Giardia and Cryptospovidium are both recognised as important causes of infectious diarrhoea in children worldwide, and childcare centres have been shown to be a major site of infection. The incidence of infectious diarrhoea in children attending childcare centres has been estimated at between two to five times greater than in children cared for at home. Both Giardia and Cryptospovidium have a faecal-oral route of transmission that facilitates their spread in childcare environments, but can also be interrupted through the use of efficient hygiene protocols such as handwashing. Despite their importance as causes of infectious diarrhoea, there are no data on the prevalence or transmission dynamics of these parasites in Australian childcare centres. The present study was designed to determine the prevalence and incidence of both Giardia and Ciyptosporidium in children attending childcare centres in Perth,Western Australia. Data were collected on asymptomatic infection, seasonal trends, the transmission dynamics of the parasites and risk factors for infection. The second part of the study involved the development, implementation and evaluation of a health intervention package designed to interrupt the transmission of causative organisms of infectious diarrhoea in childcare centres. This intervention was based on appropriate and effective handwashing. Over a period of 23 months, 1 172 faecal samples were collected from non-toilet-trained children (n=306) attending 14 childcare centres in Perth, Western Australia. Where possible, family and contacts of infected children were also sampled to determine the dynamics of infection in the community. Information on syrnptomology of infections and risk factors for infection was obtained by the administration of a questionnaire to parents of all the children in the study. Over all the childcare centres in the study, 7.8% of children were positive for Giardia and 10.8% were positive for Cryptosporidiunz. Of these, 37.5% of the Giardia-positive children returned positive samples on two to four occasions, but not always consecutively, suggesting either continuous or repeated infection with the parasite. Only 12% of children who were Cryptosporidiurn-positive were infected for two consecutive months. The major findings of this study included a significant seasonal peak in the prevalence of both Giardia and Cryptosporidium, with 50% of Giardia and 73% of Cryptosporidiurn infections occurring during the autumn months of March, April and May, and a high proportion of asymptomatic Giardia infections (45%), compared with only 13.5% of asymptomatic Cryptosporidiurn infections. There was evidence for the transmission of both Giardia and Cryptosporidium infections to household contacts of infected children. Of the children who were found to be positive, faecal samples were also collected from 28 family members of those children with Giardia and from 14 family members of those with Cryptosporidiunz. Of these, 17.9% family members of the children with Giardia and 28.6% of family members of the Cryptosporidiuin children returned positive faecal samples. The only significant risk factor for Giardia infection was the number of adults living in the household, with infection more likely to occur in children who had a greater number of adults in the household. Significant risk factors for Clyptosporidium infection included the age of the child: the mean age of the positive children (20.6 months) was higher than in the negative children (16.6 months), and the length of time enrolled at the centre. Children who were positive had attended for a longer time than those who were negative (1 1.2 and 7.8 months respectively). These results have important implications for the control of infection with these organisms, both within and beyond childcare centres. Since these parasites can be readily transmitted by an asymptomatic carrier, the high percentage of asymptomatic cases in this study, particularly of Giardia, strengthens the argument for health interventions which are directed at interrupting the trailsmission of the parasite. A health intervention programme was developed that focused on handwashing procedures and was targeted at the carers, the children and the parents of the children in the centres. It was designed to be a low-cost programme both financially, and in the time and effort required to implement the programme within the childcare centre, to enhance compliance with the intervention. The success of the programme in changing the knowledge, attitudes and practices of carers was evaluated through a pre- and posttest questionnaire. This showed that the programme successfully improved the knowledge of the carers in the test centres in several important areas of infection control. These included knowledge about specific organisms causing infectious diarrhoea in childcare centres, transmission of these by asymptomatic individuals and increased knowledge about effective handwashing technique. Because it has been repeatedly shown that increased knowledge does not always translate into improved practices, and that interventions are not always successful in maintaining an improvement in the desired practices, a subjective evaluation was also performed. This was designed to determine how effective the intervention was perceived to be by the carers themselves, and whether they would continue to use the intervention over time. The results showed that the majority of the carers (>88%) found the intervention appropriate and useful in teaching both the carers and the children within the centres, the importance of handwashing. Twelve months after the intervention had first been implemented, 57% of the centres in the study were still using the intervention at least once per month and a further 2996, while using it less than this, still continued to use it occasionally. This is important information, since an intervention can only be useful if it is actually being used.

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.

