Detección de ooquistes de Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) en ejemplares de cholga Aulacomya ater, extraídas desde la costa de la Región del Bío Bío, Chile / Detection of Cryptosporidium parvum (Apicomplexan: Cryptosporidiidae) oocyst in Aulacomya ater specimen, extracted from to the coast Bio Bio Region, Chile

Resumen Introducción: El bivalvo Aulacomya ater (cholga), es uno de los moluscos de mayor consumo en la población chilena. Sin embargo, existe evidencia de contaminación fecal hídrica provocada por los cauces que llegan al mar, aumentando la probabilidad de contaminación por Cryptosporidium parvum, el que genera criptosporidiosis en el ser humano. Objetivo: Determinar la presencia de C. parvum en cholgas extraídas desde la Región del Bío Bío (Chile). Material y Métodos: Se seleccionaron 55 cholgas provenientes de un centro de cultivo y de un banco natural de extracción. Estas muestras, fueron procesadas en el laboratorio y se evaluó la presencia de elementos ácido-alcohol resistentes. Las muestras positivas, se analizaron por inmunofluorescencia directa, con anticuerpo específicos contra C. parvum. Resultados: 16,4% del total de las muestras tenían ooquistes de C. parvum. Conclusiones: Por primera vez se describe C. parvum en A. ater provenientes de las costas chilenas, siendo este molusco un posible vehículo de transmisión de criptosporidiosis a la población y a sus animales depredadores. Además, la presencia de C. parvum refleja la contaminación fecal hídrica en las costas evaluadas. Actualmente estamos monitoreando otras zonas de extracción de este molusco.

Abstract Background: The bivalve Aulacomya ater (cholga), is one of the most consumed mollusks by the population. However, there is evidence of fecal water contamination caused by causes that affect the sea, increasing the probability of contamination by the Cryptosporidium parvum, which generates cryptosporidiosis in people. Aim: To determine the presence of C. parvum in cholga extracted from the Bio Bio Region (Chile). Methods: Fifty-five cholgas were selected from a cultivation center and a natural extraction bank. These samples were processed in the laboratory and the presence of acid-alcohol resistant elements was evaluated. Positive samples were analyzed by direct immunofluorescence with anti-C. parvum antibody. Results: 16.4% of the total samples were affected by the oocysts of C.parvum. Conclusions: For the first time we described C. parvum in A. ater from the Chilean coast, being this mollusk a possible vehicle for transmission of cryptosporidiosis to the population and their predatory animals. Furthermore, the presence of C. parvum reflects fecal water contamination on the evaluated coasts. We are currently monitoring other extraction areas for this mollusk.

Avances en el desarrollo de una vacuna efectiva contra Cryptosporidium parvum: una revisión de la literatura / Advances in the development of an effective vaccine against Cryptosporidium parvum: An review of the literature / Avanços no desenvolvimento de uma vacina eficaz contra Cryptosporidium parvum: uma revisão da literatura

Introducción. Cryptosporidium parvum es un parásito zoonótico altamente prevalente, asociado a enfermedad diarreica en población inmunocomprometida, niños y terneros menores de 30 días. Esta infección puede ocasionar deshidratación, alteración del estado de conciencia, retraso en el desarrollo global y, en algunos casos, la muerte del paciente. A pesar de la alta prevalencia de C. parvum, no existen medicamentos completamente efectivos ni una vacuna aprobada para prevenir dicha enfermedad. Objetivo. Realizar una revisión de la literatura sobre candidatos vacunales contra C. parvum. Método. Revisión documental mediante la búsqueda de la literatura de los últimos 20 años, disponible en las bases de datos PubMed central, WEB OF SCIENCE, Embase, REDALYC y LILACS. Resultados. Las vacunas atenuadas, recombinantes, basadas en ADN, expresadas en vectores bacterianos y sintéticas han mostrado resultados prometedores en la inducción de inmunogenicidad contra los antígenos de C. parvum, siendo el antígeno de superficie de 15 kilodaltons de Cryptosporidium parvum (cp15), el antígeno inductor de una mejor respuesta inmune celular y humoral en el modelo murino estudiado. Conclusión. Se espera que la incorporación de nuevas técnicas para la selección de antígenos promisorios y la ejecución de una gran cantidad de ensayos in vivo, favorezcan el desarrollo de una vacuna totalmente efectiva contra C. parvum. Aunque el camino para lograr este objetivo será largo y difícil, se convierte en la mejor alternativa para controlar una de las enfermedades de interés en salud pública, con mayor impacto en la población inmunocomprometida.

