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The aim of this study was to evaluate the use of a hyperimmunized plasma containing high concentration of antibodies against Clostridium difficile, Clostridium perfringens and Salmonella sp in a referral population of equine colitis cases. A prospective, blinded clinical trial was undertaken. Horses were enrolled if they were over 1 year old, duration of diarrhea at presentation was less than 72 hours, they had not received equine plasma within the last 3 months and the serum total protein was greater than 4mg/dl. Horses were randomized to receive hyperimmunized plasma, control plasma (collected from non-immunized horses) or no plasma therapy. Clinical parameters were recorded and a fecal score (2 -14) assigned (every 6 hours) based upon diarrhea frequency, volume and consistency, for a total of 72 hours. A score less than 5 was considered normal. Fecal consistency was observed until resolution, discharge or death. Complete blood counts and biochemical profiles were collected at admission, 24 and 72 hours and at admission, 24 hours and 48 hours respectively. Forty two horses were enrolled and 38 horses completed the study. At study admission clinical and clinicopathological parameters, other than fecal frequency score were comparable between the groups. Fecal frequency score was significantly different between the treatment groups (p=0.003). The mean duration of diarrhea was 40.7±9.8 hours (mean ±SEM), 119.2±56.1 hours and 72.0±24.5 hours for the hyperimmunized plasma, normal plasma and control groups respectively. This data confirms the hyperimmunized plasma used in this study decreased the time to resolution of diarrhea. / Master of Science

by Rabi Biswas. / Publication date from spine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 122-143). / PREFACE --- p.2 / ACKNOWLEDGEMENTS --- p.3 / CONTENTS --- p.4 / GLOSSARY --- p.9 / ABSTRACT --- p.11 / INTRODUCTION --- p.12 / Chapter 1.1. --- OVERVIEW --- p.12 / Chapter 1.2. --- OBJECTIVES OF THE STUDY --- p.14 / LITERATURE REVIEW --- p.15 / Chapter 2.1. --- BACKGROUND OF THE STUDY --- p.15 / Chapter 2.2. --- ESCHERICHIA COLI : OVERVIEW --- p.17 / Chapter 2.2.1. --- Morphology --- p.18 / Chapter 2.2.2. --- Cultural characteristics --- p.18 / Chapter 2.2.3. --- Biochemical reactions --- p.19 / Chapter 2.2.4. --- Antigenic Structure --- p.19 / Chapter 2.2.5. --- Identification --- p.20 / Chapter 2.2.6. --- Classification of coli --- p.20 / Chapter 2.3. --- HISTORY OF EPEC --- p.22 / Chapter 2.3.1. --- E. coli as a cause of diarrhoea --- p.22 / Chapter 2.3.2. --- The first use of the term EPEC --- p.23 / Chapter 2.3.3. --- EPEC as a separate category of E. coli --- p.24 / Chapter 2.4. --- PATHOGENESIS OF EPEC --- p.25 / Chapter 2.4.1. --- Plasmid encoded virulence properties --- p.25 / Chapter 2.4.2. --- Characteristic interaction with intestinal mucosa --- p.26 / Chapter 2.4.3. --- Production of toxins --- p.28 / Chapter 2.5. --- EPIDEMIOLOGY OF EPEC --- p.29 / Chapter 2.6. --- EPIDEMIOLOGY OF EPEC IN CHINA AND HONG KONG --- p.32 / Chapter 2.7. --- CLINICAL INFECTION BY EPEC AND MANAGEMENT --- p.33 / Chapter 2.7.1. --- Epidemiological syndromes --- p.33 / Chapter 2.7.2. --- Infective