Detection of cryptosporidium oocysts in water and environmental concentrates

Whilst current methods for the isolation and enumeration of Cryptosporidium spp. oocysts in water have provided some insight into their occurrence and significance, they are regarded as being inefficient, variable and time-consuming, with much of the interpretation being left to the expertise of the analyst. Two expectations of novel developments are to reduce the variability and subjectivity associated with the isolation and identification of oocysts. Flocculation, immunomagnetisable and flow cytometric techniques, for concentrating oocysts from water samples, should prove more reliable than current methods, whilst the development of more avid and specific monoclonal antibodies in conjunction with the use of nuclear fluorochromes will aid identification. Further insight into the viability, taxonomy, species identification, infectivity and virulence of the parasite should be forthcoming through the use of techniques such as the polymerase chain reaction, in situ hybridisation and non-uniform alternating current electrical fields. Such information is necessary in order to enable microbiologists, epidemiologists, engineers, utility operators and regulators to assess the safety of a water supply, with respect to Cryptosporidium contamination, more effectively.

Avaliação da heterogeneidade molecular de Cryptosporidium sp. em amostras clínicas

O Cryptosporidium é um parasito coccídeo reconhecido por causar diarréia em humanos e animais em todo o mundo. O gênero compreende pelo menos 20 espécies confirmadas, sendo o C. hominis e C. parvum as principais espécies causadoras de criptosporidiose em humanos. Ferramentas moleculares têm sido desenvolvidas para detectar e diferenciar espécies/genótipos e subgenótipos de Cryptosporidium. O objetivo do trabalho foi avaliar a heterogeidade molecular de Cryptosporidium sp. obtidos de amostras clínicas provenientes dos municípios do Rio de Janeiro e de Salvador, através da PCR em tempo real e seqüenciamento automático. Foram analisadas 85 amostras, distribuídas em 3 grupos distintos, sendo 45 delas do município do Rio de Janeiro e 40 amostras provenientes de Salvador, Bahia. Todas as amostras foram positivas para Cryptosporidium sp. pelo método de coloração de Kinyoun a frio. O ensaio da PCR em tempo real combinou uma reação multiplex para a detecção do gênero Cryptosporidium e da espécie C. parvum e uma reação simples para a detecção de C. hominis. Na detecção do gênero Cryptosporidium foram utilizados par de primers e uma sonda TaqMan desenhados a partir do alinhamento de seqüências conservadas do gene 18S rRNA de várias espécies de Cryptosporidium disponíveis no GenBank. Para a detecção das espécies C. parvum e C. hominis foram utilizados primers e sondas específicos obtidos a partir de seqüências de cada espécie disponíveis no GenBank. A detecção do gênero Cryptosporidium através da sonda 18S rRNA, na reação duplex, foi visualizada em 63 de 85 amostras totais. Destas, a sonda TaqMan específica para C. parvum detectou 6 amostras e a sonda TaqMan específica para C. hominis detectou 42 amostras. Quinze amostras não puderam ser detectadas pelas sondas C. hominis ou C. parvum. Nos ensaios da PCR para o gene 18S, 31 amostras foram positivas e 27 delas sequenciadas. As análises filogenéticas confirmaram a presença de C. hominis, C. parvum e C. felis nas amostras estudadas. Na análise da topologia da árvore filogenética obtida por Neighbor Joining, observou-se-se que as sequências obtidas neste estudo se agruparam com espécies do gênero Cryptosporidium já descritas, com 99% ou 100% de similaridade. Conclui-se que os dois métodos utilizados são importantes ferramentas para o diagnóstico da criptosporidiose e diferenciação de C. hominis e C. parvum em investigações epidemiológicas, assim como avaliação da heterogeneidade molecular de Cryptosporidium sp

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×106–1×102 oocysts ml-1. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Caractérisation de l’infection naturelle à Cryptosporidium spp. chez le chien et le chat vus en établissement vétérinaire