Abstract not available

Thesis (Ph.D)--Murdoch University, 2009. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 150-162)

Cryptosporidium parvum can cause debilitating disease in immunocompetent persons with cholera-like symptoms characterised by self-limiting, profuse diarrhoea; on the other hand asymptomatic infection with this organism frequently occurs. However, in immunocompromised patients, the disease is more severe and is lifethreatening. A pivotal aspect of the present survey was a comparative assessment of four commonly used staining techniques (viz. modified Ziehl-Neelsen, safranin-methylene blue, auramine phenol fluorescence and Sheather's sucrose flotation) for the detection and identification of Cryptosporidium oocysts. The Sheather's flotation method proved to be superior to the other three procedures which were not only less sensitive but also less specific. A modification of the Sheather's flotation technique was developed for use with diarrhoeal stools; this was found to be simple, reliable, costeffective and the least time consuming of the above methods; this was used exclusively in a subsequent survey of the association of Cryptosporidium infection with diarrhoea in hospitalised children. Although previous epidemiological surveys of cryptosporidiosis have been conducted in South Africa standardised methods have not been employed. This initial assessment of diagnostic techniques therefore provided a tool for accurately assessing the importance of Cryptosporidium as a causative organism of diarrhoea. In an extensive study performed on children younger than 10 years old, who were hospitalised with a primary diagnosis of diarrhoea at King Edward VIII Hospital, it was found that 9,0% (111/1229) were passing Cryptosporidium oocysts; this was the second most common enteric pathogen. In 72% (80/111) of patients with Cryptosporidium infections it was the only pathogen. The prevalence of cryptosporidiosis was highest during the months of February, March, April and May; direct correlation between the rainfall in the Durban area and the prevalence of cryptosporidiosis was demonstrated (r = 0,6125). Cryptosporidium infection was more prevalent in the 4-6 month age group (p = 0,001). The fact that Cryptosporidium infections may be symptomatic in some individuals and asymptomatic in others, suggests that strain differences in respect of pathogenic potential may occur. A prerequisite to the investigation of strain differences was to increase parasite numbers; both in vivo and in vitro culture techniques were employed. Culture in chicken embryos failed to increase the parasite population and only limited areas of the chorio-allantoic membranes showed a few developmental stages. Cell cultures proved to be more suitable for Cryptosporidium growth and parasite numbers increased proportionally with duration in culture. Attempts at infecting suckling Balb/c mice were unsuccessful; however experimental infection of immunosuppressed adult rats facilitated the examination of various developmental stages of the parasite. Isoenzyme electrophoresis is an excellent method for demonstrating polymorphism in many species. Of the five enzyme systems that were tested, glucose phosphate isomerase, malic enzyme and phosphoglucose dehydrogenase proved to be the most promising. The electrophoresis of lysates, prepared from oocysts, in an agarose gel system was found to give adequate and reproducible resolution of isoenzyme patterns. Isoenzyme polymorphism could be demonstrated in oocysts harvested from the stools of four children. Such polymorphism has not been described previously and indicates a more extensive study to investigate strain differences, and to correlate these with the clinical histories of infected subjects. This approach may be invaluable in elucidating the pathogenesis of Cryptosporidium infections in man. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1990.

Cryptosporidiosis is a common infectious diarrhoeal disease of mammalian livestock and humans worldwide. The etiological organisms responsible are intestinal apicomplexans of the genus Cryptosporidium, including C. parvum, that infect intestinal epithelial cells. Immunocompromised or malnourished hosts develop severe life-threatening disease. Immunological elimination of Cryptosporidium requires CD4+ T cells and IFN-γ. Nevertheless, studies have shown innate immune responses have a significant protective role. Importantly, in T cell-deficient mice, IFN-γ is important for control of C. parvum infection. In innate immunity natural killer (NK) cells are major producers of IFN-γ and are activated by cytokines including type I IFNs but the roles of these components in immunity to Cryptosporidium infection have not been investigated. Therefore, the purpose of this project was to study the involvement of type I IFNs and NK cells in immunity to C. parvum employing in vitro and in vivo (murine) infection models. Enterocytes were shown capable of the production of type I IFNs in response to C. parvum infection. These cytokines directly inhibited parasite development in epithelial cells. Also, in neonatal SCID mice the level of infection increased after treatment with anti-type I IFN neutralising serum. A higher level of infection was observed in Rag2-/-γc-/- mice deficient in T, B and NK cells in comparison to Rag2-/- mice with a normal NK cell population and early mortality during chronic infection of adult animals was associated with the absence of NK cells. Using cultures of SCID mouse splenocytes, NK cells were the main source of IFN-γ in response to C. parvum antigen stimulation. However, IFN-γ was also found to have a protective role in Rag2-/-γc-/- mice, implying cells other than lymphocytes produce this cytokine. In conclusion, this is the first study to indicate important protective roles for type I IFNs and NK cells in innate immunity against C. parvum.

