Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples / Validação da nested PCR em tempo real em um tubo para detecção de Cryptosporidium spp. em amostras fecais de aves

Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Resumo O objetivo deste trabalho foi validar um protocolo de nested PCR em tempo real em um tubo (nPCR-TR-1T) seguida de sequenciamento genético para detectar e caracterizar as espécies e genótipos de Cryptosporidium em aves. Um total de 443 amostras de DNA genômico, extraído de amostras fecais de aves, foi analisado pela nPCR-TR-1T e pela nested PCR convencional. Pela nPCR-TR-1T, foi observada positividade para Cryptosporidium spp. de 20,3% (90/443), em contraste com a nested PCR convencional, que apresentou positividade de 8,1% (36/443). O teste de sensibilidade analítica mostrou que a nPCR-TR-1T detecta aproximadamente 0,5 oocisto (2 esporozoítos) por reação. A avaliação da especificidade analítica não revelou amplificação de microrganismos que comumente apresentam amplificação inespecífica com primers utilizados para o diagnóstico de Cryptosporidium spp. O cálculo da repetibilidade evidenciou o mesmo resultado em 27 de 30 amostras (90%). Em relação à reprodutibilidade da nPCR-TR-1T, foi observado o mesmo resultado em 80% (24/30) das amostras examinadas. Foi possível realizar o sequenciamento em todas as 90 amostras amplificadas pela nPCR-TR-1T, com identificação de C. baileyi, C. galli, C. meleagridis, C. proventriculi e Cryptosporidium genótipo I em aves. O sequenciamento dos fragmentos amplificados pela nested PCR convencional foi possível em 10/36 (27,8%) das amostras positivas.

Prevalence of human cryptosporidiosis in the Americas: systematic review and meta-analysis

ABSTRACT Cryptosporidiosis is a disease caused by the Cryptosporidium spp parasite. As some species of Cryptosporidium have a wide host spectrum, the characterization of the pathogen at the species or genotype level is of great importance to define the sources of infection for humans and the potential for public health. This study investigated the diversity of the genus Cryptosporidium spp. in humans from all over the American continent and observed whether the method used to search for the parasite influenced the prevalence found in the Americas. This systematic review was carried out using the Pubmed, Science direct, Lilacs, Scielo, and Scopus databases with publications from January 1, 2010, to December 31, 2020. For data synthesis, the PRISMA flowchart was used and for the meta-analysis we used the MetaXL program. Of the selected publications, 57, 9 and 16 belonged to the region of South, Central and North America, respectively. The prevalence found for South, Central, and North America was 7%, 7%, and 8%, respectively, when analyzing publications that used only the microscopy method. When we analyzed the publications that used immunological and molecular methods, we found prevalences of 10%, 9%, and 21% for South, Central, and North America, respectively. The C. hominis subtype IbA10G2 was the most reported in the American continent, followed by subtype IeA11G3T3 and, for C. parvum, subtype IIaA15G2RI was the most reported. In conclusion, Cryptosporidium spp. is present throughout the American continent and its prevalence is higher when immunological and/or molecular methods are used, in addition to direct microscopic examination.

Intestinal apicomplexan parasitoses among a hospital-based population in Honduras, 2013-2019 / Parasitosis por Apicomplexa intestinales en población hospitalaria en Honduras, 2013-2019

Abstract | Introduction: Intestinal apicomplexa protozoa are a recognized cause of gastroenteritis. They are endemic in Honduras and their epidemiology varies in different population groups. Objective: To identify risk factors for cyclosporiasis, cryptosporidiosis, and cystoisosporiasis. Materials and methods: We conducted a case-control study in a hospital-based population. We performed the diagnosis using the modifed Ziehl-Neelsen staining technique and collected the information from laboratory records and clinical charts. Results: Cyclosporiasis was associated with diarrhea (OR=2.28; 95%CI: 1.10-4.89), weight loss (OR=12.7; 95%CI: 2.49-122.00), watery stools (OR=2.42; 95%CI: 1.26-4.65), and infection with another protozoan (OR=3.13; 95%CI: 1.66-5.95). Cryptosporidiosis was associated with HIV infection (OR=15.43; 95%CI: 3.34-71.22), diarrhea (OR=3.52; 95%CI: 1.40-9.40), lymphopenia (OR=6.16; 95%CI: 1.99-18.98), and green color stools (OR=3.00; 95%CI: 1.23-7.30). Cystoisosporiasis was associated with HIV infection (OR=11.20; 95%CI: 3.53-35.44), diarrhea (OR=7.30; 95%CI: 1.89-28.52), leukopenia (OR=4.28; 95%CI: 1.33-13.75), green color stools (OR=11.59; 95%CI: 1.16-558.60), and Charcot-Leyden crystals (OR=11.59; 95%CI: 1.16-558.60). Conclusions: In this hospital-based population from Honduras, HIV infection was a risk factor for cryptosporidiosis and cystoisosporiasis, but not for cyclosporiasis.

