Margarida Alves

Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal

ABSTRACT Cryptosporidium parvum and Cryptosporidium hominis isolates from human immunodeficiency virus-infected patients, cattle, and wild ruminants were characterized by PCR and DNA sequencing analysis of the 60-kDa glycoprotein gene. Seven alleles were identified, three corresponding to C. hominis and four corresponding to C. parvum. One new allele was found (IId), and one (IIb) had only been found in Portugal. Isolates from cattle and wild ruminants clustered in two alleles. In contrast, human isolates clustered in seven alleles, showing extensive allelic diversity.

Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal

To investigate the transmission of cryptosporidiosis in Portugal, Cryptosporidium hominis and Cryptosporidium parvum from HIV-infected patients, cattle, and wild ruminants were characterized by sequence analysis of the 60-kDa glycoprotein (GP60) gene. Fourteen subtypes within nine subtype families were identified, and three of the subtype families (If, IIb, and IId) were restricted or largely limited to Portugal. Parasites from cattle from various regions in Portugal and wild ruminants in Lisbon showed limited genetic heterogeneity (only two subtype families). All wild ruminants had the same subtype, which was also the predominant subtype in cattle all over Portugal and was found in nine HIV-infected patients in Lisbon. Two other C. parvum subtypes were only restricted to limited locations. In contrast, human parasites displayed 13 subtypes in nine subtype families, with most of the infections caused by parasites in Ib, IIa, IIc, and IId families. Two of the C. parvum subtype families (IIc and IIb) had only been found in humans. The high overall parasite diversity and high percentage of C. hominis infections attributable to Ib and C. parvum infections to IId represent unique characteristics of Cryptosporidium transmission in humans in Portugal.

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the β-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Occurrence and molecular characterization of Cryptosporidium spp. in mammals and reptiles at the Lisbon Zoo

The presence of Cryptosporidium parasites in mammals and reptiles kept at the Lisbon Zoo was investigated. A total of 274 stool samples were collected from 100 mammals and 29 reptiles. The species and genotype of the isolates identified by light microscopy were determined by nested PCR and sequence analysis of a fragment of the small subunit rRNA gene. Cryptosporidium oocysts were found in one black wildebeest (Connochaetes gnou), one Prairie bison (Bison bison bison) and in one Indian star tortoise (Geochelone elegans). The PCR and sequence analysis of these three isolates showed that those excreted by the Prairie bison were Cryptosporidium mouse genotype, those from the black wildebeest were from a new Cryptosporidium genotype and those infecting the Indian star tortoise were Cryptosporidium tortoise genotype. The present work reports a new Cryptosporidium genotype in a black wildebeest and the first finding of the Cryptosporidium mouse genotype in a ruminant.

MULTILOCUS GENOTYPING OF CRYPTOSPORIDIUM ISOLATES FROM HUMAN HIV-INFECTED AND ANIMAL HOSTS

’he genus Cryptosporidiwn comprises parasites of several species that infect mammals and other vertebrates. Molecular studies have drown that Cryptosporidiwn parvwn, the most common species infecting man, comprises the “human” genotype (H) found exclusively m humans and the “cattle” genotype (C) found in both humans and livestock [8]. Other genotypes, until now considered to be host specific, have also been found in mice, pigs, ferrets and marsupials [5, 141. Recently, FCR-RFLP and DNA sequence analysis have also identified C. felis, C. meleagridis and a Cryptosporidiwn “dog type” in HIVinfected patients as well as in immunmpetent persons [4, 6, 7. 9,

Microsatellite Analysis of Cryptosporidium hominis and C. parvum in Portugal: a Preliminary Study

The most important causative agents of human cryptosporidiosis are Cryptosporidium parvum found in domestic livestock, wild animals and humans and C. hominis found almost exclusively in humans [1, 2, 8, 9, 10]. Since several genotypes comprise the species C. parvum, the occurrence in humans of both C. hominis and C. parvum (especially the bovine genotype) is evidence that both anthroponotic and zoonotic cycles occur in human infections [8]. Microsatellites are a rich source of polymorphisms that can be used in epidemiological studies as fingerprints for the identification of sources of infection and transmission routes of cryptosporidiosis [4– 7]. In this study we characterized Portuguese Cryptosporidium isolates from human and bovine origins by fragment analysis of the microsatellite locus ML2.

Distribution of Cryptosporidium species and subtypes in water samples in Portugal: a preliminary study.

MARGARIDA ALVES, ANA M. RIBEIRO, CÉLIA NETO, ELISABETE FERREIRA, MARIA J. BENOLIEL, FRANCISCO ANTUNES and OLGA MATOS Unidade de Protozoários Oportunistas/VIH e Outras Protozooses, UPMM, Instituto de Higiene e Medicina Tropical, Rua da Junqueira, n. 96, 1349-008 Lisboa, Portugal, and Laboratório Central da EPAL, Unidade de Análises, Equipa de Microbiologia, Lisboa, Portugal, and Clı́nica Universitária de Doenças Infecciosas, Faculdade de Medicina (Hospital de Santa Maria), Lisboa, Portugal