dose --- p.33 / Chapter 2.7.3. --- Incubation period --- p.33 / Chapter 2.7.4. --- Host factors --- p.33 / Chapter 2.7.5. --- Reservoirs of infection --- p.33 / Chapter 2.7.6. --- Routes of transmission --- p.34 / Chapter 2.7.7. --- Seasonal variation --- p.34 / Chapter 2.7.8. --- Mechanism of diarrhoea --- p.34 / Chapter 2.7.9. --- Histology --- p.35 / Chapter 2.7.10. --- Clinical features --- p.35 / Chapter 2.7.11. --- Treatment --- p.35 / Chapter 2.7.12. --- Prevention --- p.36 / Chapter 2.8. --- DETECTION OF EPEC: LABORATORY METHODS --- p.36 / Chapter 2.8.1. --- O/H Serotyping --- p.36 / Chapter 2.8.2. --- Adhesion assay --- p.37 / Chapter 2.8.3. --- EAF probe --- p.37 / Chapter 2.8.4. --- FAS (Fluorescein Actin Staining) test --- p.37 / Chapter 2.8.5. --- ELISA (Enzyme Linked Immunosorbent Assay) --- p.38 / Chapter 2.8.6. --- eaeA gene probe --- p.38 / Chapter 2.8.7. --- bfpA probe --- p.39 / Chapter 2.8.8. --- PCR (Polymerase Chain Reaction) --- p.39 / MATERIALS AND METHODS --- p.41 / Chapter 3.1. --- PATIENT RECRUITMENT AND DATA COLLECTION --- p.41 / Chapter 3.1.1. --- Study site --- p.41 / Chapter 3.1.2. --- Study design --- p.41 / Chapter 3.1.3. --- Study period --- p.42 / Chapter 3.1.4. --- Study population --- p.42 / Chapter 3.1.5. --- Selection of patients --- p.42 / Chapter 3.1.6. --- Inclusion criteria for cases --- p.43 / Chapter 3.1.7. --- Exclusion criteria --- p.43 / Chapter 3.1.8. --- Selection of control group --- p.43 / Chapter 3.1.9. --- Collection of stool specimens --- p.44 / Chapter 3.1.10. --- Treatment of the study-patients --- p.45 / Chapter 3.1.11. --- Collection of date --- p.45 / Chapter 3.1.12. --- Ethical approval --- p.46 / Chapter 3.2. --- LABORATORY METHODS --- p.47 / Chapter 3.2.1. --- IN PWHLABORATORY --- p.47 / Chapter 3.2.2. --- IN AFRIMS LABORATORY --- p.48 / Chapter 3.3. --- DATA MANAGEMENT AND STATISTICAL METHODS --- p.63 / RESULT --- p.64 / Chapter 4.1. --- DEMOGRAPHY OF THE PATIENTS --- p.64 / Chapter 4.1.1. --- Age distribution of the patients --- p.64 / Chapter 4.1.2. --- Sex distribution of the patients --- p.64 / Chapter 4.1.3. --- Ethnic origin of the patients --- p.65 / Chapter 4.1.4. --- Distribution of area of abode in Hong Kong --- p.66 / Chapter 4.1.5. --- School attendance of the patients --- p.66 / Chapter 4.2. --- PREDISPOSING FACTORS FOR DIARRHOEA --- p.67 / Chapter 4.2.1. --- History of breast feeding of the patients --- p.67 / Chapter 4.2.2. --- History of contact with diarrhoea --- p.68 / Chapter 4.2.3. --- Travel history within last two weeks preceding onset of diarrhoea --- p.68 / Chapter 4.2.4. --- Source of drinking water --- p.69 / Chapter 4.3. --- CLINICAL FEATURES --- p.70 / Chapter 4.3.1. --- Duration of diarrhoea at the time of admission --- p.70 / Chapter 4.3.2. --- Frequency of stool --- p.71 / Chapter 4.3.3. --- Nature and contents of stool --- p.72 / Chapter 4.3.4. --- Condition of the perineum --- p.72 / Chapter 4.3.5. --- Duration of vomiting at the time of admission --- p.73 / Chapter 4.3.6. --- Frequency of vomiting --- p.73 / Chapter 4.3.7. --- Level of dehydration in cases --- p.74 / Chapter 4.3.8. --- Urine output during illness --- p.74 / Chapter 4.3.9. --- Fever associated with illness --- p.75 / Chapter 4.4. --- HISTORY OF HOME- MANAGEMENT --- p.77 / Chapter 4.4.1. --- Main food taken at home during illness --- p.77 / Chapter 4.4.2. --- Supplementary fluid taken at home during illness --- p.77 / Chapter 4.4.3. --- Duration of hospital stay --- p.78 / Chapter 4.4.4. --- Recruitment of patients in different months --- p.79 / Chapter 4.5. --- RESULTS OF GENE PROBING FOR E. COLI --- p.80 / Chapter 4.6. --- DETAILS OF THE EAF+ EPEC CASES --- p.82 / Chapter 4.6.1. --- Associated infections in EAF+ cases --- p.83 / Chapter 4.7. --- AETIOLOGY OF DIARRHOEA --- p.84 / Chapter 4.7.1. --- Age distribution --- p.85 / Chapter 4.7.2. --- Seasonal distribution --- p.86 / Chapter 4.7.3. --- Clinical features --- p.87 / Chapter 4.7.4. --- Different groups of Salmonella --- p.88 / Chapter 4.7.5. --- Dual infection among enteropathogens isolated --- p.89 / DISCUSSION --- p.90 / Chapter 5.1. --- RISK FACTORS ASSOCIATED WITH DIARRHOEA --- p.91 / Chapter 5.1.1. --- Age and sex of the patients --- p.91 / Chapter 5.1.2. --- Nutritional status --- p.91 / Chapter 5.1.3. --- Breast feeding --- p.92 / Chapter 5.1.4. --- Travelling --- p.93 / Chapter 5.2. --- SEVERITY OF DIARRHOEA IN HONG KONG --- p.93 / Chapter 5.3. --- MOLECULAR EPIDEMIOLOGY OF EPEC IN HONG KONG --- p.94 / Chapter 5.3.1. --- EAF probe --- p.94 / Chapter 5.3.2. --- EAF and EPEC virulence --- p.95 / Chapter 5.3.3. --- eaeA probe --- p.96 / Chapter 5.3.4. --- FAS test --- p.97 / Chapter 5.3.5. --- bfpA probe --- p.97 / Chapter 5.3.6. --- Comparison and contrast among the probes --- p.97 / Chapter 5.3.7. --- Probes in the present study --- p.99 / Chapter 5.3.8. --- Role of serogrouping at present --- p.100 / Chapter 5.3.9. --- EPEC in Hong Kong --- p.100 / Chapter 5.4. --- PREVALENCE OF OTHER CATEGORIES OF E. COLI --- p.101 / Chapter 5.5. --- COMMON AETIOLOGY OF DIARRHOEA IN HONG KONG --- p.102 / Chapter 5.5.1. --- Rotavirus as the most common cause --- p.102 / Chapter 5.5.2. --- Non-typhoid Salmonella as the major bacterial pathogen --- p.102 / Chapter 5.5.3. --- Campylobacter and Shigella as cause of diarrhoea --- p.103 / Chapter 5.5.4. --- Role of parasites in childhood diarrhoea in Hong Kong --- p.104 / Chapter 5.6. --- CONTROL MEASURES FOR DIARRHOEAL DISEASES --- p.105 / Chapter 5.6.1. --- Prevention of diarrhoea through improved nutrition --- p.105 / Chapter 5.6.2. --- Fluid supplementation in diarrhoea --- p.106 / Chapter 5.6.3. --- Strategies to control diarrhoea --- p.106 / Chapter 5.6.4. --- Health education --- p.107 / CONCLUSION --- p.108 / APPENDIX --- p.109 / Chapter 7.1. --- QUESTIONNAIRE --- p.109 / Chapter 7.2. --- LABORATORY METHODS --- p.111 / Chapter 7.2.1. --- Routine culture of stool specimens for bacteria --- p.111 / Chapter 7.2.2. --- "Serotyping of Salmonella, Shigella and Vibrio cholerae" --- p.114 / Chapter 7.2.3. --- Microscopic examination of ova and cysts --- p.117 / Chapter 7.2.4. --- Laboratory diagnosis of rotavirus --- p.118 / Chapter 7.3. --- INVESTIGATION REQUISITION FORM --- p.121 / REFERENCES --- p.122 / GRADUATE SEMINARS & PUBLICATIONS --- p.144 / Chapter a. --- Graduate seminars --- p.144 / Chapter b. --- Publications --- p.144

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser.

