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S Cacciò

Publications and source records attributed to S Cacciò.

At least 19 recordsLinked to original sources
PubMed2003

Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland.

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

Animals↗
PubMed2001

Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum.

The genetic polymorphism among 57 Cryptosporidium parvum isolates belonging to genotype 'C' was studied by PCR amplification and the sequencing of two microsatellite loci (ML1 and ML2). A comparative analysis of DNA sequences showed the presence of three (ML1-238, ML1-226, and ML1-220) and seven (ML2-231, ML2-229, ML2-227, ML2-213, ML2-193, ML2-191, and ML2-187) different alleles at these two loci. Alleles differed by expansions/contractions of the microsatellite repeats that generated length polymorphisms. Some alleles were found to be associated with infections of all examined hosts (calf, kid, lamb, and human), whereas others were either associated with a single host, or were geographically restricted. When considering the information from both loci, some preferential associations between alleles are apparent. These data confirm the utility of microsatellite markers for the molecular identification of C. parvum, which is of particular relevance in the investigation of the source of infection of outbreaks and single cases, as well as for genetic studies.

Animals↗
PubMed2001

Molecular identification of food-borne and water-borne protozoa.

Cryptosporidium and Giardia can be transmitted to humans by contaminated food and water, resulting in large outbreaks of diarrheal disease. Sensitive methods for detecting these parasites are needed to control and prevent infection. However, this issue is complicated by the fact that there is still uncertainty about the role played by different species/genotypes with respect to human disease. We are in the process of collecting samples from clinical cases (both sporadic and outbreak-related human infections) and from the environment (tap and waste water samples from different geographic regions), to test the efficacy of methods for detection and genotyping. Concerning Cryptosporidium parvum, we have developed new genotyping methods based on highly polymorphic microsatellite markers. The use of microsatellite markers allows the route of transmission to be traced; these methods can also be used not only to distinguish between anthroponotic and zoonotic transmission but also to identify the source(s) of infection. Regarding Giardia, which was found very frequently in environmental water samples, we are testing the beta-giardin gene as a marker to discriminate among species/genotypes.

Animals↗
PubMed2000

The beta-tubulin gene of Babesia and Theileria parasites is an informative marker for species discrimination.

A fragment of the beta-tubulin gene was polymerase chain reaction (PCR) amplified from genomic DNAs of Babesia bovis, Babesia bigemina, Babesia divergens, Babesia major, Babesia caballi, Babesia equi, Babesia microti, Theileria annulata and Theileria sergenti. Single amplification products were obtained for each of these species, but the size of the amplicons varied from 310 to 460 bp. Sequence analysis revealed that this variation is due to the presence of a single intron, which ranged from 20 to 170 bp. The extensive genetic variability at the beta-tubulin locus has been exploited to develop two types of species identification assays. The first assay can be used on samples containing mostly parasite DNA, like those prepared from infected erythrocytes. Following PCR amplification, the species identification is obtained directly from the size of the products (for Babesia species infecting human or horse) or using a simple PCR-restriction fragment length polymorphism (RFLP) protocol (for Babesia species infecting cattle). The second assay can be used on samples prepared from whole blood, that contain both parasite and host DNAs. In this case, due to the strong conservation of the beta-tubulin gene, co-amplification of a gene fragment from the host DNA was observed. A nested PCR assay was developed for the specific amplification of parasite DNA, using a primer designed to span the exon-intron boundary. Direct identification of Babesia species infecting human and horse is again obtained after the electrophoretic separation of the amplification products, while for Babesia and Theileria species infecting cattle, differentiation is based on a nested PCR-RFLP protocol. These methods may be used for the simultaneous identification of horses and cattle carrying multiple parasites by means of a single PCR or using the PCR-RFLP protocol.

Animals↗
PubMed2000

A microsatellite marker reveals population heterogeneity within human and animal genotypes of cryptosporidium parvum.

