Thesis (Ph.D.)--Murdoch University, 2004. / Thesis submitted to the Division of Health Sciences. Includes bibliographical references (leaves 145-167).

Orientador: Marcelo Vasconcelos Meireles / Resumo: Cryptosporidium spp. são parasitos protozoários que infectam principalmente as células epiteliais do trato digestivo em vários animais, incluindo humanos. Este protozoário causa doença entérica, que se caracteriza como diarréia auto-limitante em indivíduos saudáveis, ou mesmo doença fatal em pacientes imunodeficientes. Cryptosporidium parvum é a principal causa de infecção em mamíferos e é o principal responsável pela criptosporidiose zoonótica. Novos métodos de detecção, prevenção, tratamento e estratégias de controle para criptosporidiose em seres humanos são cada vez mais necessários. Anticorpos e proteínas recombinantes têm potencial para o desenvolvimento de medicamentos, vacinas ou podem ser usados para desenvolver técnicas de diagnóstico mais acessíveis com alta sensibilidade e especificidade. Neste estudo, o objetivo principal foi a expressão da proteína recombinante CP41 de C. parvum em Escherichia coli e produção de um anticorpo específico para CP41 recombinante (rCP41), visando a detecção de oocistos de Cryptosporidium por imunofluorescência. / Doutor

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-21Bitstream added on 2014-06-13T19:26:14Z : No. of bitstreams: 1 matos_dj_me_araca.pdf: 760484 bytes, checksum: c06dae3728a9ffd470f98e2c615bdd6d (MD5) / O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo. / The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms.

Les parasites du genre Cryptosporidium comprennent des espèces infectant le tractus gastro-intestinal ou respiratoire d’un grand nombre de vertébrés y compris l'homme. Ces protistes intracellulaires sont les agents d’une zoonose cosmopolite à transmission oro-fécale, la cryptosporidiose. Au vu des travaux de notre laboratoire, nous savons à présent que Cryptosporidium parvum est également capable d’induire des néoplasies digestives chez un modèle murin SCID (Severe Combined Immunodeficiency mice), traitées ou pas par la dexaméthasone. Alors que C. muris, une autre espèce de Cryptosporidium, induit une infection chronique non associée à des transformations néoplasiques.Pour toutes ces raisons, il nous est apparu intéressant d’effectuer un travail de thèse articulé autour de trois axes principaux, l’épidémiologie, la transmission et la pathogénicité du parasite Cryptosporidium. Nous nous sommes intéressés dans un premier temps à l’épidémiologie moléculaire et la biodiversité génétique de Cryptosporidium dans des populations humaines de la région du Nord-Liban. Ceci nous a permis de mettre en évidence une prévalence de 5% de Cryptosporidium chez la population générale avec une prédominance de C. hominis. Ce qui constituait les premières données épidémiologiques de la cryptosporidiose au Liban. Ensuite d’autres études nous ont permis de montrer que cette prévalence pouvait atteindre même 10% chez les patients symptomatiques et les enfants.Dans un second temps, nous avons voulu étudier le mode de transmission du parasite et les facteurs de risque pouvant y être associés. Pour ce faire, une recherche du parasite a été réalisée aussi bien au Liban qu’en France chez des animaux d’élevage, sauvages, de compagnie et en captivité. Une première étude a été réalisée chez des patients et des bovins du Nord-Liban. L’ensemble des données rapportées nous permettent de suggérer un mode de transmission de la cryptosporidiose majoritairement anthroponotique au Liban, mais les résultats du génotypage ne permettent pas d’exclure la présence d’une transmission zoonotique. D’autres études réalisées en France, notamment sur des échantillons de selles collectées auprès des zoos de la Palmyre (à Royan) et de Lille ont montré un taux de prévalence de Cryptosporidium spp inférieur à 1%. Ces animaux ne semblent donc pas être un réservoir potentiel de cette infection. Alors que chez les poissons sauvages, nous avons pu identifier la présence entre autre de l’espèce zoonotique C. parvum dans l’estomac et l’intestin des poissons. Ceci nous permet de considérer les poissons comme étant une source de contamination potentiel pour l’homme, l’animal mais également pour l’environnement.Enfin le troisième axe avait pour but d’étudier la pathogénicité de ce parasite. Pour commencer nous avons voulu explorer les mécanismes de la cancérogénèse induite par la souche IOWA de C. parvum au niveau de la région iléocæcale des souris SCID traitées par la dexaméthasone (SCID-D). Pour ce faire nous nous sommes intéressés à quatre marqueurs de voies de signalisation cellulaires impliquées dans la survenue de cancers colorectaux (APC, Bêta-caténine, P53 et K‐ras). Nous avons ainsi pu montrer que la voie Wnt était impliquée dans ce processus. Ensuite nous avons voulu étudier l’association entre la pathologie cancéreuse et le parasitisme par Cryptosporidium chez l’homme. Une recherche du parasite a donc été réalisée dans des biopsies d’origines coliques et gastriques inclues en paraffine appartenant à des patients atteints ou non de cancers digestifs. Une différence significative a été rapportée entre la prévalence de la cryptosporidiose retrouvée chez la population de patients présentant des lésions cancéreuses (17%) et celle du groupe control constitué de patients non cancéreux mais présentant des symptômes (7%) p-value = 0.03. L’ensemble de ces données obtenues chez l’animal et chez l’homme montre que ce parasite a un impact important en santé humaine et animale. / Parasites of the genus Cryptosporidium comprise species infecting the gastrointestinal or respiratory tract of a wide variety of vertebrates including humans. These intracellular protists are the agents of a cosmopolitan zoonosis, with féco-oral transmission, cryptosporidiosis. Recent work from our laboratory, showed that the zoonotic species Cryptosporidium parvum is capable to induce digestive neoplasia in a SCID Severe Combined Immunodeficiency Mice (SCID) model, treated or not with dexamethasone. However C. muris, another species of Cryptosporidium, induces chronic infection in this rodent model but is not associated with neoplastic transformation.For all these reasons, it seemed interesting to carry out a thesis project articulated around three different axes: epidemiology, transmission and pathogenesis of the Cryptosporidium infection. We focused initially on the molecular epidemiology and genetic biodiversity of this parasite among human populations in North Lebanon. We found a Cryptosporidium prevalence of 5% among the general population, being C. hominis the predominant species. This prevalence could reach until 10% in symptomatic patients and children. This is the first epidemiological data about cryptosporidiosis in this country.Secondly, we studied the transmission routes and the main risk factors associated with the transmission of this parasite. To do this, a first study was conducted in parallel among animal populations in North Lebanon and France. The reported data suggest a predominance of an anthroponotic route of transmission for cryptosporidiosis in Lebanon, but the results of genotyping does not exclude the presence of zoonotic transmission. Other studies conducted in France, especially based on collection of stool samples in the zoos of Palmyre (Royan) and Lille showed that Cryptosporidium spp were present in less than 1% of captivity animals. The low prevalence strongly demonstrates that these animals play a negligible role as potential reservoirs of infection. While in wild fish, we could identify the presence of C. parvum, a zoonotic species, in the stomach and the gut of fish. These data suggest that the fish could be a natural host of C. parvum and a potential source of contamination for humans, animals but also for the environment.Finally, the third topic aimed to study the pathogenicity of this parasite. Firstly, we explored metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis in the SCID model treated with dexamethasone (SCID-D). We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as β-catenin, Apc, E-cadherin, Kras and p53. We were able to show that the Wnt pathway was involved in this process. Finally, we wanted to study the association between digestive cancer and parasitism with Cryptosporidium in humans. Cryptosporidium molecular detection was therefore carried out in colonic and gastric biopsies belonging to patients with and without digestive cancers of recent diagnosis collected in North Lebanon. A statistically significant difference was observed between the prevalence of cryptosporidiosis found among the population of patients with digestive cancer (17%) and that of the control group consisting of non-cancer patients but with digestive symptoms (7%) (p-value = 0.03). All these data obtained in animals and humans strengthens the importance of this parasite in public health.