This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.

PhD (Microbiology) / Department of Microbiology / Background: Diarrheal diseases have a major effect on human health, Globally; it is second only to pneumonia as a leading cause of death among children under five. They are due to a variety of infectious and non-infectious agents; including Entamoeba spp. Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Amebiasis is frequent in communities without clean water and poor sanitation, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica inclusive of all ages, sexes and HIV status is uncertain. Diagnosis of the parasite is usually by microscopy. However, microscopy lacks sensitivity and specificity, therefore it is not reliable. Fortunately, molecular diagnostic tests have been developed to detect different Entamoeba species in humans. It is known that the parasite E. histolytica causes asymptomatic and symptomatic diseases. However, the transition from colonization to disease is still unclear. While parasite and host factors, as well as environmental conditions influence the infection outcome, there is currently no clear explanation of wide variation in the presentation of the disease. This could suggest that there are other factors affecting the disease outcome. A better understanding of these factors as well as their role in disease remains target objectives of modern scientists and it will definitely help in the fight against the disease. In spite of the emerging evidence that the host microbiome, parasite burden and the inflammatory response contribute to the virulence of E. histolytica, their roles have never been defined in developing regions such as Giyani and Pretoria. In addition, the present study hypothesized that co-infections with E. histolytica and secretion of extracellular vesicles/exosomes have a significant impact on the virulence of E. histolytica. Little has been explored or elucidated about responses triggered by other enteropathogens/ameba interplay that could be important in the induction of tissue invasion and disease and also how E. histolytica/enteropathogens interplay in these infections has not been determined. Therefore, the knowledge of this interplay could help in understanding how this modifies disease manifestations by modulating pathogen virulence and the host response. The use of secretion systems is an essential biological process exploited by pathogenic microorganisms to promote survival and spread of the pathogen, which in turn exacerbate the infection. The study of extracellular vesicles (EVs) released by pathogens is a new and exciting field that may realistically contribute to a better understanding of the pathogenic process of E. histolytica and provide alternate control strategies. Aim and objective of the study: The overall aim of the study was to determine the impact of enteric pathogens and secreted extracellular vesicles on amebic virulence and the outcome of infection. This aim was addressed in through a series of six primary objectives, which were: a. To investigate the distribution and prevalence of protozoan parasites in South Africa. b. To investigate novel species of Entamoeba circulating in the South African population. ix c. To elucidate the impact of gut microbiota and immune response during amebic infection. d. To determine the role of Entamoeba histolytica macrophage inhibitory factor (EhMIF) during amebic infection. e. To investigate the impact of co-infections on the outcome of amebiasis. f. To determine the presence of secreted extracellular vesicles/exosomes in Entamoeba histolytica. Brief methodology and results: A modified and validated Taqman qPCR assay (with taqman probes and genus specific primers) was used for amplification and target detection. This assay was used to investigate the distribution and prevalence of protozoan parasites (Cryptosporidium spp and Giardia lamblia) in South Africa, the assay was considered superior for this project because it is more sensitive than conventional PCR and it can be used to detect multiple infection targets. This assay allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. A total of 484 stool samples collected from diarrheal and non-diarrheal patients from rural and urban communities of South Africa were studied. The overall prevalence of parasites (Giardia lamblia and Cryptosporidium spp) in rural and urban patients were found to be 49% (112/227) and 21% (54/257) respectively (p= < 0.0001). The