Introduction. Cryptosporidium parvum is a highly prevalent zoonotic parasite, associated with diarrheal disease in immunocompromised population, children and calves under 30 days. This infection is associa- ted to dehydration, delayed global development and, in some cases, the death of the patient. Despite the high prevalence of C. parvum, there are no fully effective medications and an approved vaccine to prevent such disease. Objective. To conduct a thorough review of the literature on vaccine candidates against C. parvum. Method Documentary review by searching the literature of the last 20 years, available in the central PubMed, WEB OF SCIENCE, Embase, REDALYC and LILACS databases. Results. Attenuated, recombinant, DNA-based, expressed in bacterial vectors and synthetic vaccines have shown promising results in inducing immunogenicity against C. parvum, being the Cryptospori- dium parvum 15 kiloDalton surface antigen (cp15), the antigen inducer of a better cellular and humoral immune response in the murine model studied. Conclusion. It is expected that the incorporation of new techniques for the selection of promising antigens and the execution of a large number of in vivo assays will favor the development of a fully effective vaccine against C. parvum. Although the way to achieve this goal will be long and difficult, it will become the best alternative to control one of the diseases with the greatest impact on the immu- nocompromised population.

Introdução. O Cryptosporidium parvum é um parasita zoonótico de alta prevalência associado à doença diarreica em populações imunocomprometidas, crianças e bezerros com menos de 30 dias. Essa infecção pode causar desidratação, alteração do estado de consciência, atraso no desenvolvi- mento global e, em alguns casos, a morte do paciente. Apesar da alta prevalência de C. parvum, não existem medicamentos totalmente eficazes e uma vacina aprovada para prevenir a doença. Objetivo. Realizar uma revisão literária dos candidatos à vacina contra C. parvum. Método. Revisão documental, mediante pesquisa da literatura dos últimos 20 anos, disponível nas bases de dados PubMed central, WEB OF SCIENCE, Embase, REDALYC e LILACS. Resultados. Vacinas atenuadas, recombinantes e baseadas em DNA, expressas em vetores bacteria- nos e sintéticos, mostraram resultados promissores na indução de imunogenicidade contra antígenos de C. parvum, sendo o antígeno de superfície de 15 kilodaltons de Cryptosporidium parvum (cp15) o antígeno indutor de uma melhor resposta imune celular e humoral no modelo murino estudado. Conclusão. Se espera que a incorporação de novas técnicas para a seleção de antígenos promissores e a execução de um grande número de ensaios in vivo favoreçam o desenvolvimento de uma vacina totalmente eficaz contra C. parvum. Embora o caminho para alcançar este objetivo seja longo e difícil, torna-se a melhor alternativa para controlar uma das doenças de interesse na saúde pública com maior impacto na população imunocomprometida.

Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata: Planorbidae) under experimental conditions

Abstract INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

Molecular Prevalence and Genotypes of Cryptosporidium parvum and Giardia duodenalis in Patients with Acute Diarrhea in Korea, 2013–2016

Cryptosporidium parvum and Giardia duodenalis are the main diarrhea-causing parasitic pathogens; however, their prevalence in Korea is unknown. Here, we conducted a survey to determine the prevalence and genotype distribution of these 2 pathogens causing acute diarrhea in 8,571 patients hospitalized in 17 Regional Institute of Health Environment sites in Korea, during 2013–2016. C. parvum and G. duodenalis were detected and genotyped by nested PCR, and the isolate were molecularly characterized by sequencing the glycoprotein 60 (Gp60) and β-giardin genes, respectively. The overall prevalence of C. parvum and G. duodenalis was 0.37% (n=32) and 0.55% (n=47), respectively, and both pathogens were more prevalent in children under 9 years old. Molecular epidemiological analysis showed that the C. parvum isolates belonged to the IIa family and were subtyped as IIaA13G2R1, IIaA14G2R1, IIaA15G2R1, and IIaA18G3R1. Analysis of the β-giardin gene fragment from G. duodenalis showed that all positive strains belong to assemblage A. This is the first report on the molecular epidemiology and subtyping of C. parvum and G. duodenalis in such a large number of diarrheal patients in Korea. These results highlight the need for continuous monitoring of these zoonotic pathogens and provide a basis for implementing control and prevention strategies. Further, the results might be useful for epidemiological investigation of the source of outbreak.

Ocorrência e identificação de Cryptosporidium e Giardia em amostras de água superficial destinada ao abastecimento público do estado de São Paulo / Occurrence and identification of Cryptosporidium and Giardia from surface water catchment in Sao Paulo, Brazil

Estudos de revisão sobre surtos associados à transmissão hídrica revelaram que os protozoários parasitas Cryptosporidium parvum e Giardia duodenalis (sinonímia: G. lamblia e G. intestinalis) são os principais responsáveis pelo maior número de casos registrados em todo o mundo. A contaminação das águas superficiais que servem ao abastecimento público por estes protozoários representa risco à saúde humana e animal, pois ambos parasitas apresentam resistência à cloração, processo convencional utilizado para desinfecção em Estações de Tratamento de Água (ETAs).Em vista desta lacuna, o presente estudo propõe identificar espécies e genótipos de Cryptosporidium e Giardia a partir de 128 amostras de águas superficiais de 11 mananciais do estado de São Paulo, de acordo com o Método 1623.1 (USEPA, 2012). Para identificar estes parasitas, foi realizada a recuperação dos (oo) cistos a partir de lâminas raspadas, seguindo o protocolo adaptado da USEPA, em seguida, utilizou-se o PCR em tempo real para identificar os genes 18S rRNA para Cryptosporidium e SSU para Giardia. Os resultados mostraram que a frequência de ocorrência desses protozoários nos pontos de captação foi de 29,7% para Giardia e 30,4% para Cryptosporidium. Os cistos estavam presentes em 10 dos 11 pontos de captação com frequências que variaram de 17 a 100%, e concentrações que variaram de

Review studies on waterborne outbreaks have been showing that Cryptosporidium parvum and Giardia duodenalis (synonym: G. lamblia and G. intestinalis) are the primarily responsible for the highest number of the cases recorded worldwide. Contamination of surface waters catchments by these protozoa is a risk factor to human health because both parasites are resistant to chlorination, which is a conventional process used for disinfection in water treatment plants (WTP). The present study aimed to identify species of Cryptosporidium and Giardia recovered from surface water catchment samples from 11 municipalities from the State of São Paulo, totalizing 128 samples. Quantification of both parasites was carried out according to method 1623.1 (USEPA, 2012). In order to identify parasites, the recovering of (oo)cysts from slides followed USEPA´s protocol by scraping slides, then Real Time PCR using the 18S rRNA genes for Cryptosporidium and SSU for Giardia were carried out. Results showed that the frequency of occurrence of these protozoa at the catchment points was 29,7% for Giardia and 30,4% for Cryptosporidium. Cysts were present in 10 of 11 catchments points with frequencies varying from 17 to 100% with concentrations ranging from