Resumen | Introducción. Los protozoos Apicomplexa intestinales son causa reconocida de gastroenteritis. Estas parasitosis son endémicas en Honduras y su epidemiologia varia según los grupos poblacionales. Objetivo. Identifcar los factores de riesgo para ciclosporiasis, criptosporidiosis y cistoisosporiasis. Materiales y métodos. Se hizo un estudio de casos y controles en población hospitalaria. El diagnóstico se hizo utilizando la coloración modifcada de Ziehl-Neelsen. La información se obtuvo del registro de laboratorio y las historias clínicas. Resultados. La ciclosporiasis se asoció con diarrea (OR=2,28; IC95% 1,10-4.89), pérdida de peso (OR=12,7; IC95% 2,49-122), heces líquidas (OR=2,42; IC95% 1,26-4,65), infección con otros protozoos (OR=3,13; IC95% 1,66-5,95). La criptosporidiosis se asoció con el HIV (OR=15,43; IC95% 3,34-71,22), la diarrea (OR=3,52; IC95% 1,40-9,40), la linfopenia (OR=6,16; IC 95% 1,99-18,98), las heces de color verde (OR=3,00; IC95% 1,23-7,30). La cistoisosporiasis se asoció con el HIV (OR=11,20; IC95% 3,53-35,44), la diarrea (OR=7,30; IC95% 1,89-28,52), la leucopenia (OR=4,28; IC95% 1,33-13,75), las heces de color verde (OR=11,59; IC95% 1,16- 558,60), y los cristales de Charcot-Leyden (OR=11,59; IC95% 1,16-558,60). Conclusiones. En este estudio de base hospitalaria en Honduras, el HIV fue un factor de riesgo para la criptosporidiosis y la cistoisosporiasis, pero no así para la ciclosporiasis.

Prevalence of cryptosporidiosis in animals in Iran: A systematic review and metaanalysis

Objective: To review the prevalence of cryptosporidiosis among animal population of Iran. Methods: Data were systematically gathered from 1 January 2000 to 1 January 2020 in the Islamic Republic of Iran from the following electronic databases: PubMed, Springer, Google Scholar, Science Direct, Scopus, Web of Science, Magiran, and Scientific Information Database (SID). According to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) and inclusion criteria, 88 eligible studies were obtained. Results: The pooled prevalence of cryptosporidiosis using random and fixed effects model according to heterogeneity among animals was as follows: rodents 18.8% (95% CI 12.6%-25.0%), camels 17.1% (95% CI 8.6%-25.7%), cattle 16.8% (95% CI 13.4%-20.1%), goats 14.1% (95% CI 5.2%-23.0%), horses 12.2% (95% CI 8.3%- 16.2%), birds 10.5% (95% CI 7.6%-13.4%), sheep 9.9% (95% CI 2.4%-4.9%), cats 8.8% (95% CI 4.8%-12.8%) and dogs 3.7% (95% CI 7.0%-12.8%). Conclusions: Cryptosporidiosis has been reported and present in a wide range of animals in Iran over the years and has a high prevalence in most of these species.

Identification of potential targets and synergistic mechanism of Kushen Decoction for the treatment of cryptosporidiosis / 中国血吸虫病防治杂志

Objective To explore the potential targets and synergistic mechanisms of Kushen Decoction for the treatment of cryptosporidiosis using network pharmacology and molecular docking methods. Methods The main active ingredients of Kushen Decoction were captured from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TC-MSP) and the Universal Protein Resource (UniProt) database, and the potential targets were predicted. In addition, the active ingredients of Kushen Decoction that were not included in the TCMSP database were retrieved in CNKI, WanFang Data, CBM, PubMed and Web of Science databases, and the target genes of all supplemented active ingredients were predicted using the online TargetNet database. Network construction and analysis were performed using the Cytoscape software, and cryptosporidiosis-related targets were retrieved in the Comparative Toxicogenomics Database and GeneCards database. The protein-protein interaction (PPI) network was created using the STRING database, and the DAVID database was used for GO enrichment and KEGG pathway analyses. The tissue distribution of key targets was investigated using the BioGPS database, and the AutoDockTools software was employed to verify the molecular docking results. Results A total of 38 active ingredients of Kushen Decoction were screened, and the core ingredients included quercetin, (+)-14α-hydroxymatrine and apigenin. A total of 831 targets of Kushen Decoction and 512 cryptosporidiosis-related targets were predicted, and PPI network analysis revealed 69 key targets, including AKT1, TNF and IL-6. There were 303 biological processes, 46 molecular functions and 29 cellular components involved in the treatment of cryptosporidiosis with Kushen Decoction, and 13 KEGG pathways played a therapeutic role in the synergistic mechanisms of multiple targets, such as Toll-like receptor (TLR), nuclear factor kappa B(NF)-κB, nucleotide binding oligomerization domain like receptor (NLR) signal pathways. The core targets were mainly distributed in the hematologic and immune systems. Molecular docking analysis showed that the binding energy between active ingredients and key targets were all less than 0 kJ/mol, indicating the strong binding of ligands to receptors. Conclusions The active ingredients of Kushen Decoction, such as quercetin, (+)-14α-hydroxymatrine and apigenin, may act on targets like AKT1, TNF, IL-6 to modulate TLR, NLR and NF-κB signaling pathways to play a synergistic role in the treatment of cryptosporidiosis in the hematologic and immune system.