Isolates of the protozoan parasite Cryptosporidium parvum have been differentiated into 2 genotypes: genotype 'H', which is associated only with human infections, and genotype 'C', which is associated with both human and animal infections. To date, the analysis of polymorphisms of genes and of the small subunit ribosomal DNA have revealed no heterogeneity within the 2 genotypes. In the present study, a locus containing simple sequence repeats (microsatellites) was PCR amplified and sequenced from 94 C. parvum isolates, which were collected from humans (immunocompetent and immunocompromized individuals, outbreak and single cases) and from several animal hosts in 3 continents. The analysis revealed that genotype 'H' can be further differentiated into 2 subgenotypes, and genotype 'C' can be further differentiated into 4 subgenotypes. The 6 subgenotypes differ in terms of expansions/contractions of the microsatellite repeats and by point mutations. Some subgenotypes showed a wide geographical distribution, whereas others were restricted to specific regions. Therefore, microsatellites are informative markers for more defined studies on the epidemiology, the transmission routes, and the population structure of this parasite.

Animals↗
PubMed2000

Telomeres in warm-blooded vertebrates are composed of GC-rich isochores.

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.

Animals↗
PubMed1999

Genetic polymorphism at the beta-tubulin locus among human and animal isolates of Cryptosporidium parvum.

Sequence analysis of a fragment of the beta-tubulin gene was performed on 13 isolates of the parasite Cryptosporidium parvum, eight from humans and five from animals. A total of 12 synonymous substitutions and a deletion of two bases within the intron sequence were found. This genetic variation defined two alleles at the beta-tubulin locus, which can be identified by a simple polymerase chain reaction-restriction fragment length polymorphism assay. A total of 20 isolates were also tested using four available molecular markers. These analyses showed congruently that the C. parvum isolates segregate into two groups, one found exclusively in humans and the other found in both humans and animals. Since no recombinant genotypes were observed, the results are consistent with the hypothesis of a substantially clonal reproduction in this parasite.

Animals↗
PubMed1999

Identification and characterisation of three antigenic proteins from Cryptosporidium parvum sporozoites using a DNA library expressing poly-histidine tagged peptides.

To identify antigenic peptides of the parasite Cryptosporidium parvum, an expression library that allows for the production of chimeric proteins fused with a 6-histidine tag was made. The library was screened with C. parvum sporozoite rabbit anti-serum, and three positive clones (sa20, sa35, and sa40) were identified. The corresponding recombinant proteins (SA20, SA35, and SA40) were expressed in Escherichia coli and purified by metal-affinity chromatography. The sequence of sa20 and sa35 clones did not show any homology with known genes or proteins, whereas the 5' end of the sa40 clone showed homology with two previously identified C. parvum sequences. Hybridisations to intact chromosomes fractionated by pulsed-field gel electrophoresis revealed that the sa35 and sa40 sequences are localised on chromosome VII, whereas the sa20 sequence is localised on chromosome VI. Reverse transcriptase-PCR experiments showed the presence of mRNAs for sa35 and sa40 in the oocyst, whereas the sa20 mRNA was undetectable in this stage. The serological response to the three proteins was assayed in C. parvum-immunised rabbits and in immunocompetent individuals with cryptosporidiosis. The Western blot results indicated that rabbits, challenged with a sporozoite crude antigen or with an oocyst crude antigen, were highly responsive to these three antigens. Human serum samples showed a response to the three proteins, although the response to SA20 appeared to be unrelated to a recent C. parvum infection. These results suggest that the SA35 and the SA40 proteins may be useful in detecting C. parvum infections.

Amino Acid Sequence↗
PubMed1998

Establishing the Cryptosporidium parvum karyotype by NotI and SfiI restriction analysis and Southern hybridization.

The molecular karyotype of the coccidian parasite Cryptosporidium parvum has proven difficult to study because chromosomes of similar sizes migrate together when submitted to pulsed-field gel electrophoresis (PFGE). In the present work, the karyotype was studied by restriction of chromosome-sized DNA with the rare-cutting enzymes NotI and SfiI, followed by PFGE separation of the restriction fragments and Southern hybridization. These experiments showed that the C. parvum karyotype is formed by eight chromosomes, ranging in size from approximately 0.95 to 1.45 million base pairs (Mbp), accounting for a genome size of 9.6Mbp. As a first step towards the construction of a physical map of the C. parvum genome, a total of 20 probes, including 16 genes and the ribosomal DNA (rDNA) sequence, was mapped to intact chromosomes and to their restriction fragments. In this way, all chromosomes, but one, were identified by specific markers. A comparison of mapping data of homologous genes from different species belonging to the phylum Apicomplexa showed differences in the distribution of rDNA sequences and in the chromosomal localization of alpha- and beta-tubulin genes. The variation in genome size among these parasites is also discussed.

