Opening a can of worms: Archived canned fish fillets reveal 40 years of change in parasite burden for four Alaskan salmon species.

How has parasitism changed for Alaskan salmon over the past several decades? Parasitological assessments of salmon are inconsistent across time, and though parasite data are sometimes noted when processing fillets for the market, those data are not retained for more than a few years. The landscape of parasite risk is changing for salmon, and long-term data are needed to quantify this change. Parasitic nematodes of the family Anisakidae (anisakids) use salmonid fishes as intermediate or paratenic hosts in life cycles that terminate in marine mammal definitive hosts. Alaskan marine mammals have been protected since the 1970s, and as populations recover, the density of definitive hosts in this region has increased. To assess whether the anisakid burden has changed in salmonids over time, we used a novel data source: salmon that were caught, canned, and thermally processed for human consumption in Alaska, USA. We examined canned fillets of chum (Oncorhynchus keta, n = 42), coho (Oncorhynchus kisutch, n = 22), pink (Oncorhynchus gorbuscha, n = 62), and sockeye salmon (Oncorhynchus nerka, n = 52) processed between 1979 and 2019. We dissected each fillet and quantified the number of worms per gram of salmon tissue. Anisakid burden increased over time in chum and pink salmon, but there was no change in sockeye or coho salmon. This difference may be due to differences in the prey preferences of each species, or to differences in the parasite species detected across hosts. Canned fish serve as a window into the past, providing information that would otherwise be lost, including information on changes over time in the parasite burden of commercially, culturally, and ecologically important fish species.

Rapid visual detection of anisakid nematodes using recombinase polymerase amplification and SYBR Green I.

Anisakidosis is a food-borne parasitic disease (FBPD) caused by the third-stage larvae of the family Anisakidae. Therefore, it is important to develop a simple, rapid and equipment-free detection method for anisakids in fish samples or seafood since current methods are time-consuming and require complex instruments. In this study, a recombinase polymerase amplification (RPA)-based method was established for the first time to detect anisakids by targeting the internal transcribed spacer (ITS) regions. The detection results were visualized by including SYBR Green I (SG) in the method. The sensitivity of RPA-SG assay was 102 copies per reaction of recombinant plasmid (within 20 min at 37°C), similar to quantitative real-time PCR (qPCR). The assay had high specificity for detecting anisakids against other related parasites and host fish. In addition, the assay was further used to detect fresh marine fish contaminated with anisakids and it showed high precision. These results indicate that the novel RPA-SG assay suitable for visual detection of anisakids in the field and food safety control.

Anisakidosis en peces frescos comercializados en tres ciudades de la Región del Bío Bío, Chile / Anisakidosis in fresh fish commercialized in three cities of the Bío Bío Region, Chile

INTRODUCCIÓN: La anisakidosis humana es una zoonosis transmitida por alimentos, causada por la ingestión de carne de pescado parasitada por nematodos anisákidos. Investigaciones sobre la presencia de anisákidos en pescados comercializados, sin cocción o congelación previa, en la región centro sur de Chile son escasas. OBJETIVO: Identificar nematodos anisákidos en catorce especies de peces marinos, obtenidos por pesca artesanal y comercializados en Concepción, Talcahuano y Lebu, ciudades de la Región del Bío Bío, Chile. MÉTODO: Entre los años 2018 y 2020, se examinó en busca de anisákidos a 334 pescados marinos frescos. RESULTADOS: Se identificó larvas de Anisakis spp. y Pseudoterranova sp. en ejemplares de siete y cuatro especies de peces, respectivamente. La mayoría de las larvas se encontraron vivas y ubicadas a nivel visceral. CONCLUSIÓN: La mayoría de las especies de peces que resultaron positivas son reconocidos hospederos intermediarios de estos parásitos. La presencia de anisákidos en la musculatura de estas especies indica que su preparación, en forma ahumada o cruda, es un riesgo para salud pública, recomendándose comprar estos productos en forma eviscerada y/o congelada, junto a promover su cocción.

BACKGROUND: Human anisakidosis is a food-borne zoonosis, caused by the intake of fish meat parasitized by anisakid nematodes. Research of anisakids parasites in commercialized fish, without previous cooking or freezing, in the south central region of Chile is scarce. AIM: To identify anisakid nematodes in fourteen species of marine fish, obtained by artisanal fishing and commercialized in Concepción, Talcahuano and Lebu, cities from Bío Bío Region, Chile. METHODS: During 2018 to 2020, we examinated for anisakids to 334 fresh marine fishes. RESULTS: Larvae of Anisakis spp. and Pseudoterranova sp. were identified in specimens of seven and four fish species, respectively. Most of the larvae were found alive and located at the visceral level. CONCLUSION: Most of the positive fish species are recognized intermediate hosts for these parasites. The presence of anisakids in the musculatura of these species indicates that their preparation, in smoked or raw form, is a risk to public health, and it is recommended to buy these products in gutted and / or frozen form, together with promoting their cooking.

