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Giuseppe Nascetti - One of the best experts on this subject based on the ideXlab platform.
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species specific real time pcr primers probe systems to identify fish parasites of the genera anisakis pseudoterranova and hysterothylacium nematoda Ascaridoidea
Fisheries Research, 2017Co-Authors: Arne Levsen, Michela Paoletti, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
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Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)
Fisheries Research, 2017Co-Authors: Michela Paoletti, Arne Levsen, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
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Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters and genetic markers
Systematic Parasitology, 2000Co-Authors: Lia Paggi, Simonetta Mattiucci, Giuseppe Nascetti, Bjorn Berland, Rossella Cianchi, David I. Gibson, Luciano BulliniAbstract:Five genetically distinct and reproductively isolated species have been detected previously within the morphospecies Pseudoterranova decipiens from the Arctic-Boreal, Boreal and Antarctic. Morphological analysis was carried out on male specimens identified by genetic (allozyme) markers, allowing the detection of significant differences at a number of characters between two members of the P. decipiens complex, namely P. decipiens A and B. On the basis of such differences, the nomenclatural designation for the two species is discussed. The names Pseudoterranova krabbei n. sp. and P. decipiens (sensu stricto) are proposed for species A and B, respectively. Morphological and genetic differentiation between the two species is shown using multivariate analysis. Allozyme diagnostic keys for routine identification of the four members of the P. decipiens complex, namely P. decipiens (s.s.), P. krabbei , P. bulbosa and P. azarasi , irrespective of sex and life-history stage, are provided.
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Allozyme and morphological identification of shape Anisakis, Contracaecum and Pseudoterranova from Japanese waters (Nematoda, Ascaridoidea)
Systematic Parasitology, 1998Co-Authors: Simonetta Mattiucci, Kokichi Kikuchi, Giuseppe Nascetti, Hajime Ishikura, Rossella Cianchi, Noriyuki Sato, Lia Paggi, Luciano BulliniAbstract:Allozyme markers were used to identify anisakid nematodes from marine Japanese waters, morphologically assigned to three species complexes: Anisakis simplex (s. l.), Contracaecum osculatum (s. l.) and Pseudoterranova decipiens (s. l.). Samples assigned to A. simplex (s. l.) were found to correspond genetically to A. simplex sensu stricto, those of C. osculatum (s. l.) to C. osculatum A. No morphological characters are yet available to distinguish sibling species of these two complexes. As to the P. decipiens complex, two distinct species were detected: the first corresponded to P. decipiens C, previously recovered in the northern Atlantic, the second to P. decipiens D from Japan. The two species are genetically well differentiated, with five of the 19 loci tested showing distinct fixed alleles. Their reproductive isolation was proved by the lack of hybrids or recombinants in sympatric samples recovered from the same definitive host, Erignathus barbatus. P. decipiens D was found to correspond morphologically to Porrocaecum azarasi, previously considered a synonym of P. decipiens. Accordingly, the name Pseudoterranova azarasi (Yamaguti & Arima, 1942) n. comb. is proposed for P. decipiens D. Similarly, P. decipiens C fits in general morphology, type-locality and host with Ascaris bulbosa, also previously considered a synonym of P. decipiens. The name Pseudoterranova bulbosa (Cobb, 1888) n. comb. is proposed for P. decipiens C.
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genetic and ecological data on the anisakis simplex complex with evidence for a new species nematoda Ascaridoidea anisakidae
Journal of Parasitology, 1997Co-Authors: Simonetta Mattiucci, S Damelio, Giuseppe Nascetti, Rossella Cianchi, J Brattey, L Paggi, P. Arduino, L Margolis, Stephen C Webb, P. OrecchiaAbstract:Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str. and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C + A. simplex s. str., South Africa and New Zealand for A. simplex C + A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (D Nei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average D Nei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average D Nei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity, H e = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.
Simonetta Mattiucci - One of the best experts on this subject based on the ideXlab platform.