Cryptosporidium spp. est un protozoaire parasite du système gastro-intestinal largement répandu chez les vertébrés et causant la cryptosporidiose, une zoonose occasionnant des troubles digestifs sévères pouvant entrainer la mort chez les individus immunodéficients. Au Canada, la déclaration de cette maladie est obligatoire depuis l’an 2000. Ainsi, il est pertinent de mieux comprendre l’infection chez les animaux de compagnie, puisqu’ils sont potentiellement un réservoir du parasite. Durant l’année 2008, des échantillons fécaux provenant de 1 202 chats (n = 371) et chiens (n = 831) de la province du Québec ont été analysés par comptage des ookystes de Cryptosporidium spp. au moyen de la technique de centrifugation en solution de sulfate de zinc. Dans cette étude,la prévalence de Cryptosporidium spp. chez les chats (28/371 : 7,55 %) et chez les chiens(88/831 : 10,59 %) de compagnie confirme leur potentiel en tant que réservoir du parasite. Au Québec, de par leur nombre, les chats sont potentiellement un réservoir zoonotique du parasite plus important que celui des chiens, bien qu’il n’existe pas de différence significative entre la prévalence du parasite chez le chat et le chien pour l’année 2008. L’âge (p = 0,0001) et l’infection concomitante par Giardia spp. (p = 0,0001) se sont avérés être des facteurs associés avec la présence de Cryptosporidium spp. chez le chien. Parmi l’ensemble des variables testées chez le chat (l’âge, le sexe, la saison et l’infection concomitante par Giardia spp.), aucune n’a été associée de manière significative à la présence du parasite chez le chat. Ceci peut être dû au nombre limité d’individus testés pour cette espèce. Un suivi de l’excrétion des ookystes de Cryptosporidium spp. chez deux chats suggère que l’excrétion des ookystes peut se faire sur une période de sept mois et que le taux d’excrétion varie dans le temps. Le diagnostic moléculaire des espèces et génotypes de Cryptosporidium spp. isolés à partir des échantillons de matières fécales devait être réalisé par la technique de PCR emboîtée des fragments des gènes ARNr 18S et HSP70 et du séquençage des produits de PCR. Aucun résultat positif n’a toutefois été obtenu. Afin d’augmenter la puissance statistique des analyses épidémiologiques sur la prévalence de Cryptosporidium spp., il serait nécessaire à l’avenir de travailler sur un nombre d’animaux beaucoup plus important.

Prévalence d’excrétion de Giardia et Cryptosporidium chez les humains, les animaux domestiques et les lémuriens de l’écosystème du Parc National de Ranomafana, Madagascar

L’augmentation des interactions entre humains et animaux sauvages en lisière des habitats naturels pourrait faciliter la transmission d’agents pathogènes entre les humains et les différentes espèces animales d’un écosystème et ainsi favoriser l’émergence de maladies. Nous avons effectué une étude transversale portant sur l’infection par Giardia et Cryptosporidium chez les humains, les animaux domestiques, les rongeurs et les lémuriens au sein de l’écosystème de Ranomafana, Madagascar. Des échantillons de fèces ont étés collectés de manière non invasive chez des personnes volontaires, des mammifères domestiques et des rongeurs introduits habitant trois villages situés en lisière du Parc National de Ranomafana (PNR) ainsi que quatre espèces de lémuriens (Propithecus edwardsii, Prolemur simus, Eulemur rubriventer et Microcebus rufus) du PNR. Des analyses coproscopiques par la technique d’immunofluorescence directe ont été réalisées afin de détecter la présence de Cryptosporidium et Giardia. Leur prévalence a été estimée et certaines variables reliées à l’infection par les parasites ont été identifiées. Cryptosporidium et Giardia ont été détectés avec une prévalence estimée à 22,9 % et 13,6 % respectivement chez les humains. La prévalence de ces deux parasites variait de 0 % à 60 % chez les animaux domestiques et les rongeurs au sein des villages. L’espèce hôte, l’âge ainsi que la co-infection par un autre protozoaire sont les seules variables associées à l’infection par Cryptosporidium et Giardia dans cet écosystème tandis qu’aucune association avec une coinfection par un ordre de nématode n’a été détecté. De plus, Cryptosporidium a été détecté chez 10,5 % des lémuriens du PNR. Cette étude documente pour la première fois la présence de Cryptosporidium chez deux espèces de lémuriens du PNR. Par contre, Giardia n’a pas été détecté dans les échantillons issus de lémuriens du PNR.

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.

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.