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

Monitoring of Noxious Protozoa for Management of Natural Water Resources

Waterborne parasitic protozoa, particularly Giardia lamblia and Cryptosporidium spp., are common causes of diarrhea and gastroenteritis worldwide. The most frequently identified source of infestation is water, and exposure involves either drinking water or recreation in swimming pools or natural bodies of water. In practice, studies on Cryptosporidium oocysts and Giardia cysts in surface water are challenging owing to the low concentrations of these microorganisms because of dilution. In this study, a 3-year monitoring of Cryptosporidium parvum, Giardia lamblia, and Naegleria fowleri was conducted from August 2014 to June 2016 at 5 surface water sites including 2 lakes, 1 river, and 2 water intake plants. A total of 50 water samples of 40 L were examined. Cryptosporidium oocysts were detected in 22% of samples and Giardia cysts in 32%. Water at the 5 sampling sites was all contaminated with Cryptosporidium oocysts (0–36/L), Giardia cysts (0–39/L), or both. The geometric mean concentrations of Cryptosporidium and Giardia were 1.14 oocysts/L and 4.62 cysts/L, respectively. Thus, effective monitoring plans must take into account the spatial and temporal parameters of contamination because they affect the prevalence and distribution of these protozoan cysts in local water resources.

Simultaneous Molecular Detection of Cryptosporidium and Cyclospora from Raw Vegetables in Korea

Cryptosporidium and Cyclospora are well-known coccidian protozoa that can cause waterborne and foodborne diarrheal illnesses. There have been a few reports regarding contamination in different vegetables with Cryptosporidium, but no data are available regarding the sources of Cyclospora infections in Korea. In the present study, we collected 6 kinds of vegetables (perilla leaves, winter-grown cabbages, chives, sprouts, blueberries, and cherry tomatoes) from July 2014 to June 2015, and investigated contamination by these 2 protozoa using multiplex quantitative real-time PCR. Among 404 vegetables, Cryptosporidium and Cyclospora were detected in 31 (7.7%) and 5 (1.2%) samples, respectively. In addition, Cryptosporidium was isolated from all 6 kinds of vegetables, whereas Cyclospora was detected in 4 kinds of vegetables (except perilla leaves and chives). Cryptosporidium (17.8%) and Cyclospora (2.9%) had the highest detection rates in chives and winter-grown cabbages, respectively. Cryptosporidium was detected all year long; however, Cyclospora was detected only from October to January. In 2 samples (sprout and blueberry), both Cryptosporidium and Cyclospora were detected. Further investigations using TaqI restriction enzyme fragmentation and nested PCR confirmed Cryptosporidium parvum and Cyclospora cayetanensis, respectively. In conclusion, we detected C. cayetanensis in vegetables for the first time in Korea. This suggests that screening should be employed to prevent these protozoal infections in Korea.

Detection of Intestinal Parasites in Diarrhea Samples Using Various Diagnostic Methods and Evaluation of the Stability of In-house Quality Control Materials for Stool Examination

BACKGROUND: Because of a lack of quality control (QC) materials, stool examination has not been standardised. This study examined intestinal parasites in diarrhea specimens to manufacture and evaluate the performance stability of QC materials for stool examination. METHODS: This study examined diarrhea specimens submitted for stool culture. Microscopic examination was performed using the direct smear and formalin-ether concentration method (Military General Laboratory, MGL). Enzyme-linked immunosorbent assay (ELISA) kits (R-Biopharm AG, Germany) and xTAG Gastrointestinal Pathogen Panel (Luminex Corp., USA) were used for the three major protozoa: Cryptosporidium parvum, Giardia lamblia, and Entamoeba histolytica. Polymerase chain reaction (PCR) was performed for Dientamoeba fragilis and Blastocystis hominis. The QC materials for stool examination were generated using Diphyllobothrium nihonkaiense ova. The manufactured QC materials were evaluated under different storage conditions, with varying preservatives, temperatures, and storage times. RESULTS: From November 2015 to April 2016, 82 diarrhea specimens were collected and tested. All results from microscopy and ELISA were negative; C. parvum (n=2) and G. lamblia (n=1) were detected by xTAG, while D. fragilis (n=10) and B. hominis (n=2) were detected by PCR. High- and low-concentration QC materials were manufactured. Using the high-concentration QC material, ova were observed in all storage conditions using MGL. Using the low-concentration QC material, the ova were observed until 14 days, but not after 3 weeks. CONCLUSIONS: It should be considered for making QC materials for stool examinations that focus on D. fragilis and B. hominis frequently found in Korea and with the caution to the low-concentration of QC materials could be unstable.