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species specific real time pcr primers probe systems to identify fish parasites of the genera anisakis pseudoterranova and hysterothylacium nematoda Ascaridoidea
Fisheries Research, 2017Co-Authors: Arne Levsen, Michela Paoletti, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
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Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)
Fisheries Research, 2017Co-Authors: Michela Paoletti, Arne Levsen, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
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Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters and genetic markers
Systematic Parasitology, 2000Co-Authors: Lia Paggi, Simonetta Mattiucci, Giuseppe Nascetti, Bjorn Berland, Rossella Cianchi, David I. Gibson, Luciano BulliniAbstract:Five genetically distinct and reproductively isolated species have been detected previously within the morphospecies Pseudoterranova decipiens from the Arctic-Boreal, Boreal and Antarctic. Morphological analysis was carried out on male specimens identified by genetic (allozyme) markers, allowing the detection of significant differences at a number of characters between two members of the P. decipiens complex, namely P. decipiens A and B. On the basis of such differences, the nomenclatural designation for the two species is discussed. The names Pseudoterranova krabbei n. sp. and P. decipiens (sensu stricto) are proposed for species A and B, respectively. Morphological and genetic differentiation between the two species is shown using multivariate analysis. Allozyme diagnostic keys for routine identification of the four members of the P. decipiens complex, namely P. decipiens (s.s.), P. krabbei , P. bulbosa and P. azarasi , irrespective of sex and life-history stage, are provided.
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Allozyme and morphological identification of shape Anisakis, Contracaecum and Pseudoterranova from Japanese waters (Nematoda, Ascaridoidea)
Systematic Parasitology, 1998Co-Authors: Simonetta Mattiucci, Kokichi Kikuchi, Giuseppe Nascetti, Hajime Ishikura, Rossella Cianchi, Noriyuki Sato, Lia Paggi, Luciano BulliniAbstract:Allozyme markers were used to identify anisakid nematodes from marine Japanese waters, morphologically assigned to three species complexes: Anisakis simplex (s. l.), Contracaecum osculatum (s. l.) and Pseudoterranova decipiens (s. l.). Samples assigned to A. simplex (s. l.) were found to correspond genetically to A. simplex sensu stricto, those of C. osculatum (s. l.) to C. osculatum A. No morphological characters are yet available to distinguish sibling species of these two complexes. As to the P. decipiens complex, two distinct species were detected: the first corresponded to P. decipiens C, previously recovered in the northern Atlantic, the second to P. decipiens D from Japan. The two species are genetically well differentiated, with five of the 19 loci tested showing distinct fixed alleles. Their reproductive isolation was proved by the lack of hybrids or recombinants in sympatric samples recovered from the same definitive host, Erignathus barbatus. P. decipiens D was found to correspond morphologically to Porrocaecum azarasi, previously considered a synonym of P. decipiens. Accordingly, the name Pseudoterranova azarasi (Yamaguti & Arima, 1942) n. comb. is proposed for P. decipiens D. Similarly, P. decipiens C fits in general morphology, type-locality and host with Ascaris bulbosa, also previously considered a synonym of P. decipiens. The name Pseudoterranova bulbosa (Cobb, 1888) n. comb. is proposed for P. decipiens C.
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genetic and ecological data on the anisakis simplex complex with evidence for a new species nematoda Ascaridoidea anisakidae
Journal of Parasitology, 1997Co-Authors: Simonetta Mattiucci, S Damelio, Giuseppe Nascetti, Rossella Cianchi, J Brattey, L Paggi, P. Arduino, L Margolis, Stephen C Webb, P. OrecchiaAbstract:Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str. and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C + A. simplex s. str., South Africa and New Zealand for A. simplex C + A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (D Nei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average D Nei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average D Nei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity, H e = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.
Einar Stromnes - One of the best experts on this subject based on the ideXlab platform.
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An in vitro study of lipid preference in whaleworm (Anisakis simplex, Nematoda, Ascaridoidea, Anisakidae) third-stage larvae
Parasitology Research, 2014Co-Authors: Einar StromnesAbstract:The behavioural response of nematodes to chemical stimuli has been extensively investigated in some free-living and plant parasitic species. However, in animal parasitic species, little is yet known, particularly in regards to marine forms such as the whaleworm ( Anisakis simplex ). Previous studies showed that A. simplex L3-larvae tend to prefer fish tissue with high lipid content. The intention of this study was to investigate the behaviour of A. simplex L3 in response to different concentrations of fish lipid in further detail. This was done by an in vitro study based on larvae from cod ( Gadus morhua ). Ten larvae were placed in each of the culture containers containing agar that was separated into three segments of equal size. Three categories of agar were used containing 0, 2 and 7 % cod liver oil. A total of 900 larvae were included. The study consisted of three parts: The purpose of experiment I was to establish whether different lipid concentrations influenced the migration pattern at all. Experiment II was intended to examine whether A. simplex L3-larvae were able to actively search for lipids. Experiment III was set up to analyse the short-distance dispersion of the L3-larvae. Experiment I indicated that the L3-larvae move randomly but do not stop randomly since the tendency to move out of the start area was inversely correlated with lipid concentration. Experiment II indicates that the larvae are almost unable to select areas of high lipid concentrations when more than a few centimetres away. Experiment III showed that the L3-larvae prefer high-fat content and can seek it out over short distances.
