Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice

Abstract Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle is necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Chlorogenic acid derived from Moringa oleifera leaf as a potential antiinflammatory agent against cryptosporidiosis in mice

@#Cryptosporidiosis is a serious illness in immunodeficient patients, and there is still no drug that can completely remove the parasite from the host. The present study represents the first report investigating the impact of the active molecule chlorogenic acid (CGA), naturally isolated from Moringa oleifera leaf extract (EMOLE), on immunosuppressed, Cryptosporidium parvum-infected BALB/c mice. Mice were divided into five groups: normal mice, infected immunosuppressed mice, and infected immunosuppressed mice treated with EMOLE, CGA, and nitazoxanide (NTZ) drugs. Parasitological, immunological, and histopathological investigations were recorded besides differences in the mice’ body weight. Infected control mice showed elevated levels of oocyst shedding throughout the study. The EMOLE- and CGA-treated groups showed 84.2% and 91.0% reductions in oocyst shedding, respectively, with no significant difference compared to the drug control. The inflammatory markers IFN-γ, IL-6, IL-1β, and TNF-α were significantly higher in the infected control group. Treatment with 300 mg/kg/day of EMOLE or 30 mg/kg/day of CGA significantly downregulated pro-inflammatory cytokine levels compared to the infected group, although they did not change significantly compared to the NTZ-treated group. Histopathology of intestinal sections showed inflammatory and pathological changes in the infected control group. Low-grade tissue changes and an obvious improvement in villi structure were seen in mice treated with CGA. This study highlighted the role of CGA, isolated and purified from EMOLE, as an effective anti-inflammatory agent in eradicating C. parvum infection.

Prevalência de la criptosporidiosis en seres humanos y terneros, y detección molecular del Cryptosporidium parvum / Prevalence of cryptosporidiosis in humans and calves and molecular detection of Cryptosporidium parvum

RESUMEN Objetivo. El objetivo de este estudio fue investigar la prevalencia de especies de Cryptosporidium en humanos y terneros en la provincia de Van, Turquía. Materiales y métodos. Se incluyeron en el estudio un total de 150 pacientes, incluidos 50 pacientes en hemodiálisis, 40 pacientes inmunosuprimidos con diarrea, 30 pacientes con diarrea solamente y 30 pacientes inmunocompetentes. Se recolectaron muestras de heces rectales de un total de 50 terneros alojados en establos y granjas en 10 aldeas centrales de Van, Turquía. Resultados. Se detectó Cryptosporidium parvum en el 17.3% de las 150 muestras de heces tomadas de seres humanos. C. parvum se observó en el 20% de los 50 pacientes en hemodiálisis, el 32.5% de los 40 pacientes inmunosuprimidos con diarrea y el 10% de los 30 pacientes con diarrea solamente, mientras que no hubo Cryptosporidium spp. detectado en los pacientes inmunocompetentes. C. parvum se observó en sólo el 6% de los 30 terneros diarreicos. Conclusiones. Claramente se entendio que la Criptosporidiosis fue detectada en una alta tasa en las muestras de los pacientes inmunosuprimidos sin y con sintomas de diarrea, y que además la especie activa que causó la enfermedad fue el agente etiologico Criptosporidium parvum. Por lo tanto, estos dos grupos de pacientes deben ser evaluados en lo que a términos de Criptosporidiosis se refiere.

ABSTRACT Objective. To investigate of the prevalence of Cryptosporidium species in humans and calves in the province of Van, Turkey. Materials and methods. Included in this research were 150 patients, comprising 50 hemodialysis patients, 40 immunosuppressed patients with diarrhea, 30 patients with diarrhea only, and 30 immunocompetent patients. Collected were stool rectal samples from 50 calves that were housed in stables and farms in 10 central villages of Van, Turkey. Results. Cryptosporidium parvum was detected in 17.3% of the 150 human stool samples. C. parvum was observed in 20% of the 50 samples from the hemodialysis patients, 32.5% of the 40 samples from the immunosuppressed patients with diarrhea, and 10% of the 30 samples from patients with diarrhea only, whereas no Cryptosporidium spp. was detected in the samples from the immunocompetent patients. C. parvum was observed in only 6% of the samples from the diarrheic 30 calves. Conclusions. It was clearly understood that cryptosporidiosis was detected at a high rate in the samples from the immunosuppressed patients and those who were immunosuppressed with diarrhea, and that the active and effective species that causes cryptosporidiosis in the Van region is C. parvum. Hence, these patient groups should be evaluated in terms of cryptosporidiosis.