Aplicación de la metodología de las fuerzas motrices y el modelo OMS de determinantes sociales de la salud tomando como ejemplo la criptosporidiosis en Colombia / Application of the driving forces methodology and the WHO model of social determinants of health taking cryptosporidiosis in Colombia as an example / Aplicação da metodologia forças motrizes e do modelo de determinantes sociais da saúde da OMS, tomando a criptosporidiose como exemplo na Colômbia

RESUMEN Objetivos Analizar la relación que existe entre la contaminación de agua por ooquistes de Cryptosporidium spp. y los efectos en la salud de las poblaciones humanas y animales en Colombia. Métodos Es un ensayo de tipo analítico que se centró en la aplicación documentada y analizada, de la metodología de las "Fuerzas Motrices" y el modelo OMS de Determinantes Sociales de la Salud tomando como ejemplo la Criptosporidiosis. Resultados Las "fuerzas motrices" involucradas en la relación entre contaminación de agua por Cryptosporidium spp. y la salud de humanos y animales fueron el aumento en la demanda de productos pecuarios y la producción pecuaria de baja calidad; la "presión" consistió en la mala implementación de prácticas de producción, la invasión de zonas protegidas y de reservas ecológicas, y la disminución de fronteras entre lo rural y lo urbano; el "estado" se basó en la contaminación del suelo y las fuentes de agua, y en la deficiencia en el manejo de las excretas; la "exposición" radicó en la entrada de ooquistes vía fecal-oral, por la contaminación del suelo y el agua, y el "efecto" consistió en una diarrea autolimitante, una deshidratación severa, el síndrome de mala absorción y la muerte. Conclusiones Los ecosistemas pueden mantener poblaciones saludables, pero cuando son mal administrados o rápidamente alterados debido a la presión humana, también pueden estar asociados con la aparición de enfermedades, como la Criptosporidiosis.(AU)

ABSTRACT Objectives To analyze the relationship between water contaminated with Cryptosporidium spp. oocysts and its effects on the health of human and animal populations in Colombia. Materials and Methods Analytical essay focused on the documented and analytical application of the Driving Forces Methodology and the WHO model of Social Determinants of Health using cryptosporidiosis as an example. Results The driving forces involved in the relationship between water contaminated with Cryptosporidium spp. oocysts and human and animal health were increased demand for livestock products and low-quality livestock production. Regarding pressure, it was related to the poor implementation of production practices, the invasion of protected areas and ecological reserves, and the reduction of rural-urban borders. On the other hand, status was associated with contamination of soil and water sources, as well as the deficient management of excreta. Finally, exposure was determined by the entry of oocysts via the fecal-oral route through contaminated soil and water, and effect consisted of self-limiting diarrhea, severe dehydration, malabsorption syndrome, and death. Conclusions Ecosystems can maintain healthy populations, but when they are mismanaged or rapidly altered by human pressure, they can also be associated with the emergence of diseases such as Cryptosporidiosis.(AU)

OBJETIVOS Analisar a relação entre contaminação da água por Oocistos de Cryptosporidium spp. e os efeitos sobre a saúde das populações humanas e animais na Colômbia. MÉTODOS Trata-se de um estudo de tipo analítico que se concentrou na aplicação documentada e analisada da metodologia "Forças de Condução" e do modelo da OMS de Determinantes Sociais da Saúde, tomando a criptosporidiose como exemplo. RESULTADOS As "forças motrizes" envolvidas na relação entre contaminação da água por Cryptosporidium spp. e saúde humana e animal foram o aumento da demanda por produtos animais e a produção animal de baixa qualidade; a "pressão" consistia na má implementação das práticas de produção, na invasão de áreas protegidas e reservas ecológicas e na redução das fronteiras entre as áreas rurais e urbanas; o "estado" se baseava na contaminação do solo e nas fontes de água e na deficiência no manejo dos excrementos; a "exposição" foi devido à entrada de oocistos pela via fecal-oral, devido à contaminação do solo e da água, e o "efeito" consistiu em diarréia autolimitada, desidratação grave, síndrome de má absorção e morte. CONCLUSÕES Os ecossistemas podem manter populações saudáveis, mas quando são mal gerenciados ou rapidamente alterados devido à pressão humana, também podem ser associados ao aparecimento de doenças, como a criptosporidiose.(AU)