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growth of whaleworm anisakis simplex nematodes Ascaridoidea anisakidae third stage larvae in paratenic fish hosts
Parasitology Research, 2003Co-Authors: Einar Stromnes, Karin AndersenAbstract:The growth of Anisakis simplex L3 larvae in the three paratenic hosts saithe (Pollachius virens), cod (Gadus morhua) and redfish (Sebastes marinus) was studied based on material collected off the island of Vega on the west coast of Norway over a period of 1 year. In all three fish species, a positive correlation between the length of larvae and the age of the fish was shown. The number of large larvae, here defined as L3>28 mm, increases with increasing age of the host. These findings clearly indicate an accumulation of large L3 larvae and larval growth in the fish host. The larvae were additionally grouped according to the tissue – muscle, liver or other viscera – they were located in. When the average lengths of the three groups of larvae were compared, the gadoids saithe and cod had somewhat higher values for L3 larvae found in the liver. Redfish, on the other hand, which store fat to a relatively higher degree in the viscera, had the highest average length of larvae in this tissue. The lowest average values in all three species were found in the musculature. These trends suggest that the growth of A. simplex L3 larvae is positively correlated with the fat content of the tissue in which it is encapsulated. Of the three host species included in this study, saithe seemed to be the best suited for A. simplex L3, as the growth rate and average length of the parasite was greatest in this species. The growth and average length of L3 was least pronounced in redfish while cod held an intermediate position.
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spring rise of whaleworm anisakis simplex nematoda Ascaridoidea third stage larvae in some fish species from norwegian waters
Parasitology Research, 2000Co-Authors: Einar Stromnes, Karin AndersenAbstract:The seasonal variation in the infection of saithe (Pollachius virens), cod (Gadus morhua), and redfish (Sebastes marinus) from a coastal area of central Norway with Anisakis simplex third-stage larvae (L3) was studied over a period of 1 year. In all three host species there was an increase in the abundance of the parasite in spring, with a peak appearing in March and April. Cod displayed the most distinct seasonal variation, showing a clear abundance peak in April. The abundance peak in April for redfish was not as pronounced. In saithe the abundance over the seasons was less prominent, with a maximum appearing in March and another increase in abundance occurring during July. It is apparent that the causes behind the pattern of infection observed in this study are complex. However, possible explanatory mechanisms are discussed. Arguments are propounded to suggest that the increased supply of parasite eggs from northward-migrating whales in addition to the general spring bloom of plankton constitute the most important factors governing the phenomenon of "spring rise" in A. simplex L3 in the study area.
L Paggi - One of the best experts on this subject based on the ideXlab platform.
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electrophoretic detection of population variation within contracaecum ogmorhini nematoda Ascaridoidea anisakidae
Electrophoresis, 2001Co-Authors: Xingquan Zhu, S Damelio, L Paggi, Robin B GasserAbstract:This study examined genetic variation among specimens of Contracaecum ogmorhini from different otariid hosts and geographical origins using a polymerase chain reaction (PCR)-based mutation detection approach. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified individually by PCR, scanned for sequence variation by single-strand conformation polymorphism (SSCP), and samples displaying variable SSCP profiles were subjected to cycle sequencing. While C. ogmorhini individuals from Arctocephalus pusillus pusillus (CoAPP) from South Africa and those from Arctocephalus pusillus doriferus (CoAPD) from Australia had very similar SSCP profiles for both ITS-1 and ITS-2, individuals of C. ogmorhini from Zalophus californianus (CoZC) from Pacific Canada could be unequivocally distinguished based on their profiles. In accordance with SSCP results, both CoAPP and CoAPD had identical ITS consensus sequences, whereas CoZC differed in sequence from both CoAPP and CoAPD populations by 0.2% (one base in the ITS-1) and 0.7% (two bases in the ITS-2). Based on the nucleotide difference in the ITS-2 sequence, a PCR-linked restriction fragment length polymorphism (RFLP) could be employed to distinguish individuals representing CoZC from those of both CoAPP and CoAPD. The findings suggest that C. ogmorhini may represent a complex of at least two species.