Cryptosporidiosis, a public health challenge: A combined 3D shape-based virtual screening, docking study, and molecular dynamics simulation approach to identify inhibitors with novel scaffolds for the treatment of cryptosporidiosis

Cryptosporidiosis is a neglected tropical disease caused by the protozoan parasite Cryptosporidium parvum. Limited therapeutic options, limitation in in vitro parasite culture, and lack of a reliable animal model of parasite for replication of in vivo life cycle and drug testing demand alternative methods for drug development. The in silico methods of drug discovery prove a crucial process in such conditions.Recent research reported a limited number of small molecules for drug development. Purine nucleotide biosynthesis in Cryptosporidium species is dependent on the IMPDH (CpIMPDH) enzyme, so distortion of parasite IMPDH has been pursued as a compelling strategy for curbing Cryptosporidium infection due to its different kinetics from the host enzyme. Our study's primary aim was to discover novel ligand molecules with noticeable activity against Cryptosporidium parvum IMPDH. For this purpose, we selected 18 previously discovered ligands to understand the interaction feature between ligand and receptor, and their shape and electronic features are employed as a template for shape-based virtual screening of the ZINC database (drug-like subset) search approach via Schrodinger-2019 (Maestro 11.9). The obtained hits were subsequently subjected to structure-based screening, quantum polarized ligand docking (QPLD), and molecular dynamics simulations to fetch potential small molecules with the highest binding affinity for CpIMPDH protein. Further ligand binding energy and pharmacokinetic analysis were also taken into consideration as filtering criteria for selecting the most promising drug-like compounds. On this experimentation analysis, three top-ranked (ZINC24855054, ZINC58171263, and ZINC08000072) molecules were found to have appropriate pharmacokinetic properties along with surpassing in silico inhibitory potential towards the CpIMPDH compared to known inhibitors. The molecular docking and molecular dynamics simulation analysis results satisfactorily confirmed the inhibitory action. Therefore, these new scaffolds deduced by the presented computational methodology could recommend lead molecules for designing promising anti-cryptosporidial drugs targeting CpIMPDH protein.

Potential effect of methanol leaf extracts from three Podocarpus species on experimental cryptosporidiosis

@#Cryptosporidiosis causes diarrhea in both immunocompetent and immunocompromised individuals, with acute manifestations occurring particularly in children and the elderly. Up till now, there is no curative therapy for cryptosporidiosis, so discovery of new classes of drugs are of great importance. This study aimed to examine the effect of methanol leaves extracts of the three Podocarpus species; P. macrophyllus (Thunb.), P. gracilior (Pilg.) and P. elongatus (Aiton) L’ Hér. ex Pers and their combination on Cryptosporidium parvum (C. parvum) in experimentally infected mice in comparison with the commercially used drug, Nitazoxanide. As well as spectrophotometric estimation of the total phenolic and flavonoid content of these extracts was done. Results revealed that treatment with these three Podocarpus extracts and their combination showed a significant reduction of the number of C. parvum oocyst shed in the stool of infected mice compared to infected control group and Nitazoxanideinfected treated group at P < 0.001. The combination of the three Podocarpus extracts was the most effective treatment showing the lowest number of oocysts shedding in comparison with other used extracts and Nitazoxanide. Histopathological inspection of sections from ilium and colon displayed signs of improvement after treatment with P. macrophyllus and P. gracilior extracts and more remarkable improvement when the three extracts were combined. It was concluded that the three Podocarpus species extracts used in this study had a promising anti-Cryptosporidium activity especially when they were combined.

Identification, molecular characterization and factors associated with occurrences of Cryptosporidium spp. in calves on dairy farms in Brazil / Identificação, caracterização molecular e fatores associados à ocorrência de Cryptosporidium spp. em bezerros de propriedades leiteiras no Brasil

Abstract Cattle are an important source of zoonotic species of Cryptosporidium for humans. The aim of this study was to investigate the presence of Cryptosporidium, identify the species and determine the risk factors relating to environment, animals and management among dairy calves in eight Brazilian states. A total of 408 fecal samples from calves aged 1-60 days were analyzed. An epidemiological questionnaire was completed. Sample screening was performed using Ziehl-Neelsen technique and the positive samples were subjected to nested PCR. Cryptosporidium species were identified by means of the PCR-RFLP technique, using SSPI, ASEI and MBOII enzymes. The Ziehl-Neelsen technique showed that 89.7% (35/39) of the farms and 52.9% (216/408) of the samples were positive. Through nested PCR, these protozoa were detected in 54.6% of the samples. The 56 samples subjected to PCR-RFLP presented Cryptosporidium parvum. There was higher prevalence of the parasite in animals aged 7 to 28 days (62.6%). Diarrhea, ages between seven and 28 days and a spring water source were factors associated with the risk of infection. The calf hutch-type management system was associated with reduced infection. These findings demonstrate the high level of Cryptosporidium spp. circulation in cattle herds and the predominance of the species C. parvum.

