structures called oocysts. Infection is self-limiting in adults with normal immune systems. However, severe disease can occur in immunocompromised individuals, or those without cellular immunity. Cryptosporidium is a leading cause of infant mortality in developing countries, due to diarrhea. There are no human vaccines and no broad effective drug treatments. Several vaccine candidates have been described: the glycoproteins Gp900, Gp40, and Gp15 and the protein Cp23, the immuno-dominant-antigen. Details about modifications to these proteins have not previously been reported. Using mass spectrometry, we identified 16 Cryptosporidium N-glycosylated proteins, including Gp900 and a possible oocyst wall protein. The observed N-glycan structures exhibited only two compositions: HexNAc2Hex5 and HexNAc2Hex6; these glycoforms had a single extended arm. The simplicity of Cryptosporidium N-glycans contrasts with the complexity of host N-glycans. Four heavily O-glycosylated proteins included Gp900, Gp40, Gp15, and a novel mucin-like protein, Gp20. Single O-HexNAc residues modified Ser/Thr in low density regions of Gp15 and Gp900, while attachment of O-HexNAc residues on tandem Ser/Thr repeats of Gp20 and Gp40 approached saturation. Identification of N-acetylgalactosamine (GalNAc) as the HexNAc released from proteins suggests that most Cryptosporidium O-glycans resemble the immunogenic Tn antigen (O-GalNAc). The immunodominant antigen Cp23, while not glycosylated, was discovered to be N-myristoylated and S-palmitoylated on the first and second residues, respectively. This is the first identification in Cryptosporidium of these modifications. Information about the N-glycans, O-glycans, and lipid modifications may be useful for design of better serodiagnostic reagents and more effective vaccines. To date, there are no vaccines against Toxoplasma infection, and the only available pharmaceutical therapies are expensive. In the second study, a novel O-fucose modification was discovered on nuclear pore-associated proteins including nucleoporins. This observation has profound implications on how the organism may regulate trafficking in/out of the nucleus by employing a system parallel to that described for O- linked N-acetylglucosamine in other organisms. In summary, the new details regarding the vaccine candidates of Cryptosporidium and the discovery of the novel O-fucose modifications in T. gondii provide information that could prove useful for development of effective drugs and vaccines. / 2018-11-01T00:00:00Z

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis

Cats are infected with a variety of internal parasites, some of which are zoonotic. Therefore, being able to effectively detect and determine prevalence of internal parasites in cats is important for both feline and human health. Some parasites are easier to detect than others. Diagnosing Giardia duodenalis and Cryptosporidium spp. can be difficult because cysts and oocysts shed in the feces are small, shed intermittently, and require a trained technician to consistently identify them. As a result, infections with these protozoan parasites can be missed. Fecal immunoassays detect antigens in feces and can have increased sensitivity when compared to traditional microscopic techniques, but still do not detect every infection. The current reference standard is an immunoassay known as the direct immunofluorescent assay, but it requires expensive equipment and a long incubation period. As a result, two prototype lateral flow fecal immunoassays, the Cryptosporidium EZ VUE and Giardia EZ VUE, designed by TECHLAB® Inc were evaluated for the ability to detect G. duodenalis and Cryptosporidium spp. infections in cats because they are cheap, easy to use, easy to store and easy to interpret. In addition, samples were examined using a 33% zinc sulfate (ZnSO4) centrifugal fecal flotation procedure and the MERIFLUOR® Cryptosporidium/Giardia direct immunofluorescent assay (IFA), which served as the reference test. Other internal parasites found on the centrifugal fecal flotation with zinc sulfate were recorded to determine prevalence. Both EZ VUE fecal immunoassays demonstrated potential in diagnosing infections in cats when compared to centrifugal fecal flotation and the reference. Additionally, a variety of other internal parasites were identified. This included several potentially zoonotic species including Spirometra mansonoides, Ancylostoma sp. and Toxocara cati, which was also the most commonly identified species of parasite. Additionally, it was determined that several factors may contribute to higher prevalence of parasites especially in cats with the status of stray or feral. / M. S.

Amylopectin granules in Apicomplexan protozoa are hypothesized to be used as an energy source to aid the parasites in surviving in the environment allow latent stages to excyst and release infective stages, allow them to be mobile, invade host cells, and to continue their life cycle. The objective of this project was to determine if parasite glycolytic enzymes: alpha-amylase, amyloglucosidase, enolase, lactate dehydrogenase, and phosphorylase could be used to decrease amylopectin stores and subsequently infectivity of Cryptosporidium parvum oocysts/sporozoites in both fresh oocysts and stored oocysts. In addition, glycolytic enzymes and substrates: glucose, glucose-1-phosphate, and glycogen synthase were investigated to determine if they can be used to increase amylopectin stores and thus increase infectivity to aid in detection/storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in 1mg/ml glycolytic enzymes or substrates (except glucose - 0.05M and glycogen synthase - 1U/ml) and electroporated. Oocysts were incubated at 37&#176;C for one hour to allow treatments to react with amylopectin followed by incubation on HCT-8 cells for 24 hours for infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity. An amylopectin assay and excystation assay was performed to determine if the enzymes degraded amylopectin and if decreased amylopectin reduced excystation. Alpha amylase and amyloglucosidase had the greatest impact on reducing both amylopectin and infectivity of fresh oocysts with reductions of 99.5% and 99.1% in infective oocysts, respectively (P<0.05). These results suggest that amylopectin may be an important factor in infection, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. In fresh oocysts, amylopectin content was correlated to excystation and infectivity with a decrease in amylopectin correlating to decreased excystation and infectivity. In contrast, there was no direct correlation for stored oocysts. When glucose, glucose-1-phosphate, or glycogen synthase was used to increase infectivity, results show that glycogen synthase had little effect, but glucose and glucose-1-phosphate significantly increased amylopectin content, excystation, and infectivity. In conclusion, amylopectin may be an important polysaccharide store of Cryptosporidium parasites to cause infection by allowing excystation of the oocysts to release infective sporozoites. / Ph. D.