First study of Cryptosporidium spp. occurrence in eared doves (Zenaida auriculata) / Primeiro estudo de ocorrência de Cryptosporidium spp. em pomba-de-bando (Zenaida auriculata)

Abstract Cryptosporidium is a protozoan parasite with a wide range of hosts, including humans. However, only a few Cryptosporidium species have been described in birds (C. meleagridis, C. baileyi, C. galli and C. avium). The aim of this study was to investigate the occurrence of Cryptosporidium spp. in feces of eared doves (Zenaida auriculata), followed by molecular characterization of the parasite. A total of 196 animals of both sexes were trap-captured; the animals were culled and the intestinal contents were collected for DNA extraction. After extraction, a nested-PCR (nPCR), which amplifies a fragment of the 18S rRNA gene of Cryptosporidium spp., was performed. The amplicons obtained were purified and sequenced. PCR analysis revealed that 30 animals (15.3%) were positive for Cryptosporidium spp. There was no significant sex-dependent enrichment of Cryptosporidium occurrence (p > 0.05). Only 15 out of the 30 positive samples were successfully sequenced and their species determined, of which, 13 (86.7%) and 2 (13.3%) were C. meleagridis and C. galli, respectively. Herein, we present for the first time a molecular characterization of Cryptosporidium from feces of eared doves (Z. auriculata) and propose that these birds are a potential source of C. meleagridis infection in humans.

Resumo Cryptosporidium é um protozoário com uma grande variedade de hospedeiros, incluindo os seres humanos. No entanto, poucas espécies têm sido descritas em aves (Cryptosporidium meleagridis, C. baileyi, C. galli e C. avium). O objetivo do presente estudo foi investigar a ocorrência de Cryptosporidium spp. em fezes de pombas-de-bando (Zenaida auriculata), e realizar a caracterização molecular dos isolados. Um total de 196 animais de ambos os sexos foram capturados, eutanasiados e o conteúdo intestinal recolhido para extração de DNA. Após a extração, realizou-se uma nested-PCR (nPCR), que amplifica um fragmento do gene 18S rRNA do Cryptosporidium spp.. Os fragmentos obtidos foram purificados e encaminhados para sequenciamento. Os resultados da n-PCR revelaram 30 animais (15.3%) positivos para Cryptosporidium spp.. Quanto ao sexo dos animais não foram observadas diferenças estatísticas significativas (p > 0.05). Somente 15 de 30 amostras positivas foram sequenciadas com sucesso e as espécies determinadas, das quais, 13 (86.7%) e 2 (13.3%) foram C. meleagridis e C. galli, respectivamente. Esse é o primeiro estudo com caracterização molecular de Cryptosporidium de fezes de pombas-de-bando (Z. auriculata), e propõe serem esses animais potenciais fonte de infecção de C. meleagridis para humanos.

Intestinal cryptosporidiosis in renal transplant recipients: Prevalence, species detection and comparative evaluation of SSU rRNA and Cryptosporidium oocyst wall protein genes

Context: Cryptosporidiosis is intestinal opportunistic infection commonly occurring in immunocompromised patients including renal transplant (RT) recipients receiving continuous immunosuppressive therapy. Knowledge about species of Cryptosporidium-infecting RT recipients is necessary to know about mode of its transmission (anthroponotic or zoonotic). Various genes such as small subunit rRNA (SSU rRNA) and Cryptosporidium oocyst wall protein (COWP) genes may help in species identification though their sensitivity and specificity are highly variable. Subjects and Methods: A total of 993 and 575 stool samples were examined for Cryptosporidium by microscopy from 358 RT recipients and 200 healthy controls, respectively. Stool samples of RT recipients and healthy controls were subjected to polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) for species identification. Results: Cryptosporidium was more commonly detected amongst RT recipients than healthy controls (30/358, 8.4% vs. 0/200, respectively; P < 0.001). The infection was more common amongst patients with diarrhoea than those without (26/162, 16.1% vs. 4/145, 2.8%; P < 0.001). Cryptosporidium parvum was identified in 10/30 (33.3%) and Cryptosporidium hominis in 20/30 (66.7%) samples. SSU gene PCR-RFLP proved to be more sensitive (100%) than COWP (90%); however, specificity of both was same (100%). Conclusions: Cryptosporidiosis is common amongst RT recipients, particularly those with diarrhoea. C. hominis is the most common species in the studied population. SSU rRNA PCR was more sensitive molecular method for the differentiation of Cryptosporidium species.