Resumo O gado é uma fonte importante de espécies zoonóticas de Cryptosporidium para o homem. O objetivo deste estudo foi investigar a presença de Cryptosporidium, identificar a espécie e determinar os fatores de risco relacionados ao meio ambiente, aos animais e ao manejo em bezerros leiteiros em oito estados brasileiros. Um total de 408 amostras fecais de bezerros, com idade entre 1 e 60 dias, foram analisadas. Um questionário epidemiológico foi preenchido. A triagem das amostras foi realizada pela técnica de Ziehl-Neelsen, e as amostras positivas foram submetidas à "nested" PCR. As espécies de Cryptosporidium foram identificadas pela técnica de PCR-RFLP, utilizando-se as enzimas SSPI, ASEI e MBOII. A técnica de Ziehl-Neelsen mostrou que 89,7% (35/39) das fazendas e 52,9% (216/408) das amostras foram positivas. Por meio de nested PCR, esses protozoários foram detectados em 54,6% das amostras. As 56 amostras submetidas à PCR-RFLP apresentaram Cryptosporidium parvum. Houve maior prevalência do parasita em animais de 7 a 28 dias (62,6%). Diarreia, idade entre sete e 28 dias, e fonte de água mineral foram fatores associados ao risco de infecção. O sistema de manejo do tipo "casinha" para bezerros foi associado à redução da infecção. Esses achados demonstram o alto nível de Cryptosporidium spp. em circulação nos rebanhos bovinos e o predomínio da espécie C. parvum.

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection

@#The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

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.

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.

Octreotide modulates expression of somatostatin receptor subtype 4 and 5 in unweaned rat jejunum inflamed by Cryptosporidium parvum / 中国药理学通报

Aim To investigate the effects of intestinal infection induced by Cryptosporidium parvum on the ex-pression pattern of SSTR4 and SSTR5 subtype, and to examine the effect of octreotide on modulating short-term and long-term SSTR4 and SSTR5 subtype expres-sion in rat jejunum. Methods Five-day-old suckling Sprague-Dawley rats were orally gavaged with 105Cryp-tosporidium oocysts to establish the model of post-infec-tion irritable bowel syndrome (PI-IBS). Rats then re-ceived 50 μg·kg-1·d-1of octreotide by intraperito-neal injection from day 10 to day 17 post-infection. Animals were sacrificed on day 17,37 and 50 post-in-fection for immunohistochemical analysis and on day 14,35 and 50 for mRNA expression analysis of SSTR4 and SSTR5 subtypes. Results Immunohistological a-nalysis of jejunum tissues demonstrated that SSTR5 was mainly expressed in submucosa while a little in crypt,while SSTR4 was mainly expressed in crypt while a lit-tle in submucosa and absorptive cells. Real-time PCR analysis indicated a significant increase of SSTR4 and SSTR5 expression in the inflamed jejunum. Octreotide treatment decreased the expression of SSTR4 and SSTR5 on day 50 post-infection. Conclusions SSTR4 and SSTR5 may be involved in the inflammatory reac-tion in PI-IBS rats induced by Cryptosporidium parvu-man, which shows an increase in SSTR4 and SSTR5 mRNA expression in jejunum. Octreotide therapy in-hibits the jejunum hypersensitivity and relieve PI-IBS symptoms, which may be related to the decrease of SSTR4 and SSTR5 expression.

Effect of Cryptosporidium parvum infection on Toll-like receptors of intesti-nal mucosa in mice / 中国血吸虫病防治杂志

Objective To investigate the mechanism of Toll-like receptor in intestinal mucosal injury induced by Cryptospo-ridium parvum infection in mice.Methods Totally 30 male BALB/c mice were randomly divided into a normal control group,1-week infection group and 2-week infection group.The mice of the 1-week and 2-week infection groups were sacrificed 7 days and 14 days after the infection respectively,and the mice of the normal control group were sacrificed 14 days after the infection.The model of intestinal infection of C.parvum in mice was built by using the immunosuppressive method and oocyst intragastric ad-ministration.The pathological changes of the intestinal mucosa of mice were observed with a light microscope and the villus height,crypt depth and ratio of villus height/crypt depth were measured.The ultrastructure of the intestinal mucosa of mice was observed by a transmission electron microscope(TEM).The expressions of TLR2 and TLR4 in the intestinal mucosa were tested by qPCR and Western blotting.Results Under the light microscope,the intestinal villi were dropsical,obviously atrophied and shortened,and the submucosal structure was dropsical.The height of chorionic villi and the ratio of villus height to crypt depth in the jejunum of the 1-week and 2-week infection groups were significantly lower than those in the normal control group(all P<0.05),while the depth of the recess of the former two was significantly increased(all P<0.05).With the extension of the infection time,the villus height and the ratio of villus height to crypt depth in the jejunum of mice decreased significantly(both P<0.05),and the crypt depth increased significantly(P<0.01).The TEM observation showed that the structure of the oocyst of C.parvum in the jejunum of the infected mouse was intact,the villi around the oocyst were abscission seriously,and the oocyst wall was fused with the epithelial cell membrane.The qPCR observation showed that compared with the normal control group,the expressions of TLR2 mRNA and TLR4 mRNA in the intestinal mucosa of the 1-week and 2-week infection groups were significantly higher(all P<0.05).In addition,the expressions of TLR2 and TLR4 mRNA in the 2-week infection group were significantly higher than those in the 1-week infection group(both P<0.05).The Western blotting showed that the expres-sions of TLR2 protein and TLR4 protein in the intestinal mucosa of the 1-week and 2-week infection groups were significantly higher than those of the normal control group(all P<0.05).Furthermore,the expressions of TLR2 and TLR4 protein in the 2-week infection group were significantly higher than those in the 1-week infection group(both P<0.05).Conclusions TLR2 and TLR4 are important receptors for intestinal mucosal recognition of C.parvum.The C.parvum infection may lead to intestinal mucosal damage possibly via the mechanisms associated with the up-regulation of TLR2 and TLR4 expressions.