Natural infection by Cryptosporidium SPP. in production animals: first description of subtype IIaa15G2R1 in goat kids and piglets in Brazil

Cryptosporidiosis is a severe enteric disease, with varied clinical manifestations. In young animals the infection is more common and may be more severe. In this study the polymerase chain reaction (PCR) was used to detect Cryptosporidium parasites in goat kids, calves, lambs, piglets and colts sharing the same environment. Fecal samples were collected directly from the rectum of 192 goat kids, 184 calves, 44 lambs, 47 piglets and 26 colts aged up to twelve months, males and females, of different breeds, from the Brazilian states of Goiás, Mato Grosso do Sul, Minas Gerais and São Paulo. PCR was used for amplifying a fragment of 18S rRNA gene and the gene encoding the surface glycoprotein GP60. Positive PCR amplification was observed in 16.7% (32/192) goat kids, 6.5% (12/184) calves and 2.1% (1/47) piglets. Based on the sequencing of 18S rRNA PCR products, all samples from goat kids were identified as C. parvum. Among calves, C. parvum was identified in 41.7% (5/12), C. andersoni in 16.7% (2/12), C. ryanae in 16.7% (2/12) and C. bovis in 25% (3/12) of the animals. All GP60 sequences were classified as genotype IIaA15G2R1 and were found in goat kids, calves and piglets sharing the same environment. This is the first description of the molecular identification and genotyping of Cryptosporidium in goat kids and piglets in Brazil. We conclude that Cryptosporidium species and C. parvum GP60 subtypes that infect livestock in Brazil, may act as sources of zoonotic infection for other animals and humans.

Serum concentrations of acute phase proteins and immunoglobulins of calves with rotavirus diarrhea / Concentração sérica de proteínas de fase aguda e imunoglobulinas em bezerros acometidos por diarreia por rotavírus

The aim of the present study was to characterize changes in acute phase protein levels according to the occurrence of rotavirus diarrhea in calves in the first month of life. Blood and fecal samples were taken before colostrum intake and at 1, 2, 7, 15, 21 and 30 days of age from 24 Holstein calves allotted in three experimental groups: calves that did not present diarrhea (group A), calves that presented diarrhea, but tested negative for rotavirus in feces (group B), and calves that presented diarrhea and tested positive for rotavirus in feces (group C) (experiment 1). When the animals presented episodes of diarrhea, blood and fecal samples were taken at 24-hour intervals until the end of clinical signs (experiment 2). Serum proteins were separated by SDS-PAGE technique and rotavirus in feces was detected by PAGE. Data of experiment 1 were analyzed by ANOVA and Tukey's test, considered significant at P<0.05. Data of experiment 2 were subjected to the HSD test. Total protein, globulins, and IgG concentrations were lower in group C than in groups A and B. Ceruloplasmin and transferrin levels were higher in group C than in groups A and B. Serum concentrations of haptoglobin and α1-acid glycoprotein did not differ significantly between groups throughout the experimental period. Calves presented diarrhea between 10.4 and 14.6 days of age in group B, and between 10.3 and 14.6 days of age in group C. In the moments of diarrhea manifestation, least square means of IgA, haptoglobin and α1-acid glycoprotein concentrations did not differ significantly between groups B and C, but ceruloplasmin and transferrin concentrations were higher in group C than in group B, as opposed to what occurred with IgG levels. These findings show that optimizing passive immunity transfer of immunoglobulins decrease the likelihood of calves developing diarrhea caused by rotavirus. In addition, ceruloplasmin presents characteristics of a biomarker of rotavirus infection in calves.(AU)