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

Orientador: Katia Denise Saraiva Bresciani / Resumo: Os protozoários do gênero Cryptosporidium spp. são parasitos intracelulares obrigatórios, pertencentes ao filo Apicomplexa. Estes parasitos são capazes de se desenvolver nas microvilosidades das células epiteliais do intestino delgado de hospedeiros vertebrados, sendo potencialmente letais em adultos e crianças imunodeficientes. Não existem medicamentos eficazes para controlar essa doença e o desenvolvimento de medicamentos é dificultado pela falta de métodos de cultura eficazes que atuam permitindo detecção completa do ciclo de vida do parasito. Uma lacuna fundamental existe em relação ao entendimento de como os esporozoítos de Cryptosporidium spp. interagem com as células para iniciar a invasão e sua replicação. Assim, foi estudada essa lacuna para explicar a distinção entre aspectos da biologia do Cryptosporidium spp. e a especificidade do hospedeiro. Por meio de um ensaio de imunofluorescência detectou-se separadamente parasitos ligados e que invadiram às células e qual foi sua evolução ao longo de 2, 24 e 48 horas do parasitismo nas células hospedeiras. Com base nos genes mais significativos nas análises LDA (análise linear discriminante) e RDA (análise de redundância) e com maior valor FPKM (fragmentos por quilobase de transcritos por milhão de leituras mapeadas), pode-se perceber uma diferença significativa na expressão gênica desse parasito extracelular e intracelular. Os genes que mais se expressaram em células incubadas por 48 horas após a infecção foram analisados ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The protozoa of the genus Cryptosporidium spp. are obligate intracellular parasites that belongs to the phylum Apicomplexa. These parasites are capable to develop in the microvilli of epithelial cells of the gastrointestinal tract of vertebrate hosts, being potentially lethal in immunodeficient adults and children. There are no effective medications to control cryptosporidiosis, and drug development is hampered by the lack of effective culture methods that act to allow the complete life cycle of the parasite. It is fundamental to understand how Cryptosporidium spp. sporozoites interact with cells to initiate invasion and replication. Thus, this subject has been studied to explain the distinction between Cryptosporidium spp. biology and host specificity. By an immunofluorescence assay we detected bounded parasites invading host cells and their evolution over two, 24 and 48 hours. Based on the most significant genes in the LDA (Linear Discriminant Analysis) and RDA (redundancy analysis) analysis and with higher FPKM value (fragments per kilobase of transcribed per million mapped readings), we can perceive a significant difference in the expression of this extracellular and intracellular parasite. The genes that most expressed in cells incubated for 48 hours after infection were analyzed for the biological species of Ontology of Genes (GO) that show significantly the enriched processes and functions of the intracellular parasite. The knowledge of the genes that are most expresse... (Complete abstract click electronic access below) / Doutor