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.

Molecular epidemiology of Cryptosporidium infection in infants with diar-rhea in Wuhan City / 中国血吸虫病防治杂志

Objective To understand the prevalence of Cryptosporidium infection in diarrhea infants under 2 years old in Wuhan City,so as to provide the epidemiological evidence for the prevention and treatment of cryptosporidiosis. Methods The fecal samples from infants under 2 years old with diarrhea were collected in Hubei General Hospital and Central South Hospital in Wuhan City,Hubei Province from August 2014 to July 2015. The fecal samples were stored in 2.5%potassium dichromate at 4℃after filtered. The DNA was extracted from the fecal pellets with the phenol-chloroform method. The Cryptosporidium species were detected by a nested PCR assay targeting the SSU rRNA gene of the parasite. All the positive PCR products were sequenced on ABI 3100 automated sequencer,and the amplified sequences were compared to homologous sequences in the NCBI database by using the Basic Local Alignment Search Tool(BLAST). Phylogenetic analyses were performed by using the software MEGA (version 4.0)based on the Neighbour-Joining method. Results The human stool specimens(n=298)were screened for the presence of Cryptosporidium by nested PCR. The infection rate of Cryptosporidium was 3.02%(9/298). The infection rate of Cryp-tosporidium was 5.93%(7/118)in the infants between 1-2 years old,and the infection rate was 1.11%(2/180)in the infants un-der 1 year old,and there was a significant difference between the two groups(χ2 =4.13,P<0.05). The nine samples which were positive by nested PCR were successfully sequenced and compared with the reference sequences in GenBank. The results revealed the nine positive specimens were all infected with C. parvum,and two of them were co-infected with C. hominis. Neigh-bor-joining trees were constructed from the aligned partial SSU rRNA sequences of these nine isolates,and in the SSU rRNA lo-cus,the nine isolates were grouped with C. parvum. Conclusion There exists Cryptosporidium infection in the infants under 2 years old with diarrhea in Wuhan City,and the main species of Cryptosporidium is C. parvum.

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.

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.

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.

A Study Of The Prevalence Of Cryptosporidium Parvum In Stool Samples Of Patients Of Tertiary Care Hospital, Ahmedabad.

Abstracts: Background: Intestinal parasitic infections remain a serious public health problem globally.Although there could be many other causes of diarrhoea, the enteric protozoa Cryptosporidium parvumhave been recognized as important causes of both out-break-related and sporadic diarrhoea in humans. Both immunocompetent and immunocompromised individuals could be the victims but immunocompromised peoples are likely to be most seriously affected. This study was done to determine the prevalence of Cryptosporidium parvumin Stool samples. Methodology: A 100 Stool samples of patients visiting General Hospital, Sola, Ahmedabad from December 2013 to March 2014 were followed for Stool microscopy for demonstration of cyst of Cryptosporidium parvum.Modified ZN stain done from direct smear from stool sample but their concentration is increased by formal ether concentration technique. Results: Out of total 100 stool samples were examined in which 85 were positive for bacteriological infections and 15 for parasitic infection. Prevalence of Cryptosporidium parvum infection in our study is 5 %. Among 100 patients only 3 were positive for Cryptosporidium infection in 96 immunocompetent patients and 2 were positive for Cryptosporidium infection in 4 immunocompromised patients. So higher rate of prevalence of Cryptosporidium parvum infection seen in immunocompromised patients. Conclusion: Cryptosporidium infection is transmitted by feco-oral route & water borne, so proper sanitation and disinfection of water reduce the prevalence of infection. Cryptosporidium parvum diarrhoea is self-limiting illness and cured by fluid therapy. Drug therapy is only for severe infection. In immunocompromised patients like HIV antiretroviral therapy and fluid therapy is necessary for Cryptosporidium infection.