Orientador: Marcelo Vasconcelos Meireles / Resumo: Existem poucos estudos relacionados à epidemiologia e aspectos clínicos da infecção por Cryptosporidium spp. em suínos, especialmente no Brasil. Os suínos apresentam infecções clínicas ou subclínicas e são mais comumente infectados por Cryptosporidium suis e Cryptosporidium scrofarum. O objetivo deste trabalho é determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em suínos originados de quatro fazendas com sistemas de produção semi-intensivos no Estado de São Paulo, Brasil. Duzentas amostras fecais foram coletadas de suínos, de março a agosto de 2018, e analisadas para verificar a ocorrência de Cryptosporidium spp. por nested PCR visando a amplificação de fragmento parcial do gene da subunidade 18S do rRNA (18S rRNA) e sequenciamento genético. Resultados positivos para Cryptosporidium spp. foram detectados em 17% (34/200) das amostras. O sequenciamento dos fragmentos amplificados identificou C. scrofarum em 10 das 11 amostras sequenciadas. Dessa forma, verificamos que a espécie zoonótica C. scrofarum está presente em fazendas de suínos com sistemas de produção semi-intensivos no Estado de São Paulo e que não houve diferença significativa entre as taxas de prevalência entre suínos de <3 meses e >3 meses de idade. / Mestre

To determine the antigenic relatedness of the different developmental stages of Cryptosporidium parvum, monoclonal IgG3 antibody (mAb), Cmg-3, was produced by immunizing mice with partially purified merozoites. The monoclonal Cmg-3 reacted with a 3.5 kDa antigen of sporozoites in western blots and appeared to react with cell surface antigens of air-dried merozoites and sporozoites using immunofluorescence (IF). Additional mAbs, C6B6 (IgG1) and C4A1 (IgM), which react with a 20 kDa and multiple sporozoite antigens, respectively, were employed for immunoelectron microscopic studies with Cmg-3. These mAbs showed similar (surface/cytoplasmic) immunoelectron microscopic colloidal gold labeling patterns with all C. parvum life cycle stages. The three mAbs were also examined for potential modulation of cryptosporidial infections in vivo by daily oral mAb administration to oocyst-inoculated neonatal mice. Monoclonal-treated neonatal mice were sacrificed four and eight days post infection (pi). Differences in infection rates were observed among the treatment groups (p < .05). Suckling mice treated daily with orally administered mixtures of mAbs (ascitic fluids) showed significantly reduced parasite loads compared to control mice at four and eight days pi, while suckling mice receiving mAb Cmg-3 alone showed significant differences only at four days pi. Passive transfer of immunity using hyperimmune bovine colostrum was performed to determine the therapeutic and prophylactic efficacy of daily oral administration of anti-C. parvum antibody on the manifestation of cryptosporidial disease in neonatal mice as a model for treating cryptosporidiosis in immunocompromised patients. Hyperimmune colostrum was found to provide therapeutic and prophylactic efficacy against cryptosporidiosis in neonatal mice. Significantly fewer (p < 0.05) stages of C. parvum were found in mice that received hyperimmune skim colostrum (HSC) or hyperimmune original colostrum (HC) than in those treated with control colostrum (CC) or saline. Using IF, antigen-specific IgG in HSC and HC to C. parvum was 35 times greater than that of CC. There was no significant difference between groups treated with HSC or HC (p < .05), which suggests that the immunoglobulins, other biologically active factors such as cytokines, or both, might be active factors of immunity against cryptosporidiosis.

