Serena Cavallero

Interactions between parasites and microbial communities in the human gut

The interactions between intestinal microbiota, immune system, and pathogens describe the human gut as a complex ecosystem, where all components play a relevant role in modulating each other and in the maintenance of homeostasis. The balance among the gut microbiota and the human body appear to be crucial for health maintenance. Intestinal parasites, both protozoans and helminths, interact with the microbial community modifying the balance between host and commensal microbiota. On the other hand, gut microbiota represents a relevant factor that may strongly interfere with the pathophysiology of the infections. In addition to the function that gut commensal microbiota may have in the processes that determine the survival and the outcome of many parasitic infections, including the production of nutritive macromolecules, also probiotics can play an important role in reducing the pathogenicity of many parasites. On these bases, there is a growing interest in explaining the rationale on the possible interactions between the microbiota, immune response, inflammatory processes, and intestinal parasites.

Molecular characterization and phylogeny of anisakid nematodes from cetaceans from southeastern Atlantic coasts of USA, Gulf of Mexico, and Caribbean Sea

In the present study, 407 anisakid nematodes, collected from 11 different species of cetaceans of the families Delphinidae, Kogiidae, Physeteridae, and Ziphiidae, from the southeastern Atlantic coasts of USA, the Gulf of Mexico, and the Caribbean Sea, were examined morphologically and genetically characterized by PCR restriction fragment length polymorphism to identify them to species level, assess their relative frequencies in definitive hosts, and determine any host preference. Sequence data from nuclear ribosomal internal transcribed spacer and mitochondrial cox2 genes were analysed by maximum parsimony and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. The results revealed a highly diverse ascaridoid community. Seven Anisakis species and Pseudoterranova species were recovered as adult parasites. Larval forms of Contracaecum multipapillatum were also found in a coastal population of bottlenose dolphins. The phylogenetic trees obtained from the combined dataset (and most individual datasets) revealed the existence of distinct clades, the first including species of the Anisakis simplex complex (A. simplex s.s., Anisakis pegreffii, A. simplex C), (Anisakis nascettii, Anisakis ziphidarum) and the second including Pseudoterranova ceticola ((Anisakis paggiae, (Anisakis physeteris, Anisakis brevispiculata)). This finding, excluding the relationship of P. ceticola, is consistent with the morphology of adult and larval specimens. Considering the presence versus absence of an intestinal cecum, the relationship of P. ceticola with the members of the second clade of Anisakis appears inconsistent with morphological evidences but consistent with host preference. The position of Anisakis typica as the sister group to the two main anisakid clades indicates that it represents a third distinct lineage.

Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean).

The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status.

Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

Background The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the worlds most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples. Methodology 151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions. Principal Findings No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum. Conclusions/Significance These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.

Presence of Anisakid Larvae in the European Anchovy, Engraulis encrasicolus, Fished Off the Tyrrhenian Coast of Central Italy

A survey was carried out to determine the prevalence of anisakid nematode larvae in European anchovy (Engraulis encrasicolus) fished off the Tyrrhenian coast of central Italy. From February through July 2012, 1,490 specimens of E. encrasicolus caught in three different fishing areas (off Civitavecchia, Anzio, and Gaeta in the northern, central, and southern Lazio region of Italy, respectively) were tested for the presence of anisakid larvae, both by visual microscopic inspection and enzymatic digestion. In each of the three fishing areas, each of two sampling times produced 250 fish (with the exception of one sampling time in Gaeta that produced 240 fish). Larvae of the family Anisakidae were detected with an overall estimated prevalence of 2.3%, and each positive fish harbored a single larva. No anisakid larvae were detected in fish caught off Gaeta. Fish with larvae were significantly longer (standard length) than fish without larvae. Twenty-six larvae (74.3%) were detected by visual inspection of the viscera, eight larvae (22.8%) were detected by visual inspection of the fillets, and one larva (2.8%) was detected after digestion of pooled fillets. Molecular analysis to fully characterize the 35 detected larvae revealed 15 specimens of Anisakis pegreffii, 10 specimens of Hysterothylacium aduncum, and one hybrid genotype of A. pegreffii × Anisakis simplex. For nine specimens, no visible product was obtained after PCR amplification. The overall prevalence for A. pegreffii and H. aduncum was 1.0 and 0.7%, respectively. A comparison between fishes harboring A. pegreffii larvae and those harboring H. aduncum revealed that those with A. pegreffii were significantly heavier. The prevalence of anisakid larvae found in the present study is lower then that reported previously in E. encrasicolus collected in the Mediterranean Sea.

Survey of Anisakis sp. and Hysterothylacium sp. in sardines and anchovies from the North Adriatic Sea.