Animals↗
PubMed1997

Evolutionary changes in CpG and methylation levels in the genome of vertebrates.

We have analysed the levels of 5-methylcytosine (5mC) in DNAs from 42 vertebrates, and compiled, including data from literature, a table of genomic 5mC and GC levels (as well as the available c-values, i.e., the haploid genome sizes) of 87 species from all vertebrate classes. An analysis of the data indicates that (i) two positive correlations hold between the 5mC and GC levels of the genomes of fishes/amphibians and mammals/birds, respectively; (ii) the genomes of fishes and amphibians are, on average, about twice as methylated as those of mammals, birds and reptiles, this difference being unrelated to the amounts of repetitive DNA sequences; (iii) the 5mC and CpG observed/expected values show no overlap between the two groups of vertebrates and suggest the existence of two equilibria. The transition separating the two equilibria appears to have taken place at the time of appearance of reptiles. Its possible cause(s) and its implications are discussed.

Animals↗
PubMed1997

Methylation patterns in the isochores of vertebrate genomes.

5-Methylcytosine (5mC) levels were determined in compositional DNA fractions corresponding to different isochore families from the genomes of Xenopus, chicken, mouse and human, four vertebrates which show different isochore patterns. The results obtained indicate that: (i) positive correlations exist between the 5mC levels and the GC levels of isochores within any given genome; and (ii) DNA from Xenopus isochore families is twice as methylated as DNA from the isochores having the same GC levels from mouse, human and chicken. Moreover, the positive correlations holding between CpG levels and the GC3 levels of coding sequences of warm-blooded vertebrates were shown to comprise two regions with a border at approx. 75% GC3. The correlation corresponding to the higher region (which comprises only very rare high GC3 values in the case of Xenopus) has a higher slope than that corresponding to the lower GC3 values, a phenomenon due in all likelihood, to the increasing contribution of CpG islands. Finally, the observed/expected CpG ratio is higher in Xenopus than in warm-blooded vertebrates.

Animals↗
PubMed1996

Identification of the gene-richest bands in human chromosomes.

The human genome is a mosaic of isochores, long DNA segments which are compositionally homogeneous and which can be partitioned into five families, L1, L2, H1, H2 and H3, characterized by increasing GC levels and by increasing gene concentrations. Previous investigations showed that in situ hybridization with a DNA fraction derived from the GC-richest and gene-richest isochores of the H3 family produced the highest concentration of signals on 25 R(everse) bands that include the 22 most thermal-denaturation-resistant T(elomeric) bands, a subset of R bands. Using an improved protocol for in situ hybridization and cloned H3 isochore DNA, we have now shown (i) that the number of bands which are characterized by strong hybridization signals, and which are here called T or H3+, is 28; (ii) that 31 additional R bands, here called T'or H3* bands, also contain H3 isochores, although at a lower concentration than H3+ bands; and (iii) that the remaining R bands (about 140 out of 200, at a resolution of 400 bands), here called R" or H3- bands, do not contain any detectable H3 isochores. H3+ and H3* bands contain all the gene-richest isochores of the human genome. The existence of three distinct sets of R bands is further supported (i) by the different compositional features of genes located in them; (ii) by the very low gene density of chromosomes 13 and 18, in which all R bands are H3- bands; (iii) by the compositional map of a H3* band, Xq28; (iv) by the overwhelming presence of GC-rich and GC-poor long (> 50 kb) DNA sequences in H3+/H3* and in H3-/G bands, respectively; and (v) by the large degree of coincidence of H3+ and H3* bands with CpG island-positive bands. These observations have implications for our understanding of the causes of chromosome banding and provide a classification of chromosomal bands that is related to GC level (and to gene concentration).