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mutation scanning for sequence variation in three mitochondrial dna regions for members of the contracaecum osculatum nematoda Ascaridoidea complex
Electrophoresis, 2001Co-Authors: S Damelio, L Paggi, Xingquan Zhu, Robin B GasserAbstract:Anisakid nematodes of seals from different geographical origins, previously identified by multilocus enzyme electrophoresis as Contracaecum osculatum A (CoA), C. osculatum B (CoB), C. osculatum C (CoC), C. osculatum D (CoD), C. osculatum E (CoE) and C. osculatum baicalensis (Cob), were characterised genetically using a mutation scanning approach, in order to define genetic markers for their specific identification and differentiation. Three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit I (COI), and the small and large subunits of rRNA (ssrRNA and IsrRNA, respectively) were amplified separately from individual nematodes by polymerase chain reaction (PCR), analysed by single-strand conformation polymorphism (SSCP), and samples displaying sequence variability were subjected to sequencing. Forty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except CoD and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRNA and/or IsrRNA sequences. For all three mtDNA regions, 4 (10.5%), 7 (18.4%), 15 (39.5%) and 11 (28.9%) of 38 nucleotide positions were considered diagnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Cob. The lack of an unequivocal nucleotide difference in any of the three mtDNA sequences between CoD and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic data. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetically more similar to Cob than each was to CoC, similar to previous findings. In spite of not being able to distinguish among all six taxa of C. osculatum, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic structures of species of parasitic nematodes.
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phylogenetic relationships among species of contracaecum railliet henry 1912 and phocascaris host 1932 nematoda Ascaridoidea based on nuclear rdna sequence data
Parasitology, 2000Co-Authors: Steven A Nadler, S Damelio, B Berland, Hanspeter Fagerholm, L PaggiAbstract:Nuclear-encoded large-subunit ribosomal DNA sequences were used to infer a phylogenetic hypothesis for 17 taxa (16 nominal species) of the genera Contracaecum and Phocascaris. Phylogenetic trees based on these data have been used to assess the validity of the taxonomic distinction between these genera, which was based on the presence or absence of certain structural features, rather than on explicit hypotheses of evolutionary history. Phylogenetic hypotheses based on parsimony, likelihood, and neighbor-joining analyses of these sequence data strongly support the hypothesis that species of Phocascaris are nested within the clade of Contracaecum species hosted by phocid seals, and are more closely related to species of the Contracaecum osculatum complex than to other Contracaecum species. Alternative tree topologies representing Phocascaris as not nested within the C. osculatum complex were significantly worse interpretations of these sequence data. Phylogenetic analysis also provides strong support for the monophyly of all taxa (Contracaecum and Phocascaris) from phocid seals, which is consistent with Berland's (1964) proposal that such species form a natural group; however, his proposal to recognize all species in phocid seals as Phocascaris, with all species from birds as Contracaecum would result in a paraphyletic Contracaecum, according to the molecular phylogenetic hypothesis.
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genetic and ecological data on the anisakis simplex complex with evidence for a new species nematoda Ascaridoidea anisakidae
Journal of Parasitology, 1997Co-Authors: Simonetta Mattiucci, S Damelio, Giuseppe Nascetti, Rossella Cianchi, J Brattey, L Paggi, P. Arduino, L Margolis, Stephen C Webb, P. OrecchiaAbstract:Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str. and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C + A. simplex s. str., South Africa and New Zealand for A. simplex C + A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (D Nei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average D Nei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average D Nei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity, H e = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.
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genetic and ecological data on the anisakis simplex complex with evidence for a new species nematoda Ascaridoidea anisakidae
Journal of Parasitology, 1997Co-Authors: Simonetta Mattiucci, S Damelio, Giuseppe Nascetti, J Brattey, L Paggi, P. Arduino, L Margolis, Stephen C Webb, R Clanchi, P. OrecchiaAbstract:Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C+A. simplex s. str., South Africa and New Zealand for A. simplex C+A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (DNei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average DNei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average DNei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity. Hr = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.
Michela Paoletti - One of the best experts on this subject based on the ideXlab platform.
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species specific real time pcr primers probe systems to identify fish parasites of the genera anisakis pseudoterranova and hysterothylacium nematoda Ascaridoidea
Fisheries Research, 2017Co-Authors: Arne Levsen, Michela Paoletti, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
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Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)
Fisheries Research, 2017Co-Authors: Michela Paoletti, Arne Levsen, Simonetta Mattiucci, Alessandra Colantoni, Giuseppe NascettiAbstract:Abstract Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.