O objetivo do presente estudo foi avaliar alterações nos teores de proteínas de fase aguda de acordo com a ocorrência de diarreia por rotavírus em bezerros no decorrer do primeiro mês de vida. Amostras de sangue e fezes de 24 bezerros da raça Holandesa foram coletadas antes da ingestão de colostro e com um, dois, sete, quinze, vinte um e trinta dias de idade, sendo os bezerros alocados em três grupos: bezerros que não apresentaram diarreia (grupo A), bezerros que apresentaram diarreia, mas foram negativos para a detecção de rotavírus nas fezes (grupo B) e bezerros que apresentaram diarreia e foram positivos para detecção de rotavírus nas fezes (grupo C) (experimento 1). Sempre que os animais apresentavam episódio de diarreia, amostras de sangue e fezes eram coletadas em intervalos de 24 horas até o término dos sinais clínicos (experimento 2). As proteínas séricas foram separadas por meio da técnica de SDS-PAGE e a pesquisa de rotavírus nas fezes foi realizada por meio da técnica de PAGE. Os resultados do experimento 1 foram analisados por meio de ANOVA e do teste de Tukey, considerado significativo quando P<0,05. Os dados do experimento 2 foram submetidos ao teste HSD. Os teores de proteína total, globulinas e IgG foram menores no grupo C que nos grupos A e B, os teores de ceruloplasmina e transferrina foram maiores no grupo C que nos grupos A e B e as concentrações séricas de haptoglobina e α1-glicoproteína ácida não diferiram significativamente entre grupos. Os bezerros manifestaram diarreia, em média, com 10,4 a 14,6 dias de idade no grupo B e com 10,3 a 14,6 dias de idade no grupo C. Nos momentos de manifestação de diarreia, os teores de IgA, haptoglobina e α1-glicoproteína ácida não diferiram significativamente entre os grupos B e C, mas os teores de ceruloplasmina e transferrina foram maiores no grupo C que no grupo B, oposto ao verificado para o teor de IgG. Esses achados mostram que a otimização da transferência de imunidade passiva de imunoglobulinas reduz a probabilidade de os animais apresentarem diarreia por rotavírus. Adicionalmente, a ceruloplasmina apresenta características de um biomarcador da infecção por rotavírus em bezerros.(AU)

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Molecular Identification of Cryptosporidium Species from Pet Snakes in Thailand

Cryptosporidium is an important pathogen causing gastrointestinal disease in snakes and is distributed worldwide. The main objectives of this study were to detect and identify Cryptosporidium species in captive snakes from exotic pet shops and snake farms in Thailand. In total, 165 fecal samples were examined from 8 snake species, boa constrictor (Boa constrictor constrictor), corn snake (Elaphe guttata), ball python (Python regius), milk snake (Lampropeltis triangulum), king snake (Lampropeltis getula), rock python (Python sebae), rainbow boa (Epicrates cenchria), and carpet python (Morelia spilota). Cryptosporidium oocysts were examined using the dimethyl sulfoxide (DMSO)-modified acid-fast staining and a molecular method based on nested-PCR, PCR-RFLP analysis, and sequencing amplification of the SSU rRNA gene. DMSO-modified acid-fast staining revealed the presence of Cryptosporidium oocysts in 12 out of 165 (7.3%) samples, whereas PCR produced positive results in 40 (24.2%) samples. Molecular characterization indicated the presence of Cryptosporidium parvum (mouse genotype) as the most common species in 24 samples (60%) from 5 species of snake followed by Cryptosporidium serpentis in 9 samples (22.5%) from 2 species of snake and Cryptosporidium muris in 3 samples (7.5%) from P. regius.