distribution of specific pathogens in rural areas was Cryptosporidium spp (20%) and Giardia lamblia (14%). Our findings showed no significant difference in parasitic infections between gender and the age of the participants (Chapter 3). The discovery of novel species is of great importance to human health. We have recently discovered stools positive for Entamoeba organisms by microscopy but PCR negative for known Entamoeba species. This led to the hypothesis that novel species of Entamoeba are present in the South African population. A comprehensive assay was used which included probes to identify Entamoeba bangladeshi from diarrheal and non-diarrheal participants. A sensitive qPCR assays and amplicon sequencing was used to detect Entamoeba spp, Prevotella copri and Enterobacteriaceae. Interestingly, E. bangladeshi was identified in the South African population. Entamoeba was present in 27% (E. histolytica 8.5% (41/484), E. dispar 8% (38/484), and E. bangladeshi 4.75% (23/484) E. moshkovskii was not detected in the present study. We were also able to observe changes in the host microbiome and the parasite burden associated with E. histolytica infections in S. African diarrhea cases versus asymptomatic controls but not with E. bangladeshi or E. dispar. In E. histolytica positive samples the level of both parasite and P. copri were lower in non-diarrheal samples (p=0.0034) (Chapter 4). There is accumulating evidence that the inflammatory response contributes to injury. Little is known about the key parasite mediators of host mucosal immunopathology. This study hypothesized that migration inhibitory factor (MIF) mediates the destructive host inflammatory response seen in amebic colitis. To determine the role of EhMIF during amebic infection, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. We found that EhMIF induces IL-8 secretion from intestinal epithelial cells. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx and mucosal damage. We also found that the concentration of EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Together, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets (Chapter 5). To investigate the impact of co-infections on the outcome of amebiasis, we analyzed the co-occurence of E. histolytica with other enteropathogens known to cause diarrheal infections, such as Shigella/EIEC (IpaH), Campylobacter (cadf), Enterotoxigenic E. coli (STh), Norovirus GII and Adenovirus (Hexon). The results were compared with those obtained with E. histolytica that were not interacted with enteropathogens and with E. histolytica interacted with enteropathogens. The impact of multiple infections on the outcome of the infection was compared between nondiarrheal and diarrheal stool samples. It was found that co-infections with two pathogens were associated with diarrhea compared to single infections. Moreover, Norovirus GII, Campylobacter (Cadf) and co-infections were associated with diarrhea in the study population. This study did not show any significant impact of pathogens co-infecting with E. histolytica on the outcome of amebic infection (Chapter 6). The presence of secreted extracellular vesicles/Exosomes in Entamoeba histolytica was determined by using the Pathogenic ameba strains (HM-1:IMSS or HM-1:IMSS (Sub-strain-US) from petri’s lab to purify exosomes using the commercially available kit to isolate exosomes (total exosomes isolation kit). Our study for the first time revealed that E. histolytica does secrete Evs. This finding increases the appreciation that all organisms are likely to secrete these EVs (Chapter 7). However, the impact of these EVs on the pathogenesis of E. histolytica needs further investigations. Conclusion: This study has contributed significantly to our knowledge on infectious diarrhea and the diversity of Entamoeba species by providing new data on the rate and prevalence of Entamoeba diarrheal infections and their distribution in the South African population. Our study describes for the first time the presence of E. bangladeshi in the South African population. Furthermore, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets. This study also, for the first time revealed that E. histolytica does secrete EVs. The results from this work will undoubtedly open an exciting research to establish a deeper understanding of the function and role of these vesicles in amebic infection. We encourage public health interventions like health education programs and improvement of sanitation and hygiene in these populations. Molecular diagnostics should be used for specific diagnostic in clinical settings. / NRF

