Identification et caractérisation génétique et phénotypique de deux espèces de Cryptosporidium après divers passages chez le veau

L’étude de la variation du génotype et du phénotype fut réalisée sur deux espèces de parasites intestinaux (Cryptosporidium parvum et C. muris) via une infection expérimentale de 10 passages successifs chez le veau (Bos taurus). L’infection avec C. parvum a bien fonctionné alors qu’aucun signe clinique n’a été observé dans le cadre de cette étude avec C. muris. Pour le génotype, deux gènes (HSP70 et GP60) ont été amplifiés par double PCR puis séquencés. Les résultats ont indiqué que ces gènes n’étaient pas modifiés après 10 passages chez des veaux. Cela montre une faible évolution génétique du parasite lorsqu’il passe dans un animal hôte, facilitant ainsi les études épidémiologiques lors d’épisode de cryptosporidiose. Il faut cependant noter que les parasites utilisés ne provenaient pas de l’environnement mais d’une compagnie spécialisée en parasitologie (WaterBorne®). L’étude de la variation du phénotype a été tentée, sans succès, à l’aide d’un immuno-buvardage en point utilisant le sérum des veaux infectés. Des problèmes liés à la concentration des ookystes de C. parvum placés sur la membrane de l’immuno-buvardage en point furent suspectés.

Prevalence and determinants of Cryptosporidium spp. infection in smallholder dairy cattle in Iringa and Tanga Regions of Tanzania

The prevalence of Cryptosporidium spp. infection in a cross-sectional study of dairy cattle, from two contrasting dairying regions in Tanzania, were determined by staining smears of faecal samples with the modified Ziehl-Neelsen technique. Of the 1126 faecal samples screened, 19.7% were positive for Cr\yptosporidium spp. The prevalence was lower in Tanga Region than in Iringa Region. The prevalence of affected farms was 20% in Tanga and 21 % in Iringa. In both regions, the probability of detecting Cryptosporidium oocysts in faeces varied with animal class, but these were not consistent in both regions. In Tanga Region, Cryptosporidium oocysts were significantly more likely to be found in the faeces of milking cows. In Iringa Region, the likelihood that cattle had Cryptosporidium-positive faeces declined with age, and milking cattle were significantly less likely to have Cryptosporidium-positive faeces. In this region, 7% of cattle were housed within the family house at night, and this was marginally associated with a higher likelihood that animals had Ctyptosporidium-positive faeces. Our study suggests that even though herd sizes are small, Cryptosporidium spp. are endemic on many Tanzanian smallholder dairy farms. These protozoa may impact on animal health and production, but also on human health, given the close associations between the cattle and their keepers. Further studies are required to assess these risks in more detail, and understand the epidemiology of Cryptosporidium spp. in this management system.

Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157:H7 in young lambs

Escherichia coli O157: H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E coli O157: H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E coli O157: H7, (B) colostrum-deprived and inoculated with C. parvum and then E coli O157: H7, (C) conventionally reared and inoculated with E coli O157: H7, (D) conventionally reared and inoculated with C. parvum and then E coli O15 7: H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E coli O157: H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E coli O157: H7 per gram of faeces. E coli O157: H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E coli O157: H7 was observed (P= 0-038), with the lambs inoculated with E coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E coli O157: H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total, four animals were euthanized, two at 24 h and two at 96 h post inoculation with E coli 0 157: H7 (two conventionally reared and two colostrum-deprived). All animals euthanized were from groups pre-inoculated with C. parvum prior to challenge with E coli O157 : H7. On examination of tissues, in three of the four animals examined, multifocal attaching and effacing lesions were observed in the caecum, colon, rectum and at the recto-anal junction, and were confirmed by immunolhistochemistry to be associated with E coli O157: H7.