efficacy of three medicinal plants: garlic, ginger and mirazid and a chemical drug metronidazole against Cryptosporidium Parvum. I- immunological response

Cryptosporidisis parvum is a zoonotic protozoan parasite infects intestinal epithelial cells causing a major health problem for man and animals. Experimentally the immunologic mediated elimination of C. parvum requires CD4+ T cells and IFN-Gamma. But, the innate immune responses also have a significant protective role in both man and animals. the mucosal immune response to C. parvum in C57BL/6 neonatal and GKO mice shows a concomitant Th1 and Th2 cytokine mRNA expression, with a crucial role for IFN-Gamma in the resolution of the infection.NK cells and IFN-Gamma have been shown to be important components in immunity in T and B cell-deficient mice, but IFN-Gamma-dependent resistance is demonstrated in alymphocytic mice. Epithelial cells may play a vital role in immunity as once infected these cells have increased expression of inflammatory chemokines and cytokines and demonstrate anti-infection killing mechanisms. C. parvum immunological response was used to evaluate the efficacy of anti-cryptospori- disis agents of Garlic, Ginger, Mirazid and Metronidazole in experimentally infected mice

Antiparasitic activity of silver and copper oxide nanoparticles against Entamoeba Histolytica and Cryptosporidium Parvum cysts

Nanoparticles [NPs] have received more attention as antiparasitic agents. In the present study, silver and copper nanoparticles were synthesized and characterized using scanning electron microscopy [SEM], transmission electron microscope [TEM] and X-ray fluorescence [XRF]. The antiparasitic activity of Ag and CuO nanoparticles were tested against two of the most environmentally spread parasites in Egypt [Entamoeba histolytica and Cryptosporidium parvum]. The average sizes of synthesized Ag NPs and CuO NPs were 9 and 29 nm respectively and a significant reduction for cysts viability [p > 0.05] was observed for CuO NPs against E. histolytica cysts and Ag NPs against C. parvum oocysts. Moreover, LC[50]-3h of CuO NPs for E. histolytica and C. parvum were 0.13 and 0.72 mg/l, while Ag NPs recorded 0.34 and 0.54 mg/l respectively. Accordingly, these NPs could be suggested as a new nanoform agent for safe and effective treatment of E. histolytica and C. parvum parasites

Measurement of Elastic Modulus and Vickers Hardness of Surround Bone Implant Using Dynamic Microindentation - Parameters Definition

The clinical performance of dental implants is strongly defined by biomechanical principles. The aim of this study was to quantify the Vicker's hardness (VHN) and elastic modulus (E) surround bone to dental implant in different regions, and to discuss the parameters of dynamic microindantion test. Ten cylindrical implants with morse taper interface (Titamax CM, Neodent; 3.5 mm diameter and 7 mm a height) were inserted in rabbit tibia. The mechanical properties were analyzed using microhardness dynamic indenter with 200 mN load and 15 s penetration time. Seven continuous indentations were made distancing 0.08 mm between each other perpendicularly to the implant-bone interface towards the external surface, at the limit of low (Lp) and high implant profile (Hp). Data were analyzed by Student's t-test (a=0.05) to compare the E and VHN values obtained on both regions. Mean and standard deviation of E (GPa) were: Lp. 16.6 ± 1.7, Hp. 17.0 ± 2.5 and VHN (N/mm2): Lp. 12.6 ± 40.8, Hp. 120.1 ± 43.7. No statistical difference was found between bone mechanical properties of high and low profile of the surround bone to implant, demonstrating that the bone characterization homogeneously is pertinent. Dynamic microindantion method proved to be highly useful in the characterization of the individual peri-implant bone tissue.