Objetivos. Determinar las características epidemiológicas y clínicas de la gastroenteritis causada por Vibrio parahaemolyticus del grupo pandémico en el Perú. Materiales y métodos. Se examinó las historias clínicas y registros de laboratorio de cien casos de gastroenteritis en los cuales se aisló V. parahaemolyticus del grupo pandémico y no pandémico. Se recolectó información epidemiológica y clínica y se realizó el análisis estadístico de los datos para evaluar si la gravedad de la enfermedad se asoció con la presencia de las cepas del grupo pandémico. Resultados. Se logró colectar información epidemiológica en 85% de los casos e información clínica sólo en 37% de los casos, principalmente de los hospitalizados. Los casos del grupo pandémico tuvieron una mayor probabilidad de tener deposiciones líquidas (96,3% frente a 62,5%, p<0,05), presentar deshidratación moderada o grave (100% frente a 60%, p<0,05) y requerir atención hospitalaria (98% frente a 42,9%, p<0,0001). Fue más probable aislar una cepa pandémica en personas de 30 o más años de edad (63% frente a 39,5%, p<0,05). Conclusiones. El Vibrio parahaemolyticus del grupo pandémico causa enfermedad gastrointestinal de mayor gravedad que las cepas no pandémicas, con mayor probabilidad de requerir atención hospitalaria. Basados en este reporte, se recomienda incluir la identificación de V. parahaemolyticus en el diagnóstico etiológico de agentes causantes de gastroenteritis grave en el sistema de salud del Perú. / Objective. To determine the epidemiological and clinic characteristics of gastroenteritis caused by Vibrio parahaemolyticus strains of the pandemic group in Peru. Material and methods. Clinical and laboratory records were searched in 100 cases of gastroenteritis caused by V parahaemolyticus, either of the pandemic or non pandemic group. Clinical and epidemiological data were collected and statistical analysis was done to evaluate if the severity of illness was associated with the pandemic group. Results. Epidemiological data were collected in 85% of cases, and clinical data were only available in 37% of cases, mainly on those hospitalized. Cases associated with the pandemic strains had a higher probability of liquid stools (96.3% vs. 62.5%, p<0.05), moderate or severe dehydration (100% vs. 60%, p<0.05), and hospital care (98% vs. 42.9%, p<0.0001). Cases aged thirty or older were associated with the pandemic strains (63% vs. 39.5%, p<0.05). Conclusions. Vibrio parahaemolyticus of the pandemic group causes more severe gastrointestinal disease than none pandemic strains, with higher probability of requiring hospital care. Based on this report, it is advisable to include the identification of V. parahaemolyticus in the etiological diagnosis of agents causing severe gastroenteritis in the Peruvian health system.

Objetivos: 1) Determinar la prevalencia de incremento de eosinófilos en mucosa colónica en pacientes con colitis linfocítica (CL). 2) Determinar la coexistencia de colitis eosinofílica (CE) en pacientes con CL. Materiales y métodos: Las biopsias colónicas de pacientes adultos con diarrea crónica diagnosticados como CL en el hospital Daniel A. Carrión durante octubre 2009 a marzo 2012 fueron revisadas de forma independiente por 2 patólogos. Microscópicamente, se investigó y cuantificó la presencia de eosinófilos en mucosa colónica. Resultados: Se incluyeron 68 casos de CL, de los cuales 76,5% tuvieron eosinófilos elevados en la mucosa colónica y en 51,4% se pudo hacer el diagnóstico de CE según los criterios establecidos. Conclusión: Tres de cuatro pacientes con CL presentan eosinófilos elevados y 1 de cada 2 pacientes con CL cumple criterios para CE. / Objectives: 1) To determine the prevalence of increased number of eosinophils in colonic mucosa of patients with lymphocytic colitis (LC). 2) To determine the coexistence of eosinophilic colitis (EC) in patients with lymphocytic colitis. Materials and methods: slides of adult patients with cronic diarrhea with diagnosis of LC were reviewed between October 2009 and March 2012. The number of eosinophils was quantified. Results: Sixty eight patients with LC were included. Elevated eosinophils were found in 76.5 and in 51.4% a diagnosis of EC was established. Conclusion: 3 out of 4 patients with LC had elevated eosinophils and 1 of 2 patients with LC had criteria for EC.