Phylogenetic Analyses Based on Small Subunit rRNA and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

Ocorrência e detecção de Cryptosporidium sp e Giardia sp em águas brutas de abastecimento de formadores do Lago Guaíba

A giardíase e a criptosporidiose estão entre as enfermidades de veiculação hídrica originadas por protozoários que têm ganhado maior notoriedade nas últimas décadas. Os microrganismos causadores dessas doenças têm-se tornado um desafio para as empresas de abastecimento de água. Este trabalho teve como objetivo verificar a ocorrência dos protozoários Cryptosporidium sp e Giardia sp nas águas brutas de quatro afluentes do Lago Guaíba: Rios Taquari, Caí, dos Sinos e Gravataí. Juntos, abastecem uma população de dezenas de municípios da Região Metropolitana de Porto Alegre. Foram feitas amostragens mensais, durante doze meses, em cada afluente. Foi empregado o método de filtração em membrana para pré-concentração, centrifugação para concentração, separação imunomagnética para purificação e comparados três métodos de detecção para ambos os microrganismos: DAPI (4’,6-diamidino-2-fenilindol), Safranina e Kinyoun para Cryptosporidium e DAPI, Iodo Lugol e Iodo Tricrômico para Giardia. A avaliação microscópica e contagem de (oo)cistos foram feitas utilizando-se microscopia de imunofluorescência e contraste de fase para o DAPI e microscopia óptica para as demais colorações. Obtiveram-se os seguintes resultados: para os Rios Taquari, Caí, dos Sinos e Gravataí, respectivamente: 75, 42, 33 e 25% das amostras foram positivas para Cryptosporidium e 92, 83, 67 e 50% para Giardia. As concentrações médias de Giardia foram superiores às de Cryptosporidium em todos os quatro afluentes. Os valores médios observados foram: 80 oocistos e 176 cistos/100L no Taquari; 47 oocistos e 66 cistos/100L no Caí; 19 oocistos e 53 cistos/100L no Sinos; 16 oocistos e 92 cistos/100L no Gravataí. Estes podem estar subestimados devido à baixa recuperação dos métodos analíticos existentes e empregados. As densidades encontradas indicam que os protozoários estão disseminados nas águas superficiais dos formadores do Lago Guaíba.

Occurrence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae) in Brazil

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of São Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.

Occurrence of enteroparasites in day care centers in Botucatu (São Paulo State, Brazil) with emphasis on Cryptosporidium sp., Giardia duodenalis and Enterobius vermicularis

Verificou-se a prevalência dos enteroparasitas em 279 crianças (0 a 6 anos) de quatro creches municipais de Botucatu/SP. Foram coletadas três amostras de fezes de cada criança e processadas pelos métodos Hoffman, Faust e Ritchie e posterior coloração do esfregaço fecal pelos métodos de Auramina-O e Ziehl-Neelsen modificado para diagnóstico de Cryptosporidium sp. e método da fita gomada para diagnóstico de Enterobius vermicularis. Das crianças analisadas apresentaram-se parasitadas 53.40%, sendo que o parasita mais freqüente foi Giardia duodenalis (26.88%). Verificou-se associação significativa entre enteroparasitose, renda familiar, escolaridade materna e idade; quanto maior a renda e o grau escolar, menor a freqüência de enteroparasitas. Observou-se que G. duodenalis é mais prevalente em crianças de 0 a 4 anos e E. vermicularis em crianças entre três e quatro anos de idade. A elevada prevalência de enteroparasitas em creches sugere estrutura complexa em sua epidemiologia, onde fatores além do saneamento devem ser considerados.

Cryptosporidium spp. infection in mares and foals of the northwest region of São Paulo State, Brazil

O presente estudo teve como objetivo analisar a ocorrência da infecção por Cryptosporidium spp. em éguas e seus respectivos potros. Este estudo foi realizado em 11 fazendas localizadas nos municípios de Araçatuba, Birigui, Guararapes e Santo Antônio do Aracangua, na região Noroeste do Estado de São Paulo, de novembro de 2010 a março de 2011. Um total de 98 éguas e 98 potros de diversas raças foram analisados, sendo que, entre os filhotes, 59 eram machos e 39 fêmeas, cujas idades variavam de três até 330 dias. Fezes foram colhidas diretamente da ampola retal, purificadas e processadas pela técnica de Kinyoun modificada. A ocorrência de Cryptosporidium spp. observada foi de 21,4% (21/98) para potros e 18,4% (18/98) para éguas. A ocorrência de Cryptosporidium spp. teve uma associação significativa com a raça e a idade dos animais. A partir dos resultados obtidos, conclui-se neste estudo que potros com idade superior a dois meses e animais da raça Mangalarga foram menos susceptíveis à ocorrência de Cryptosporidium spp.