Chromosome Banding↗
PubMed1996

Human coding and noncoding DNA: compositional correlations.

As the correlations between GC levels in third codon positions (GC3) and intergenic sequence GC levels can be used to assess the distribution of genes in the human genome, they were studied in detail. Previous work from our laboratory has demonstrated the existence of linear correlations between GC levels of exons, introns, third codon positions, 5' flanking regions of genes, and long genomic DNA sequences (> or = 10 kb) or DNA molecules (50-100 kb) in which the genes are embedded. The present study confirms and extends the previous results using a larger set of data. Furthermore, an analysis of 4270 human genomic DNA and cDNA sequences has allowed us to confirm a correlation of GC3 against GC1+2. Recent additions to the sequence database have also allowed separate analyses of the 5' flanking regions of CpG island and non-CpG island genes as well as analyses of 3' flanking regions, which suggest that the GC levels of 3' flanking regions are closer to those of intergenic DNA than are those of other regions of genes.

Base Composition↗
PubMed1995

Nonrandom frequency patterns of synonymous substitutions in homologous mammalian genes.

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous positions of quartet and duet (fourfold and twofold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable) according to whether they show no change, one change, or more than one change, respectively. We observed (1) that the frequencies of conserved, intermediate, and variable positions of quartet and duet codons are different in different genes; (2) that the frequencies of the three classes are significantly different from expectations based on a random substitution process in the majority of genes (especially for GC-rich genes) for quartet codons and in a minority of genes for doublet codons; and (3) that the frequencies of the three classes of positions of quartet codons are correlated with those of duet codons, the conserved positions of quartet and duet codons being, in addition, correlated with the degree of amino acid conservation. Our main conclusions are that synonymous substitution frequencies: (1) are gene-specific; (2) are not simply the result of a stochastic process in which nucleotide substitutions accumulate at random, over time; and (3) are correlated in quartet and duet codons.

Animals↗
PubMed1995

Specific compositional patterns of synonymous positions in homologous mammalian genes.

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous (ie., third) positions of quartet (fourfold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable), according to whether they show no change, one change, and more than one change, respectively. The three classes were analyzed in their compositional patterns. In the majority of GC-rich genes, the three classes of positions (but especially conserved positions) exhibited significantly different base compositions compared to expectations based on a "random" substitution process from the "ancestral" (consensus) sequence to the present-day (actual) sequences. Significant differences were rare in GC-poor genes. An analysis of the present results indicates that natural selection plays a role in the synonymous nucleotide substitution process, especially in GC-rich genes which represent the vast majority of mammalian genes.

Animals↗
PubMed1994

Single-copy sequence homology among the GC-richest isochores of the genomes from warm-blooded vertebrates.

We have hybridized a human DNA fraction corresponding to the GC-richest and gene-richest isochore family, H3, on compositional fractions of DNAs from 12 mammalian species and three avian species, representing eight and three orders, respectively. Under conditions in which repetitive sequences are competed out, the H3 isochore probe only or predominantly hybridized on the GC-richest fractions of main-band DNA from all the species investigated. These results indicate that single-copy sequences from the human H3 isochores share homology with sequences located in the compositionally corresponding compartments of the vertebrate genomes tested. These sequences are likely to be essentially formed by conserved coding sequences. The present results add to other lines of evidence indicating that isochore patterns are highly conserved in warm-blooded vertebrate genomes. Moreover, they refine recent reports (Sabeur et al., 1993; Kadi et al., 1993), and correct them in some details and also in demonstrating that the shrew genome does not exhibit the general mammalian pattern, but a special pattern.

Animals↗
PubMed1992

Direct sequencing and bidirectional allele specific polymerase chain reaction of the bovine beta-casein B variant.

We have used the polymerase chain reaction (PCR) to amplify exon VII of the bovine beta-casein gene. The mutations responsible for the B variant were identified by direct sequencing of the amplification products. A bidirectional allele-specific PCR method (BAS-PCR) has been developed using oligonucleotides overlapping the mutation site at their 3' ends. This new procedure allows a rapid and reliable discrimination between the B and non-B alleles of beta-casein.

Alleles↗