Cryptosporidium occurrence in ruminants from the North Pioneer mesoregion of Paraná, Brazil / Ocorrência de Cryptosporidium em ruminantes da mesorregião norte pioneiro do Estado do Paraná, Brasil

Abstract The aim of this study was to investigate the occurrence of Cryptosporidium in cattle and sheep from the North Pioneer mesoregion of the state of Paraná. For this, 317 stool samples were collected from cattle and sheep on 16 properties in six municipalities in the North Pioneer mesoregion of Paraná. For detection of Cryptosporidium species, molecular analysis was performed using nested-PCR techniques targeting the 18S rRNA gene. Of the 37 beef cows and 115 calves analyzed, four (10.8%) and 14 (12.2%), respectively, were positive for Cryptosporidium. Of the 12 cows and 52 calves, one (8.3%) and 14 (26.9%), respectively, were positive for Cryptosporidium; and of the 42 ewes and 59 lambs, six (14.3%) and 12 (20.3%), respectively were positive for Cryptosporidium. Cattle (15.3%) and sheep (17.8%) were both susceptible to infection. All the properties of the municipalities of Assaí, Ibaiti and, Leópolis presented infected animals. The study showed that Cryptosporidium occurs in most municipalities assessed, that dairy calves had a higher risk (Odds Ratio=2,66, p-value=0,018) for infection than beef calves, and that sheep are just as susceptible to infection as are cattle, and that further Cryptosporidium studies are developed.

Resumo O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium em bovinos e ovinos da mesorregião norte pioneiro do Estado do Paraná. Para tanto, 317 amostras de fezes destes ruminantes foram colhidas de 16 propriedades de seis municípios do Norte Pioneiro do Paraná. Para detecção de Cryptosporidium spp foi realizada análise molecular pela Técnica de nested-PCR direcionada ao gene 18S rRNA. Das 37 vacas de corte e 115 bezerros de corte analisados, quatro (10,8%) e 14 (12,2%) foram respectivamente positivos para Cryptosporidium . Das 12 vacas e 52 bezerros de leite, um (8,3%) e 14 (26,9%) foram positivos para Cryptosporidium e das 42 ovelhas e 59 cordeiros avaliados, seis (14,3%) e 12 (20,3%) amostras estavam positivas para Cryptosporidium, respectivamente. Bovinos (15,3%) e ovinos (17,8%) foram igualmente suscetíveis à infecção. Todas as propriedades dos municípios de Assaí, Ibaiti e Leópolis apresentaram animais infectados. Este estudo demonstrou que Cryptosporidium ocorre na maioria dos municípios avaliados, sendo que os bezerros de leite apresentam maior risco (Razão de chances=2,66, p-value=0,018) à infecção que os bezerros de corte e que os ovinos são tão suscetíveis à infecção quanto os bovinos e por isso, estudos nesta espécie animal devem ser mais desenvolvidos.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul / Fatores de risco e infecção por Cryptosporidium spp. em cães e gatos no sul do Rio Grande do Sul

Abstract This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Resumo Este estudo verificou a frequência de oocistos de Cryptosporidium spp. em fezes de cães e gatos em cinco municípios da região sul do Rio Grande do Sul e fatores de risco associados à infecção. Amostras de fezes de 110 cães e 18 gatos foram coradas pelo método de auramina. No momento da coleta de fezes aplicou-se um questionário aos tutores dos animais com questões semiabertas e os dados foram submetidos à análise estatística. A frequência verdadeira de oocistos de Cryptosporidium spp. foi de 24,63% (27 cães e 2 gatos). Apenas quatro amostras de fezes caninas eram diarreicas e todas sem oocistos. As variáveis que representaram fatores de risco para a infecção foram: alimentos de preparo caseiro, água não tratada, circulação dos animais em terreno gramíneo e convivência com outros animais, principalmente bovinos. Os resultados sugerem que a frequência de cães parasitados por Cryptosporidium spp. é relevante, reforçando o caráter assintomático da infeção. Destaca-se a importância da adoção de medidas de controle, particularmente das variáveis que representaram fatores de risco à infecção.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods / Detecção e caracterização molecular de Cryptosporidium spp. em canários mantidos em cativeiro (Serinus canaria) por diferentes métodos de diagnóstico

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Resumo Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.