Resumo: Um total de 196 amostras fecais de bezerros leiteiros mestiços, de 7 a 30 dias de idade de ambos o sexos foram colhidas em 31 propriedades com o objetivo de determinar a ocorrência de Cryptosporidium na região Noroeste do Estado de São Paulo, assim como caracterizar molecularmente as espécies envolvidas nesta parasitose. Realizou-se a análise microscópica pela técnica de coloração negativa com verde malaquita em todas as amostras de fezes. Para identificação molecular de Cryptosporidium, utilizou-se a reação de nested PCR, utilizando primers específicos para amplificação de fragmentos do gene codificador da subunidade 18S do RNA ribossômico e do gene da glicoproteína GP 60, submetendo o produto da PCR a sequenciamento e análise filogenética. Por meio de exame de fezes, foi observada positividade de 2% (4/196), por meio de microscopia, e de 10,7% (21/196) pela nested PCR. Como resultado do sequenciamento, foram identificadas quatro espécies de Cryptosporidium: C. parvum (subtipo IIa15G2R1) C. ryanae, C. bovis e C. andersoni. Esta descoberta de C. parvum subtipo IIaA15G2R1 na região estudada ilustra a utilidade da subtipagem. Embora estes resultados iniciais baseados em subtipos com GP60 sejam úteis para compreender a dinâmica de transmissão das espécies de Cryptosporidium, o número de amostras analisadas é relativamente pequeno e muito mais ainda pode ser pesquisado. É interessante notar que as informações de tipagem molecular com o gene 18S rRNA e a subtipagem com o gene GP60, permitem que os epidemiologistas possam rastrear as fontes de surtos das diferentes espécies de Cryptosporidium. / Abstract: A total of 196 fecal samples from crossbred calves, 7-30 days old of both sexes were collected in 31 properties with the objective of determining the prevalence of Cryptosporidium in the northwestern region of São Paulo, as well as characterizing the molecular species involved in this parasite. We performed microscopic analysis by the technique of negative staining with malachite green in all stool samples. For molecular identification of Cryptosporidium, we used the reaction of nested PCR using specific primers for amplification of gene fragments coding for the 18S subunit ribosomal RNA gene and the glycoprotein GP 60 by subjecting the PCR product sequencing and phylogenetic analysis. By stool examination, positivity was observed 2% (4 / 196) by means of microscopy, and 10.7% (21/196) by nested PCR. As a result of sequencing, we identified four species of Cryptosporidium: C. parvum (subtype IIa15G2R1) C. Ryanair, C. bovis and C. andersoni. This discovery of C. IIaA15G2R1 parvum subtype in the studied region illustrates the usefulness of subtyping. Although based on these initial results with GP60 subtypes are useful for understanding the transmission dynamics of the species of Cryptosporidium, the number of samples is relatively small and much more can still be searched. It is interesting to note that the information of molecular typing with 18S rRNA gene and subtyping with the GP60 gene, allow epidemiologists can track sources of outbreaks of different species of Cryptosporidium. / Orientador: Luiz Claudio Nogueira Mendes / Coorientador: Marcelo Vasconcelos Meireles / Banca: Ricardo Velludo Gomes de Soutello / Banca: Katia Denise Saraiva Bresciani / Mestre

Cryprosporidium parvum is a coccidian protozoan that colonizes epithelial cells lining respiratory and digestive tracts of animals and humans. Cryptosporidiosis is a well-recognized zoonotic disease infecting primarily neonates and immunocompromised hosts, including human immunodeficiency virus-infected patients. Clinical disease is manifested as a chronic diarrheal illness that is self-limiting in immunocompetent hosts and prolonged and often life-threatening in hosts with compromised immune systems.The lack of a suitable small animal model for screening anti-cryptosporidial drugs and for examining the pathogenicity and immunobiology of chronic cryptosporidiosis was the impetus for this research effort. The objectives of the present study were three-fold: to characterize chronic Cryptosporidium parvum infections in dexamethasone-immunosuppressed mice; evaluate the effects of Cryprosporidium parvum and dexamethasone on B and T lymphocyte proliferation; and determine the effects of the immunomodulator dehydroepiandrosterone on oocyst shedding intensities of mice infected with Cryptosporidium parvum. Adult C57BL/6N mice were immunosuppressed with the synthetic glucocorticoid dexamethasone, then infected with Cryprosporidium parvum (106 oocysts/mouse) investigated for their ability to sustain a four-month chronic infection. Dexamethasone was administered intraperitoneally (125 Jlg/mouse/day) or orally (8 Jlg/ml) in the drinking water ad libitum. Infection chronicity was characterized by evaluating mouse monality, oocyst excretion in the feces, tissue distribution of the parasite, and parasite-induced pathology. A progressive infection with Cryptosporidium parvum occurred in mice immunosuppressed intraperitoneally and orally as long as dexameth sone was administered. Mice receiving dexamethasone given intraperitoneally had a shoner prepatent period and a more consistent, although cyclic, oocyst shedding pattern when compared with mice given dexamethasone orally. Mice given dexamethasone orally exhibited a delayed prepatent period, with a steady increase in oocyst shedding. All mice receiving dexamethasone orally died within three months following oocyst inoculation. Clinical signs included dehydration, icterus, and reduction in spleen and body weights. Clinical signs were more abrupt in mice receiving oral dexamethasone. Parasite colonization involved the entire intestinal tract, including the pyloric ring and Peyer's patches, but was the heaviest in the terminal ileum. Parasites were present in the lungs, gallbladder, and pancreatic ducts. Pathologic abnormalities were isolated to the terminal small intestine and included blunting and fusion of intestinal villi and crypt hyperplasia. Cryptosporidium parvum and dexamethasone administered in vivo reduced B and T lymphocyte responses to the mitogens lipopolysaccharide and concanavalin A. Dehydroepiandrosterone and dehydroepiandrosterone-sulfate resulted in no significant reductions in cryptosporidial activity as determined by oocyst shedding in the feces.