Rotavírus têm sido identificados mundialmente como o mais importante agente etiológico de diarréias agudas não-bacterianas em animais jovens de várias espécies, incluindo a humana. Foi desenvolvido e avaliado um método de ELISA tipo “duplo-sanduíche" para a detecção de rotavírus a partir de material fecal. Para tanto, a amostra NCDV de rotavírus do grupo A foi propagada em cultivo celular com células MA-104. O vírus foi concentrado por ultracentrifugação e inoculado em coelhos e carneiros. Em seguida, as frações IgG, oriundas de amostras de soro dos animais, foram purificadas por cromatografia de troca iônica e absorvidas com soro total de ambas espécies animais, utilizando-se polímero de glutaraldeído, de modo a eliminar reações inespecíficas. A presença do rotavírus foi detectada pela IgG de carneiros e revelada pela IgG de coelho, usando como conjugado IgG de cabra anti-IgG de coelho conjugada à peroxidase. Os valores de diluição dos componentes do ELISA e o valor do ponto-de-corte foram definidos usando-se 26 amostras fecais (13 positivas e 13 negativas) de leitões, tendo como prova padrão a eletroforese em gel de poliacrilamida (PAGE). Aplicado a um painel constituído de 86 amostras fecais diarréicas de leitões, os resultados do ELISA foram: 100% de sensibilidade; 98,79% de especificidade, com uma concordância de 98,83%. A variância entre 86 repetições da mesma amostra foram 0,001 (para a amostra positiva) e 0,0002 (para a amostra negativa). Estes resultados demonstram que este ELISA é um teste sensível e específico para o diagnóstico de rotavírus a partir de material fecal. / Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich" antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material.

Orientador: Iveraldo dos Santos Dutra / Banca: Julio Cesar de Freitas / Banca: Tereza Cristina Cardoso da Silva / Resumo: A diarréia neonatal é uma das principais causas de perdas na bovinocultura. O Clostridium perfringens é um enteropatógeno amplamente distribuído na natureza e responsável por várias doenças nos animais, dentre elas a diarréia neonatal. Foram examinadas 141 amostras fecais de bezerros com diarréia e 129 amostras de animais sadios, com até 28 dias de idade e pertencentes a três rebanhos distintos. Do cultivo bacteriológico em anaerobiose foi possível isolar 36,2% e 30,2% amostras suspeitas de Clostridium perfringens dos animais enfermos e dos animais sadios, respectivamente. A genotipagem bacteriana foi efetuada empregando-se a técnica de PCR multiplex com os primers dos genes codificadores das toxinas alfa (cpa), beta (cpb), épsilon (etx), iota (itxA), enterotoxina (cpe) e toxina beta2 (cpb2). Dentre as amostras isoladas, 17/51 (33,3%) e 17/39 (43,6%) dos animais com diarréia e sadios, respectivamente, amplificaram um ou mais genes codificadores das toxinas de C. perfringens. Dos bezerros com diarréia, quatorze apresentaram somente o gene cpa (tipo A), um apresentou o cpa e cpb2 (tipo A beta2 positivo), um amplificou o cpa, itxA, e cpb2 (tipo E, beta2 positivo) e um amplificou o cpa, etx, itxA e cpb2 (tipo D e E, um ou ambos cpb2 positivo). Dentre os bezerros sadios, 10 eram exclusivamente tipo A, um era tipo A cpb2 positivo, dois eram tipo E, três eram tipo E cpb2 positivo e um era tipo D e E cpb2 positivo. Não houve correlação entre a genotipagem dos genes codificadores das toxinas de Clostridium perfringens e a presença de diarréia neonatal nos bezerros. / Abstract: Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxinencoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 (Types D and E, one or both cpb2 positive). Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea. / Mestre