Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers : zoonotic and ecological implications

Acknowledgments The authors would like to thank M. N. Cueto and J.M. Antonio (ECOBIOMAR) for their excellent technical support and also Rodrigo López for making the map of the study area. We also thank the personal of the Vigo IEO, for providing information about shad captures at sea collected on the basis of national program (AMDES) included in the European Data Collection Framework (DCF) project. We are also grateful to Comandancia Naval de Tui for providing fishing data. M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068). This study was partially supported by a PhD grant from the Portuguese Foundation for Science and Technology (FCT) SFRH/BD/44892/2008) and partially supported by the European Regional Development Fund (ERDF) through the COMPETE—Operational Competitiveness Programme and national funds through Foundation for Science and Technology (FCT), under the project BPEst-C/MAR/ LA0015/2013. The authors thank the staff of the Station of Hydrobiology of the USC BEncoro do Con^ due their participation in the surveys. This work has been partially supported by the project 10PXIB2111059PR of the Xunta de Galicia and the project MIGRANET of the Interreg IV BSUDOE (South-West Europe) Territorial Cooperation Programme (SOE2/P2/E288). D.J. Nachón is supported by a PhD grant from the Xunta de Galicia (PRE/2011/198)Peer reviewedPostprin

The Mediterranean European hake, Merluccius merluccius: Detecting drivers influencing the Anisakis spp. larvae distribution

The European hake Merluccius merluccius is one of the most commercially important and widely distributed fish species, occurring both in European and Mediterranean Sea fisheries. We analyzed the distribution and infection rates of different species of Anisakis in M. merluccius (N = 1130 hakes), by site of infection in the fish host (viscera, dorsal and ventral fillets) from 13 different fishing grounds of the Mediterranean Sea (FAO area 37). The fillets were examined using the UV-Press method. A large number of Anisakis specimens (N = 877) were identified by diagnostic allozymes, sequence analysis of the partial EF1 α-1 region of nDNA and mtDNA cox2 gene. Among these, 813 larvae corresponded to A. pegreffii, 62 to A. physeteris, 1 to A. simplex (s. s.), whereas one resulted as a F1 hybrid between A. pegreffii and A. simplex (s. s.). Remarkably high levels of infection with A. pegreffii were recorded in hakes from the Adriatic/Ionian Sea compared to the fish of similar length obtained from the western Mediterranean fishing grounds. A positive correlation between fish length and abundance of A. pegreffii was observed. Concerning the localization of A. pegreffii larvae in the fish, 28.3% were detected in the liver, 62.9% in the rest of the viscera, 6.6% in the ventral part of the flesh, whereas 2.1% in the dorsal flesh

Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

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

Anisakis pegreffii impacts differentiation and function of human dendritic cells

Human dendritic cells (DCs) show remarkably phenotypic changes when matured in presence of helminth-derived products. These modifications frequently elicited a polarization towards Th2 cells and regulatory T cells thus contributing to immunological tolerance against these pathogens. In this study, the interaction between DCs and larvae of the zoonotic anisakid nematode Anisakis pegreffii was investigated. A. pegreffii larvae were collected from fish hosts and monocyte derived DCs were co-cultured in the presence of the live larvae (L) or its crude extracts (CE). In both experimental conditions A. pegreffii impacted DC viability, hampered DC maturation by reducing the expression of molecules involved in antigen presentation and migration (i.e. HLA-DR, CD86, CD83 and CCR7), increased the phagosomal ROS levels and modulated the phosphorylation of ERK1,2 pathway. These biological changes were accompanied by the impairment of DCs to activate a T cell mediated IFN. Interestingly, live larva appeared to differently modulate DC secretion of cytokines and chemokines as compared to CE. These results demonstrate for the first time the immunomodulatory role of A. pegreffi on DCs biology and functions. In addition, they suggest a dynamic contribution of DCs to the induction and maintenance of the inflammatory response against A. pegreffi

Infection levels and species diversity of ascaridoid nematodes in Atlantic cod, Gadus morhua, are correlated with geographic area and fish size

Atlantic cod (Gadus morhua) is among the most important commercial fish species on the world market. Its infection by ascaridoid nematodes has long been known, Pseudoterranova even being named cod worm. In the present study, 755 individuals were sampled in the Barents, Baltic and North Seas during 2012–2014. Prevalences for Anisakis in whole fish and in fillets in the different fishing areas varied from 16 to 100% and from 12 to 90% respectively. Abundance was also greatly influenced by the sampling area. Generalized additive model results indicate higher numbers of Anisakis in the North Sea, even after the larger body size was accounted for. Numbers and prevalence of Anisakis were positively related to fish length or weight. The prevalence of parasites in whole fish and in fillets was also influenced by the season, with the spring displaying a peak for the prevalence in whole fish and, at the same time, a drop for the prevalence in fillets. Whereas 46% of cod had Anisakis larvae in their fillets, the majority (39%) had parasites mainly in the ventral part of the fillet and only 12% had parasites in their dorsal part. This observation is of importance for the processing of the fish. Indeed, the trimming of the ventral part of the cod fillet would allow the almost total elimination of ascaridoids except for cod from the Baltic Sea where there was no difference between the dorsal and the ventral part. The presence of other ascaridoid genera was also noticeable in some areas. For Pseudoterranova, the highest prevalence (45%) in whole fish was observed in the Northern North Sea, whereas the other areas had prevalences between 3 and 16%. Contracaecum was present in every commercial size cod sampled in the Baltic Sea with an intensity of up to 96 worms but no Contracaecum was isolated from the Central North Sea. Non-zoonotic Hysterothylacium was absent from the Baltic Sea but with a prevalence of 83% in the Barents and the Northern North Sea. A subsample of worms was identified with genetic-molecular tools and assigned to the species A. simplex (s.s.), A. pegreffii, P. decipiens (s.s.), P. krabbei, C. osculatum and H. aduncum. In addition to high prevalence and abundance values, the cod sampled in this study presented a diversity of ascaridoid nematodes with a majority of fish displaying a co-infection. Out of 295 whole infected fish, 269 were co-infected by at least 2 genera