Orientador: Marcelo Vasconcelos Meireles / Banca: Sérgio Diniz Garcia / Banca: Weslen Fabrício Pires Teixeira / Resumo: Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. e comparar três métodos de detecção deste protozoário em amostras fecais de canários (Serinus canaria) criados em cativeiro nas regiões Sul e Sudeste do Brasil. Um total de 498 amostras foi purificado por centrífugo-flutuação em solução de Sheather. A detecção de Cryptosporidium spp. foi realizada utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR (gene 18S rRNA), seguida de sequenciamento dos fragmentos amplificados, e PCR em duplex em tempo real (gene 18S rRNA) específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%;15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp.. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries. / Mestre

Resumo: O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo. / Abstract: The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms. / Orientador: Katia Denise Saraiva Bresciani / Coorientador: Marcelo Vasconcelos Meireles / Banca: Fernanda Paiva / Banca: Carlos Noriyuki Kaneto / Mestre

Cryptosporidiosis is a common diarrheal disease caused by intestinal infection with the apicomplexan parasite Cryptosporidium, in humans usually either with C. hominis or C. parvum. Unfortunately, given a large burden of disease in children and immunocompromised people like AIDS patients, the only currently approved treatment, nitazoxanide, is unreliable for these patient populations. To address the urgent need for new drugs for the most vulnerable populations, large phenotypic screening efforts have been established to identify anti-Cryptosporidium growth inhibitors in vitro (hits). However, in the absence of a gold standard drug, the in vitro and in vivo characteristics that should be used to prioritize screening hits are not known. This thesis is focused on identifying promising anti-Cryptosporidium hits and drug leads, and using them to establish validated methods to guide hit-to-lead studies for anti-Cryptosporidium drug development. A re-analysis of our phenotypic screen of the Medicines for Malaria Venture Open Access Malaria Box identified a promising C. parvum growth inhibitor, MMV665917. It had similar in vitro activity against C. hominis, C. parvum Iowa, and C. parvum field strains, and it was amenable to preliminary structural activity relationship studies using commercially available variants, with one variant demonstrating nanomolar potency. Furthermore, MMV665917 was effective in vivo in an acute interferon-γ mouse model of cryptosporidiosis; and it appeared to cure an established infection in the chronic NOD SCID gamma (NSG) mouse model, unlike nitazoxanide, paromomycin, and clofazimine. We hypothesized that anti-Cryptosporidium activity in the highly immunocompromised chronic NSG mouse model might relate to compounds being capable of killing and eliminating parasites (cidal), rather than only preventing growth (static). To test this, we developed a novel in vitro parasite persistence assay that showed that MMV665917 was potentially cidal, whereas nitazoxanide, paromomycin and clofazimine appeared static. This pharmacodynamic assay also provided the concentration of compound required to maximize rate of parasite elimination, which could help design in vivo experiments. To further characterize compounds based on mechanism of action, we developed a range of in vitro medium-throughput life-stage assays. To validate and gain value from the assays, a “learner set” of compounds from our in-house screens and collaborations were tested in all of the in vitro assays and in the in vivo NSG mouse model. Using these assays, it was possible to group molecules based on chemical class/mechanism of action. Because compounds from distinct groups showed activity in the NSG mouse model, these methods could be used to obtain a diverse set of early-stage Cryptosporidium inhibitors for prioritization. Furthermore, compounds that appeared static in the in vitro parasite persistence assay did not have activity in the NSG mouse model. In summary, we report the identification and development of a highly promising initial lead, MMV665917, and report a range of in vitro assays that can be used to prioritize anti-Cryptosporidium hits and leads.