Simone M. Cacciò

Sequence analysis of the β-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples

The flagellate parasite Giardia duodenalis is a major cause of diarrhoea in humans and in animals worldwide. Molecular techniques are particularly useful for studying the taxonomy, the population structure, the zoonotic potential of animal isolates, and the correlation between the genetic variability of the parasite and the range of clinical symptoms observed in humans. In this work, a new PCR assay that targets the beta-giardin gene was tested on 21 Giardia duodenalis reference strains representing Assemblages A, B and E, which are associated with infections of humans and other mammals. The assay was then applied to 30 faecal samples collected from Italian persons. The sequence analysis of 31 PCR products from both reference strains and clinical samples showed that each Assemblage is clearly distinct from the others on the basis of specific substitutions; the sequence diversity was approximately 5%, and all substitutions occurred at the third codon positions of the gene. The analysis of the intra-Assemblage variability allowed for the identification of three genotypes within Assemblage A, and of four genotypes within Assemblage B. Interestingly, two genotypes were identified only in the clinical samples and not in reference strains. Finally, a simple PCR-restriction fragment length polymorphism method was developed for the rapid discrimination of Assemblages and applied for the direct genetic analysis of cysts present in human faecal samples.

Identification of Zoonotic Genotypes of Giardia duodenalis

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

Variation in Giardia: towards a taxonomic revision of the genus

Taxonomic uncertainty has had a negative impact on our understanding of the epidemiology of Giardia infections, particularly the role of wild and domestic animals as sources of human infection. The lack of morphological criteria for species identification and the failure of cross-infection experiments to unequivocally determine host specificity have largely contributed to this uncertainty. However, over the past ten years, it has been possible not only to demonstrate extensive genetic heterogeneity among Giardia isolates from mammals but also to confirm levels of host specificity that were recognized by early taxonomists when they proposed a series of host-related species that we consider should now be re-established.

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs.

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.

Giardia Cysts in Wastewater Treatment Plants in Italy

ABSTRACT Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O2 and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P = 0.05, analysis of variance). To characterize the cysts at the molecular level, the β-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.

Genotyping of Giardia duodenalis From Humans and Dogs From Mexico Using a β-Giardin Nested Polymerase Chain Reaction Assay

Cysts of Giardia duodenalis were collected in Mexico from symptomatic children (n = 9) and from pet dogs (n = 5), and they were directly characterized by nested polymerase chain reaction (PCR) amplification of the β-giardin gene. Eight isolates of human origin established as in vitro cultures and 2 reference strains, representing assemblages A and B of G. duodenalis, were also analyzed. PCR–restriction fragment length polymorphism showed that all isolates belonged to assemblage A. Sequence analyses indicated that the large majority of isolates were of the A1 genotype; interestingly, 2 human isolates displayed the A3 genotype, which has been previously identified in human isolates from Italy. The presence of cysts of the A1 and A3 genotypes in isolates from pet dogs is consistent with their role as reservoirs for human infection, although further studies are needed to confirm the occurrence of zoonotic transmission. Remarkably, cysts of assemblage B have not been found in any of the Mexican isolates studied to date.

Human Infection with Cryptosporidium felis: Case Report and Literature Review

An infection with Cryptosporidium felis in an HIV-positive man from Italy was successfully treated with paromomycin, despite the patient’s having a CD4+ cell count of 31/mL. Fourteen cases of human infection with C. felis have been described, all in the past 3 years, emphasizing the public health importance of Cryptosporidium parasites other than C. parvum.

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.

Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium

Stool samples from Belgian patients suffering from abdominal pain and/or diarrhoea were examined for Cryptosporidium and Giardia. Cryptosporidium-positive samples were genotyped using the 70 kDa heat shock protein and the 60 kDa glycoprotein (GP60) genes: C. hominis was identified in 54.2% and C. parvum in 45.8% of the samples. Sequencing at the GP60 locus indicated that subgenotype IbA10G2 of C. hominis and subgenotype IIaA15G2R1 of C. parvum were the most prevalent, although several other subgenotypes were identified. For Giardia, sequencing at the beta-giardin, triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) genes revealed assemblage B as the most prevalent (74.4%) in human patients. A high degree of heterogeneity was found, especially on the beta-giardin gene, and to a lesser extent on the GDH gene. Furthermore, using a novel species-specific PCR based on the TPI gene, mixed infections with both assemblage A and B were detected in a large number (32.4%) of human patients, which might have important epidemiological implications.