Infections and molecular characterization of anisakid nematodes from two species of marine fish northwest Arabian gulf (original) (raw)
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The Journal of Basic and Applied Zoology
Background: Nematodes of the family Anisakidae are parasites of many fishes and aquatic invertebrates which act as intermediate or paratenic hosts, while mammals and fish-eating birds are definitive hosts. Infective L3 larvae may be incidentally taken by human through eating raw or undercooked fish meat, causing anisakidosis. The main purpose of this study is to provide a basis for the future investigations to discover the genetic diversity of this widely distributed parasite nematodes of fishes and fish eating animals and their effect on fisheries and public health in Egypt and worldwide. Results: One thousand, one hundred and fifteen specimens belong to nine fish species were collected from Lake Nasser, Egypt, and examined for infection with Anisakid larvae. Four fish species (Oreochromis niloticus, Tilapia galilaea, Lates niloticus, and Hydrocynus forskahlii) were found infected with third stage larvae of Contracaecum spp. No other Anisakid nematodes were detected. Larvae were found in the body cavity adhering to mesenteries by a thin membrane, except in Oreochromis niloticus and Tilapia galilaea were found free in branchial chambers. The highest prevalence was recorded in L. niloticus (100%) and H. forskahlii (82%). The mean intensity of infections were 0.17-4.12 and 5.1-10.3 in L. niloticus and H. forskahlii respectively. For further identification, the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA from isolated larvae (n = 54) were amplified by PCR, followed by single-strand conformation polymorphism (SSCP) analysis which revealed five possible profiles. Conclusion: Light and scanning electron microscope studies revealed that all anisakid larvae in the present study showed the most typical features of the genus Contracaecum. The sequencing (n = 28) and sequence and phylogenetic analyses showed that the present nematode larvae are likely belonging to C. multipapillatum.
Anisakidosis is a zoonotic infection caused by members of the family Anisakidae. The presence of anisakid larvae in fish poses risk for humans and dissuade consumers from purchasing infected products. Although fish constitute important component of Egyptian diet, the prevalence of anisakid larvae in marketed fish in Egypt is not well described. Furthermore, the species of anisakid larvae is not defined in most of the available studies due to the over reliance on morphological analyses. The aim of the current work was to assess the prevalence and intensity of anisakid larvae in three common marketed fish in Egypt (Atlantic herring, Mediterranean horse mackerel and Atlantic mackerel) and to determine the species of the isolated larvae using morphological and molecular methods. Light and scanning electron microscope (SEM) analyses revealed the details of the isolated larvae. However, partial sequencing of cytochrome oxidase subunite-1 (mt cox1) gene revealed that all larvae isolated from Atlantic herring and Mediterranean horse mackerel belonged to Anisakis simplex sensu stricto with prevalence of 87.1% and 83.3%, respectively, whereas Atlantic mackerel harbored Anisakis typica with a prevalence of 42.8%. The Mediterranean horse mackerel demonstrated the highest larval mean intensity (n = 20 larvae/infected fish). This study highlights the importance of these fish as potential reservoirs for human anisakiasis in Egypt and possibly in other coastal countries. Keywords A. simplex Á Sequencing Á Atlantic mackerel Á Mediterranean horse mackerel Á Atlantic herring Electronic supplementary material The online version of this article (
PLoS ONE, 2012
Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank.
Journal of nematology
Total DNA w;rs isolated frorn itrtlividual i i e n l a~o d e s ol' t h e spccics I.orr~ydor~i,.v I / P /~Y~~(. I~. T , I.. III(I(.~Y).SOIYI,N, I.. ~/~llro)r.\i.\, I.. firofrrndorrrrn, I,. ~long(~/rr.\, atrtl I.. ~/~.~l i i i collected ill Swit~crlancl. T l i e ITS region and 111-112 e~l>arisiotr scgrrrcnts of' tlrc 2(iS rUNA wcl-c ;~mplilietl atrtl clolicrl. Tlie sequelices o b t a i n e d were alignctl in 01-tlcr t o investigate. sccltlcnc,c3 tliversicy a r~t l t o ilrli't. tlrc pllylogt~tretic rclationslrips anlorig t h r six I,orrg~clrlrIor~w spccics. 1) ]-l):! scqltc1rces wet-c. Inor(, c.otisel.ved tllari lire ITS ac-clt~c~nccs illat varied widely ill prirtiary strllcture and Ierigth, a n d n o consensus w;rs o b s c~v c d. l'hylogerietic arialyscs llsing t h e nciglrl)ot~joitii~ig, ~i i a x i r n l~r n parsinro~ry ant1 m a x i r n i~r n likcliliood mctliods w e r e pc.~-li)~-mcd with t h r e e cliil'er.c.nt scqtlcncc d;lt;r sets: IFI'SI-I.l'S2. 5.8s-DI-D2, a11t1 c o l n h i n i n g ITSl-ITS2t5.8S-I>1-1~2 scqtrerrccs. All rn~~lcil>le aligrltnerrts yieltlcd sirnilar basic tl.cc.s s u p p o r t i n g t h e existencc of t h e six species cstablislictl using nrorpIiologic;tl c11i1r.acler.s. Tliese scqrlcncc d a t a also p~.ovitlcrl cvitlcncc t h ;~t tlic differetit regions of' t h e I.I)NA a r c (,liar-acttcr-izct by diffrl-errt e v o l r~~i o n t.;~tcs ant1 11y different ('ictors ;~ssoci;~t(.d willi tlrc gc.nc.~-;tlio~r of' e x t r e m e size vat.ialiotl.