O desempenho clínico de implantes dentais é fortemente definido por princípios biomecânicos. Este trabalho objetivou quantificar a Dureza Vickers (VHN) e módulo de elasticidade (E) do osso periimplantar e discutir parâmetros metodológicos de ensaio dinâmico de indentação. Foram utilizados 10 implantes de corpo cilíndrico com interface cone morse, (Titamax CM; Neodent, Curitiba, PR, Brasil), diâmetro de 3.5 mm e altura de 7 mm inseridos em tíbia de coelho recém obtidas após abate dos animais. As propriedades mecânicas foram analisadas usando penetrador dinâmico de microdureza Vickers (CSM Micro-Hardness Tester; CSM Instruments, Peseux, Switzerland) com carga de 200 mN e tempo de penetração de 15s. Foram feitas 7 indentações no osso cortical na base da rosca (Br) e na ponta da rosca (Pr) na direção perpendicular ao implante, com distância entre elas de 0,08 mm perpendicular a interface osso implante em direção a superfície esterna. Os dados foram analisados por meio de teste t-Student (P<0,05). O valores médios e desvio padrão de E (GPa) foram: Br. 16,6 ± 1,7A; Pr. 17,0 ± 2,5A e VHN (N/mm2): Br. 125,6 ± 40,8A; Pr. 120,1 ± 43,7A. Não houve diferença significativa entre as propriedades mecânicas avaliadas no osso na base e na ponta da rosca do implante, demonstrando que a caracterização desta estrutura de forma homogênea em análises computacionais é pertinente. O método de indentação dinâmica mostrou ser altamente útil na caracterização individualizada do tecido ósseo periimplantar.

Fatal cryptosporidiosis in a calf / 대한수의학회지

A calf suffering from diarrhea was admitted to the Animal and Plant Quarantine Agency for diagnostic evaluation. Postmortem examination revealed that the mesenteric lymph node was enlarged and small intestine wall was thin. Microscopically, a large number of small round organisms were attached to the small intestine villi. Villous atrophy and proprial neutrophil infiltration were also observed. Based on modified Ziehl-Neelsen staining, electron microscopy, and ELISA results, the calf was diagnosed with fatal cryptosporidiosis.

Detection of Cryptosporidium parvum in Environmental Soil and Vegetables

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidial enteritis. Numerous outbreaks of cryptosporidiosis have been reported worldwide. Cryptosporidium is transmitted to hosts via consumption of contaminated water and food but also by direct contact with contaminated soil or infected hosts. The present study investigated farm soil collected from 34 locations along the western Korean peninsula and 24 vegetables purchased from local grocery markets in Seoul. The soil and vegetable samples were examined by real-time polymerase chain reaction (qPCR) to estimate the risk of infection. Eleven of 34 locations (32.4%) and 3 of 24 vegetable samples (12.5%) were contaminated with Cryptosporidium parvum, as confirmed by TaqI enzyme digestion of qPCR products and DNA sequencing. It is suggested that Cryptosporidium infection can be mediated via farm soil and vegetables. Therefore, it is necessary to reduce contamination of this organism in view of public health.

Epidemiological Characteristics of the First Water-Borne Outbreak of Cryptosporidiosis in Seoul, Korea

The first case of human cryptosporidiosis was reported in Korea in 1995; however, an outbreak of Cryptosporidium has not been reported in Korea until now. This paper describes the first outbreak of cryptosporidiosis in Korea. On May 24, 2012, a local public health center filed a report on 126 residents with gastrointestinal symptoms in an old apartment complex in Seoul. Epidemiological investigations were implemented on 125 of the 126 patients. The patients were reported continuously over a period of 22 days. Diarrhea was the most common clinical symptom, and lasted for 5 days on average. The tap water was the only common exposure of the patients. During the environmental investigation it was discovered that the water and septic tanks were situated closely and that the waste water pipes were corroded where they passed over the water pipes. Cryptosporidium parvum was detected in 3 of the 7 stool specimens by PCR-RFLP. A number of Cryptosporidium oocysts were also detected in the water specimens from the water tank. In conclusion, Cryptosporidium parvum was the key causal pathogen of this outbreak. It is presumed that the tap water was contaminated by a sewage leak from the aged pipelines.

Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.