Criptosporidiosis y una salud / Cryptosporidiosis and one health

RESUMEN Criptosporidiosis es una enfermedad diarreica (zoonótica) causada por el parásito Cryptosporidium, el cual infecta al intestino. Se producen tanto en el ser humano como en diferentes animales de granja, mascotas y animales silvestres. Se subestima su papel y poco se conoce sobre su epidemiología, debido al escaso interés que suscita y la limitada disponibilidad de métodos de diagnóstico. La salud de las poblaciones tanto humana como animal, están relacionadas con una estrecha interacción de tres factores: el ambiente, los agentes patógenos, y las poblaciones, lo cual determina el estado de salud y enfermedad. El enfoque, conocido como «Una Salud¼, es una estrategia global en crecimiento que se está adoptando por una diversidad de organizaciones y responsables de las políticas en respuesta a la necesidad de enfoques integrados. Este enfoque puede ser relevante para una amplia gama de objetivos mundiales de desarrollo. Cada vez se habla más de salud integral, de salud holística. Parece que somos más conscientes de que la salud no se reduce a algo puramente biológico, sino que afecta a toda la persona. No es sólo ver al enfermo globalmente, es partir de la complejidad del ser humano y del mundo entero atravesado por la vulnerabilidad e interaccionar con la totalidad de los sujetos.(AU)

ABSTRACT Cryptosporidiosis is a diarrheal disease (zoonotic) caused by the parasite Cryptosporidium, which infects the intestine. It occurs in humans and in different pets, farm and wild animals. Its role is underestimated and little is known about its epidemiology, due to the low interest it draws and the limited availability of diagnostic methods. The population health, both human and animal, is related to a close interaction of three factors: the environment, pathogens, and population, which determines the state of health and disease; some of the diseases considered as emerging, that appeared in the last years. The approach, known as "One Health", is a growing global strategy being adopted by a diversity of organizations and policy makers in response to the need for integrated approaches. This approach may be relevant to a wide range of global development goals. There is more and more talk about holistic health. It seems that we are more aware that health is not reduced to something purely biological, but affects the whole person. It is not only seeing the patient globally, it is starting from the complexity of the human being and the whole world crossed by vulnerability and interacting with the totality of the subjects.(AU)

Molecular characterization of Cryptosporidium parvum and Cryptosporidium hominis GP60 subtypes worldwide / Caracterización molecular de los subtipos de la GP60 de Cryptosporidium parvum y Cryptosporidium hominis alrededor del mundo

ABSTRACT Cryptosporidium is a zoonotic parasite very important in animal health as well as in public health. It is because this is one of the main causes of diarrhea in children, calves, lambs and other variety of youth mammalians in a lot of countries. The globalization has enabled the exchange of biological material in different regions worldwide, encouraging the spread of diseases and exposure to these biological agents to different environmental conditions, inducing adaptation through genetic changes. Based in the polymorphism of the gene for GP60, this review intended to present the distribution of Cryptosporidium parvum and Cryptosporidium hominis in humans and calves worldwide. The subtype that affects cattle more frequently corresponds to IIaA15G2R; while the subtype most frequently isolated from human samples is IaA19G2.

RESUMEN Cryptosporidium es un parásito zoonótico muy importante en salud animal así como en salud pública. Esto se debe a que el parásito se constituye en una de las principales causas de diarrea en niños, terneros, corderos y una gran variedad de mamíferos jóvenes en una gran cantidad de países. Debido a que la globalización ha permitido el intercambio de material biológico en diferentes regiones alrededor del mundo, se ha favorecido la propagación de enfermedades y se han expuesto a los agentes biológicos a diferentes condiciones ambientales, induciendo así la adaptación a través de cambios genéticos. Con base en el polimorfismo del gen GP60, esta revisión pretende presentar la distribución de Cryptosporidium parvum y Cryptosporidium hominis en humanos y terneros alrededor del mundo. El subtipo que afecta con mayor frecuencia al ganado vacuno corresponde a IIaA15G2R1; en tanto que el subtipo aislado con mayor frecuencia a partir de las muestras humanas es IaA19G2.

The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil / Primeiro estudo de prevalência molecular e caracterização de espécies de Cryptosporidium em galinhas (Gallus gallus domesticus) colonial/caipiras do Brasil

Abstract Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% - 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.