This study characterized anisakid nematodes in estuarine and near-shore species of fish in southern Western Australia. A total of 108 fish representing 13 species were examined for anisakid larvae. For the molecular characterization of anisakid larvae (n=218), we used PCR-coupled mutation scanning-sequencing-phylogenetic analyses of sequence variation in the internal transcribed spacers of nuclear ribosomal DNA. With the exception of Sillaginoides punctatus and Sillago schomburgkii, all the fish species examined (Aldrichetta forsteri, Arripis georgianus, Hyporhamphus regularis, Mugil cephalus, Platycephalus speculator, Pomatomus saltatrix, Pseudocaranx dentex, Pseudocaranx wrighti, Thysanophrys cirronatus, Trachurus novaezeelandiae and Upeneichthys lineatus) harboured at least one species of anisakid. Mutation scanning analysis identified 11 different genotypes of anisakid larvae. Phylogenetic analyses of the sequence data, employing reference sequence data for a wide range of anisakids (31 species) from public databases, revealed the presence of Anisakis pegreffii (n=3), Contracaecum multipapillatum (49), Contracaecum ogmorhini (1), Hysterothylacium larval type IV (82), Hysterothylacium larval type Vb , Hysterothylacium larval type VIII (3), Hysterothylacium larval type X (65), and Terranova type I (1) in the fish examined. The present study provides valuable information on the diversity of anisakids in southern Western Australia and also a basis for future investigations to assess the public health significance of these parasites.
Parasitology Research, 2005
Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8-5.5% (ITS-1) and 5.1-12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.
Parasitology, 2002
The anisakid nematodes morphologically corresponding with Pseudoterranova decipiens sensu lato (s.l.) (Krabbe, 1878) from different seal or sea lion hosts and geographical origins, previously identified as Pseudoterranova krabbei, P. decipiens (s.s.), P. bulbosa, P. azarasi and P. cattani by multilocus enzyme electrophoresis, were characterized using a DNA approach. Also a population of P. decipiens (s.l.) from Chaenocephalus aceratus, the blackfin icefish, from Antarctica and another from Osmerus eperlanus, the European smelt, from Germany were included in the study. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by PCR from individual nematodes and analysed by single-strand conformation polymorphism (SSCP), followed by selective sequencing. While no variation in single-stranded ITS-1 and ITS-2 profiles was detected among samples representing each of the species or populations (with the exception of slight microheterogeneity), SSCP analysis of the ITS-2 amplicons allowed the unequivocal differentiation of all of the 5 sibling species of P. decipiens (s.l.) examined, which was supported by sequence differences in ITS rDNA. Samples representing the P. decipiens (s.l.) population from O. eperlanus had the same SSCP profile as those of P. decipiens (s.s.), which was supported by a lack of nucleotide difference in the ITS between them, suggesting that the former represented P. decipiens (s.s.). Based on SSCP results and ITS sequence data, P. decipiens (s.l.) from C. aceratus was genetically most distinct with respect to all other members of Pseudoterranova examined, which indicated that it may represent P. decipiens E (based on geographical origin) or a distinct species. These findings and the molecular approach taken should have important implications for studying the life-cycles, transmission patterns, epidemiology and population genetics of these anisakid nematodes, and the diagnosis of their infections.
Infection, Genetics and Evolution, 2003
The anisakid nematode populations collected from fish and stranded cetaceans along from Iberian Peninsula waters were morphologically identified as corresponding to the Anisakis simplex complex. In order to realise their molecular identification and to analyse the extent of genetic variation, the entire ITS (ITS1, 5.8S rDNA gene and ITS2) and the mitochondrial small subunit of rRNA were pcr-amplified and sequenced. Digestions of the amplified its region with HinfI and HhaI allowed the identification of three different genotypes, belonging to A. simplex s.s., A. pegreffii and a yet not described recombinant genotype. The ITS sequences of the recombinant genotypes showed the presence of heterozygotes C/T at position 240 and 256 of the aligned sequence. Otherwise, the analysis of mtDNA sequences showed the existence of a different parental origin for recombinant genotypes. In order to check if they can be the products of a polymorphism normally occurring both in A. pegreffii and in A. simplex s.s., and/or the existence of an incomplete concerted evolution, three samples were also collected as controls in isolated geographic areas, where sympatric coexistence between A. simplex s.s. and A. pegreffii does not occur. The results supports the hypothesis that the recombinant individuals may be a product of interspecific hybridisation, and describe the Iberian Peninsula waters as a hybrid zone for the two sibling species.