Molecular identification of Cryptosporidium spp. from fecal samples of felines, canines and bovines in the state of São Paulo, Brazil

The objective of this study was to obtain information of epidemiological nature through genotypic characterization of Cryptosporidium isolates from dogs, cats and bovines from the state of São Paulo, Brazil. The extraction of DNA from oocysts was carried out and polymerase chain reaction was accomplished using specific primers to 18S rRNA gene. The amplicons were directed sequenced. Seven cat samples, nine dog samples and nine bovine samples were analysed. From the seven cat samples the genotypic analyses revealed Cryptosporidium felis in all. These were the first genotypic characterization of Cryptosporidium from domestic felines in Brazil. In nine sequenced samples from dogs, genotypic identities compatible with Cryptosporidium canis were revealed in all samples. The genotypic analyses in bovines revealed Cryptosporidium parvum in eight samples and Cryptosporidium bovis in another sample, the last one being a non-zoonotic species, not related to clinical symptoms and described for the first time in Brazil. (c) 2007 Elsevier B.V. All rights reserved.

Biological studies and molecular characterization of a Cryptosporidium isolate from ostriches (Struthio camelus)

There are many reports of cryptosporidial infection in ostriches, but none with molecular characterization of the isolates. A study was undertaken for the characterization of a Brazilian Cryptosporidium sp. ostrich isolate by using molecular phylogenetic analysis of fragments of the 18S ribosonial DNA. heat-shock Protein (lisp) 70 coding gene, and actin coding gene. Biological studies were accomplished by the experimental inoculation of chickens via oral or intratracheal routes with fresh ostrich Cryptosporidium sp. oocysts. Molecular analysis of nuceotide sequences of the 3 genes by using neighbor-joining and parsimony methods grouped the ostrich isolate as a sister taxon of Crypiosporidium badeyi and showed that the os(rich isolate is genetically distinct from all other known Cryptosporidium species or genotypes. None of the inoculated chickens developed infection as determined by mucosal smears. histology, and fecal screening for oocysts. Although biological and molecular Studies indicate that the ostrich Cryptosporidium is a new species, further Studies regarding morphological. biological, and molecular characteristics of other ostrich isolates are required to confirm the species status of the ostrich Cryprosporidium.

Cryptosporidium infection in Brazil: implications for veterinary medicine and public health

O objetivo deste trabalho foi relatar, por meio de revisão de literatura, os resultados de pesquisas sobre a criptosporidiose no Brasil, com ênfase em sua ocorrência em animais e suas implicações em medicina veterinária e em saúde pública. Um número crescente de trabalhos sobre a infecção por Cryptosporidium spp. no Brasil está disponível na literatura nacional e internacional. Nestes trabalhos, são abordados principalmente aspectos relacionados à ocorrência de Cryptosporidium spp. em alimentos, amostras ambientais, no homem e em diversas espécies animais, particularmente em aves, bovinos, cães e gatos. Por meio de técnicas de biologia molecular, a maioria das espécies e alguns genótipos identificados em outros países foram descritos no Brasil. em mamíferos, houve identificação de C. bovis, C. canis, C. felis, C. meleagridis, C. parvum e o genótipo cervídeo; em diversas espécies de aves, foi descrita infecção por C. baileyi, C. galli, C. meleagridis, C. parvum e pelos genótipos I, II e III de aves. Várias espécies foram descritas no homem, como C. parvum e C. hominis, além de algumas espécies adaptadas a hospedeiros animais, como C. canis, C. felis e C. meleagridis.

Ocorrência e caracterização molecular de Cryptosporidium em cordeiros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Importância de Cryptosporidium spp. como causa de diarréia em bezerros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil

A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of Sdo Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife. (c) 2007 Elsevier B.V. All rights reserved.