Resumo A criação de galinhas no estilo colonial/caipira é baseada em padrões de alimentação de pastagem, o que as torna potenciais contaminantes ambientais de oocistos de Cryptosporidium para humanos e outros animais. Portanto, o presente estudo teve como objetivo avaliar a prevalência molecular de Cryptosporidium spp. em galinhas criadas em sistema colonial/caipira. Um total de 351 amostras de fezes de frangos foram examinadas em 20 fazendas. Para a detecção de Cryptosporidium spp., os fragmentos do gene rRNA 18S foram amplificados utilizando-se a reação de nested-PCR. A prevalência global de Cryposporidium foi de 25,6% (IC 95% = 21,2% - 30,6%). O sequenciamento dos fragmentos amplificados permitiu a identificação de três espécies que infectam aves: C. meleagridis em 57 (62,6%), C. baileyi em 15 (16,4%), C. parvum em 3 (3,2%) amostras, bem como, um novo genótipo de Cryptosporidium (C. genótipo BrPR1) foi identificado em 3 (3,2%) amostras. Cryptosporidium genotipo BrPR1 não foi ainda classificado como uma espécie, e seu espectro de hospedeiros é desconhecido. O presente trabalho permitiu concluir que Cryptosporidium, incluindo espécies zoonóticas, existe com alta prevalência em galinhas criadas em sistema colonial/caipira na região estudada.

Effect of Egyptian propolis on cryptosporidiosis in immunosuppressed rats with special emphasis on oocysts shedding, leukogram, protein profile and ileum histopathology

Objectives To investigate the activity of Egyptian propolis extracts (ethanol and water) on cryptosporidiosis in experimentally infected dexamethasone-immunosuppressed rats. Methods A total of 180 male rats (190–220) g BWt were randomly divided into 9 equal groups (G1–G9). Groups of rats were kept as (G1): normal control, (G2–G9): immunosuppressed with dexamethasone and (G3-G9): infected with Cryptosporidium oocysts. Rats from (G4–G9) were given orally ethanol and water extract of propolis (at a dose of 50 mg/kg BWt) and nitazoxanide (standard anti-cryptosporidial drug at a dose of 100 mg/kg BWt) to infected rats with different regimes. Faecal pellets were collected from all groups to monitor oocysts shedding from the 2nd to the 15th day post infection. At the end of the experiment, blood was collected from all groups for determination of leukogram and serum proteins. Ileum specimens were also examined histopathologically. Results The highest reduction of oocysts shedding in faecal samples was 88% in rats prophylactically treated with propolis ethanol extract at the 4th dpi, and in rats prophylactically treated with water extract of propolis, was 91% at the 6th dpi. There was a marked increase in neutrophils count and α

Epidemiology status of some new emerging Cryptosporidium species and genotypes in humans / 中国人兽共患病学报

Cryptosporidium spp.are protozoan parasites that infect the epithelial cells of the gstrointestinal tract of hosts.In humans,cryptosporidiosis is usually a self-limiting infection in immunocompetent individuals,but severe diarrhea and dissemination to extra-intestinal sites can occur in high-risk individuals,such as the very young,the elderly,immunedeficiency individuals,particularly in HIV-positive patients.So far,molecular epidemiological data have confirmed the presence of 30 species and over 40 genotypes with genus Cryptosporidium,with 21 species and genotypes being found in humans.The majority of human cryptosporidiosis cases are responsible for C.hominis and C.parvum.Human cases caused by C.meleagridis,C.ubiquitum,C.felis and C.canis have been increasing.Besides that,with data accumulation of molecular epidemiology of human cryptosporidiosis,some more Cryptosporidium species and genotypes were newly identified in humans.This paper mainly reviews epidemiology status of these new emerging Cryptosporidium species and genotypes in humans.

Effect of Egyptian propolis on cryptosporidiosis in immunosuppressed rats with special emphasis on oocysts shedding, leukogram, protein profile and ileum histopathology

OBJECTIVES@#To investigate the activity of Egyptian propolis extracts (ethanol and water) on cryptosporidiosis in experimentally infected dexamethasone-immunosuppressed rats.@*METHODS@#A total of 180 male rats (190-220) g BWt were randomly divided into 9 equal groups (G1-G9). Groups of rats were kept as (G1): normal control, (G2-G9): immunosuppressed with dexamethasone and (G3-G9): infected with Cryptosporidium oocysts. Rats from (G4-G9) were given orally ethanol and water extract of propolis (at a dose of 50 mg/kg BWt) and nitazoxanide (standard anti-cryptosporidial drug at a dose of 100 mg/kg BWt) to infected rats with different regimes. Faecal pellets were collected from all groups to monitor oocysts shedding from the 2nd to the 15th day post infection. At the end of the experiment, blood was collected from all groups for determination of leukogram and serum proteins. Ileum specimens were also examined histopathologically.@*RESULTS@#The highest reduction of oocysts shedding in faecal samples was 88% in rats prophylactically treated with propolis ethanol extract at the 4th dpi, and in rats prophylactically treated with water extract of propolis, was 91% at the 6th dpi. There was a marked increase in neutrophils count and α- and β-globulins levels in infected rats treated with both extracts, while a significant decrease was detected in lymphocytes compared to the infected non treated group. β-Globulin level markedly increased in the rats administered nitazoxanide. Histopathological changes were observed in the ileum of rats infected with Cryptosporidium.@*CONCLUSIONS@#Egyptian propolis extracts have an activity on cryptosporidiosis in rats. Moreover, propolis modulated the immunity in dexamethasone-immunosuppressed rats.