No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach

A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α−1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α−1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species

Anisakiasis in italy: analysis of hospital discharge records in the years 2005-2015

Background Anisakiasis is a fish-borne zoonosis caused by the ingestion of marine food infected with Anisakis third-stage larvae, widespread marine parasitic nematodes. Gastrointestinal and/ or allergic clinical signs and symptoms are not specific. While frequently reported in countries with large raw fish consumption as Japan, the global prevalence of anisakiasis may be severely underestimated due to limitations of available diagnostic tools and to diverse clinical manifestations. Recently, infective larvae were found in the same localization with gastrointestinal tumors. The occurrence of allergic exacerbation upon secondary exposure and the possible occupational exposure, highlight the need to increase scientific evidences on anisakiasis. Methods We performed a retrospective descriptive study using analysis of Hospital Discharge Records (HDRs) from 2005 to 2015 in Italy, with particular attention to allergic manifestations. Descriptive statistics and multivariate analyses were performed using backward step-wise logistic regression models to assess spatial distribution and temporal trend as well as the variables independently associated with the allergic clinical signs and symptoms in Italian cases of anisakiasis. Results HDRs reporting the ICD-9 code for anisakiasis were retrieved (370), with a higher number of cases reported from central and southern regions, with particular regard to populations inhabiting the coastal territories. Around 40% of patients presented allergic manifestations and half of them showed serious allergic reactions. The multivariate analyses showed an independent association between allergic manifestations and features as living in southern regions and female gender, while anaphylactic episodes was independently associated only with female gender. Conclusion The present study is the first attempt to a better understanding of the epidemiological picture of anisakiasis in Italy, mining official data. A common strategy on data collection, monitoring and reporting would favor a more accurate epidemiological scenario in Italy, since the report of the diseases is not mandatory

Primer registro de anisakis sp. (nematoda, anisakidae) l3 en la cavidad corporal deatlantoraja platana (chondrichthyes, rajidae)

En la presente nota se registra por primera vez el tercer estadio larval de Anisakis sp. infectando a Atlantoraja platana (Günther, 1880). Los hospederos fueron obtenidos en desembarques pesqueros de plantas de procesado de los puertos de San Antonio Oeste (40° 44' S 64° 57' O) y San Antonio Este (40° 49' S 64° 57' O), Provincia de Río Negro, Argentina. Las larvas fueron colectadas en la cavidad visceral de los peces, cerca del órgano epigonal, un tejido linfomieloide estrechamente asociado a las gónadas y exclusivo de los peces cartilaginosos. Hay evidencias documentadas que las altas concentraciones de urea en tejidos y fluídos corporales tornan inhabitable el medioambiente celómico para ser colonizado por helmintos. Los resultados expuestos en este trabajo constituyen el primer reporte de L de Anisakis sp. en la cavidad corporal 3 de un elasmobranquio, en particular A. platana, y demuestran la capacidad de este anisákido para sobrevivir en la masa visceral de estos hospederos.This communication is the first record of the presence of a third stage larva of Anisakis sp. infecting Atlantoraja platana. The hosts were collected from fishery landings at processing plants of San Antonio Oeste (40° 44' S 64° 57' O) and San Antonio Este ports (40° 49' S 64° 57' O), Rio Negro province, Argentina. They were found in the visceral cavity near the epigonal organ, a lymphomyeloid tissue closely associated with gonads and only in cartilaginous fish. The high concentrations of urea in the body fluid and tissues of elasmobranch hosts made an inhospitable environment to the colonization of helminthes. The results produced by this work constitute the first report of L of Anisakis sp. in the body cavity of an elasmobranch, in particular 3 A. platana, and show the capability of this anisakid to survive in the visceral mass of these hosts.Fil: Moya, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Galíndez, Elena Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Di Giacomo, Edgardo Ernesto. Universidad Nacional del Comahue; ArgentinaFil: Tanzola, Rubén Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Assessing the risk of an emerging zoonosis of worldwide concern : anisakiasis

The authors sincerely thank the Biobanking platform at the PARASITE project (EU FP7 PARASITE project (GA no. 312068)) for providing host-parasite data. We thank Rosa Fernández and Cristina Martínez from CETMAR for their help during creation and divulgation of the questionnaires. We also thank Arturo del Rey Moreno (“Antequera” hospital) for his helpful comments. We are also grateful to “Subdirección General de Economía Pesquera” of “Ministerio de Agricultura, Alimentación y Medio Ambiente” (MAGRAMA) of the Spanish government for providing anchovy trade statistics for 2013. M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068).Peer reviewedPublisher PD