The occurrence of larval Anisakidae and Raphidascarididae in anchovies and sardines from the North Adriatic Sea has been estimated. Anisakis pegreffii and Hysterothylacium aduncum were reported, with low prevalence values. In brief, a total amount of 7650 fish specimens collected between September 2011 and 2012 were analysed using three different inspection analyses: a visual inspection of the coelomic cavity, an examination of the viscera exploiting the positive hydro-tropism of the larvae (modified Baermann technique) and enzymatic digestion of muscular tissue pools. Low level of infestation was reported for Anisakis sp. in both in anchovies and sardines, while higher values were reported for Hysterothylacium sp. Subsamples of nematodes collected were characterized at species level using the molecular diagnostic key based on ITS nuclear ribosomal region, and A. pegreffii and H. aduncum were identified. The low prevalence of Anisakis sp. in sardines and anchovies from the North Adriatic Sea could be related to the peculiar distribution of cetaceans and carnivorous zooplankton in the investigated region and could be used as a potential tag to define oily fishes from this specific fishing area as at low-risk for anisakiasis.

Putative hybrids between two Anisakis cryptic species: Molecular genotyping using High Resolution Melting

The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

Two new species of Contracaecum Railliet & Henry, 1912 (Nematoda: Anisakidae), C. fagerholmi n. sp. and C. rudolphii F from the brown pelican Pelecanus occidentalis in the northern Gulf of Mexico.

DNA sequencing of the nuclear ribosomal DNA internal transcribed spacers (ITS) and mitochondrial rrnS and cox2 genes, and analysis of polymorphisms in restriction profiles in the ITS and rrnS, were used to characterise anisakid nematodes belonging to Contracaecum Railliet & Henry, 1912 infecting the brown pelican Pelecanus occidentalis (L.) in Galveston Bay, Texas and Sarasota Bay, Florida. Molecular data led to the detection of two new species: Contracaecum fagerholmi n. sp., which was also supported by clear morphological evidence, and Contracaecum rudolphii F, a new cryptic species within the Contracaecum rudolphii Hartwich, 1964 complex. Bayesian phylogenetic analysis demonstrated that C. fagerholmi and C. rudolphii F form two well-separated clusters, with C. fagerholmi being closely related to Contracaecum bioccai Mattiucci et al., 2008 and C. rudolphii F being included in the C. rudolphii complex. C. fagerholmi can be readily differentiated morphologically from all of its congeners, other than C. microcephalum (Rudolphii 1809) and the five currently recognised members of the C. rudolphii complex (C. rudolphii A, B, C, D and E). C. fagerholmi differs from C. microcephalum in the length of the spicules and the shape of the distal tip of the spicules, and from C. rudolphii (sensu lato) in the shape and size of the ventro-lateral and dorsal lips and by having interlabia which are not distally bifurcate. Further studies are needed to determine which morphological characteristics can be used to distinguish the cryptic species of the C. rudolphii complex in order to assign them with formal names. The recovery of a third species, C. bioccai, from the brown pelican confirms its occurrence in this host and extends its known geographical distribution.

Genetic heterogeneity and phylogeny of Trichuris spp. from captive non-human primates based on ribosomal DNA sequence data.

Nematodes of the genus Trichuris, known as whipworms, are recognized to infect numerous mammalian species including humans and non-human primates. Several Trichuris spp. have been described and species designation/identification is traditionally based on host-affiliation, although cross-infection and hybridization events may complicate species boundaries. The main aims of the present study were to genetically characterize adult Trichuris specimens from captive Japanese macaques (Macaca fuscata) and grivets (Chlorocebus aethiops), using the ribosomal DNA (ITS) as molecular marker and to investigate the phylogeny and the extent of genetic variation also by comparison with data on isolates from other humans, non-human primates and other hosts. The phylogenetic analysis of Trichuris sequences from M. fuscata and C. aethiops provided evidences of distinct clades and subclades thus advocating the existence of additional separated taxa. Neighbor Joining and Bayesian trees suggest that specimens from M. fuscata may be distinct from, but related to Trichuris trichiura, while a close relationship is suggested between the subclade formed by the specimens from C. aethiops and the subclade formed by T. suis. The tendency to associate Trichuris sp. to host species can lead to misleading taxonomic interpretations (i.e. whipworms found in primates are identified as T. trichiura). The results here obtained confirm previous evidences suggesting the existence of Trichuris spp. other than T. trichiura infecting non-human living primates.

First case report of invasive pseudoterranoviasis in Italy

Members of the genera Anisakis and Pseudoterranova are the main causative agents of human anisakidosis: the disease is worldwide distributed, with major impact in countries with a large consumption of raw fish. Because of unspecific symptoms and limited diagnostic tools, incidence and burden of disease are probably underestimated. In Italy, all human infestations where the etiological agent has been properly identified, have been associated to the parasitic species Anisakis pegreffii, the most frequent anisakid in the Mediterranean area. Here, an invasive human case of pseudoterranoviasis is described for the first time in Italy: in 2015, a woman was found infected during a colonoscopy scheduled after the occurrence of nonspecific clinical symptoms. The nematode was found penetrating the ascending colon. The identification was performed by sequencing the mitochondrial region cox2 and by comparison to GenBank retrieved material using the BLAST search tool. The sample showed a 99% identity with Pseudoterranova decipiens sensu stricto. The record underlines the potential risk due to the consumption of raw or undercooked imported fishes.