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.

Molecular characterisation of Cryptosporidium spp. in lambs in the South Central region of the State of São Paulo / Caracterização molecular de Cryptosporidium spp. em cordeiros na região centro sul do Estado de São Paulo

Considering the proximity of sheep farmers to animals that are possibly diseased or releasing fecal oocysts into the environment and the marked pathogenicity in lambs, the aim of this study was to determine the occurrence and to molecularly characterize the infection by Cryptosporidium spp. in lambs in the South Central region of the state of São Paulo, Brazil. A total of 193 fecal samples were collected from sheep of several breeds, males and females, aged up to one year. Polymerase chain reaction (nested-PCR) was used to amplify DNA fragments from the subunit 18S rRNA gene and indicated 15% positivity; sequencing of amplified fragments was possible for 19 samples. Analysis of the obtained sequences showed that the identified species were Cryptosporidium xiaoi for 15 samples, constituting thus the first molecular characterization study of this Cryptosporidium species in Brazil. Cryptosporidium ubiquitum was identified for three samples and Cryptosporidium meleagridis for one sample; the latter two are considered zoonotic species.(AU)

Devido à proximidade de criadores de ovinos com animais possivelmente doentes e/ou eliminando oocistos fecais no ambiente e pela acentuada patogenicidade em cordeiros o objetivo foi, determinar a ocorrência e caracterizar molecularmente a infecção por Cryptosporidium spp. em cordeiros na região Centro Sul do Estado de São Paulo, Brasil. Num total de 193 amostras de fezes foram coletadas de ovinos de diversas raças, machos e fêmeas, com idade de até um ano. Por meio da reação em cadeia da polimerase (nested PCR) para a amplificação de fragmentos de DNA a partir do gene da subunidade 18S do rRNA houve positividade de 15% e o sequenciamento dos fragmentos amplificados foi possível em 19 amostras. A análise das sequências obtidas mostraram que as espécies identificadas nesses animais foram Cryptosporidium xiaoi em 15 amostras, sendo o primeiro estudo de caracterização molecular desta espécie de Cryptosporidium no Brasil. Cryptosporidium ubiquitum em três amostras, e Cryptosporidium meleagridis em uma amostra, sendo estas duas últimas consideradas espécies zoonóticas.(AU)

Ocorrência de Cryptosporidium spp. em bezerros bubalinos no estado do Pará / Occurrence of Cryptosporidium spp. in calves buffaloes in the state of Pará

A criptosporidiose é uma zoonose causada por protozoários do gênero Cryptosporidium, que acomete diversas espécies animais, inclusive o homem. Com o presente estudo objetivou-se determinar a ocorrência da infecção por Cryptosporidium spp. em bubalinos criados no estado do Pará. Para tanto, foram colhidas fezes de 253 bubalinos, de um dia até seis meses de idade. As fezes colhidas diretamente do reto foram classificadas como diarreicas e não diarreicas segundo a consistência e o aspecto, e analisadas em microscópio óptico após procedimentos de centrifugação, concentração e coloração pelo corante de Kinyoun. Observou-se uma ocorrência de Cryptosporidium spp. em 2,37% (6/253) das amostras, sendo todas não diarreicas. Não foi estabelecida relação significativa entre a infecção por Cryptosporidium spp., os sinais clínicos de diarreia e a idade dos animais (p > 0,05). Deste modo, conclui-se que a infecção pelo protozoário Cryptosporidium spp. em bubalinos no estado do Pará apresenta uma baixa ocorrência em animais na faixa etária de um dia até seis meses de idade.(AU)

Cryptosporidiosis is a zoonosis caused by opportunistic protozoa of the genus Cryptosporidium spp. that attacks various animal species, including humans. This study had the objective of determining the occurrence of infection by Cryptosporidium spp. in buffalo raised in the state of Pará, Brazil, and establishing the relations between Cryptosporidium spp., the age of the animal and the presence of clinical signs of diarrhea. A total of 253 samples of feces from one day to 6-month old buffalo were analyzed. The fecal samples, collected directly from the rectum, were classified as diarrheal and non-diarrheal and analyzed with an optical microscope after centrifuge-concentration and staining with Kinyoun stain. Laboratory analysis revealed the presence of Cryptosporidium spp. in 2.37% (6/253) samples, all of which had a firm consistency. No significant relation was established between infection by Cryptosporidium spp., the animals age from one day to 6-month and clinical symptoms of diarrhea.(AU)