Primer registro de nematodos zoonóticos de transmisión alimentaria (Nematoda: Anisakidae) en músculo de Cynoscion guatucupa (pescadilla de calada) y Macrodon ancylodon (pescadilla de red) comercializado en Uruguay / First record of foodborne zoonotic nematodes (Nematoda: Anisakidae) in muscle of fish marketed in Uruguay

Resumen En los últimos 4 años, Uruguay ha experimentado una creciente demanda por el consumo de alimentos a base de pescado crudo, tales como sushi, sashimi y ceviche. Este escenario generó la necesidad de evaluar la presencia y concentración de parásitos zoonóticos (Nematoda: Anisakidae) en porciones comestibles de especies de pescado comercializadas en el mercado local. Se analizaron 233 ejemplares de tres especies de pescado comercializadas en Uruguay: Macrodon ancylodon, Micropogonias furnieri y Cynoscion guatucupa. Se cuantificaron e identificaron, a nivel de género, nematodos en cavidad celómica y músculo, estableciendo finalmente los descriptores ecológicos para cada género encontrado y para cada especie de pescado evaluada. Dos de las especies evaluadas se encontraban parasitadas a nivel de cavidad celómica y tejido muscular, M. ancylodon y C. guatucupa. Cynoscion guatucupa resultó la especie mayormente parasitada con una prevalencia de 46%, mientras que el género de anisákido más prevalente fue Contracaecum (prevalencia de 35,1% en C. guatucupa y 32,4% en M. ancylodon). El hallazgo de anisákidos en la especie M. ancylodon representa un nuevo registro de hospedador en Uruguay. Por otra parte, se reporta por primera vez en el país la presencia de estos parásitos zoonóticos en porciones comestibles (músculo).

Abstract In the last 4 years, Uruguay has experienced a growing demand of raw fish-based foods, such as sushi, sahimi and ceviche. This scenario generated the need to assess the presence and concentration of zoonotic parasites (Nematodoes: Anisakidae) in edible parts of fish species traded in the local market. 233 fish specimens of 3 species commercialized in Uruguay were analyzed: Macrodon ancylodon, Micropogonias furnieri and Cynoscion guatucupa. The nematodes were quantified and identified at genus level, from coelomic cavity and muscle of the fishes, to finally stablish the ecological descriptors for each genus founded and for each fish species evaluated. Two of the three species of fish were parasitized with anisakids in both location (coelomic cavity and muscle), M. ancylodon and C. guatucupa. C. guatucupa was the most parasitized with a prevalence of 46%, while, the anisakid genus most prevalente was Contracaecum (prevalence of 35,1% in C. guatucupa and 32,4% in M. ancylodon). The finding of anisakids in M. ancylodon represents a report of a new host in Uruguay. Moreover, the finding for the first time in the country of these zoonotic parasites in edible parts of fish (muscle) is highlighted.

Metagenomics Analysis Reveals an Extraordinary Inner Bacterial Diversity in Anisakids (Nematoda: Anisakidae) L3 Larvae.

L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as infectious agents causing allergies and as potential vectors of pathogens and microrganisms. In spite of the close bacteria-nematode relationship very little is known of the anisakids microbiota. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of anisakids when the L3 larvae migrate through the muscles. As a consequence, the bacterial inoculum will be spread, with potential effects on the quality of the fish, and possible clinical effects cannot be discarded. A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 fishing area were studied. Bacteria were taxonomically characterized through 1803 representative operational taxonomic units (OTUs) sequences. Fourteen phyla, 31 classes, 52 orders, 129 families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity indices (Shannon and Simpson) indicate an extraordinary diversity of bacteria at an OTU level. There are clusters of anisakids individuals (samples) defined by the associated bacteria which, however, are not significantly related to fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of anisakids taxa has to be expressed by the association among bacterial OTUs or other taxonomical levels which range from OTUs to the phylum level. There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from phylum to genus level, which could also be an indicator of fish contamination or the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for defining such structures.

High occurrence of Anisakidae at retail level in cod (Gadus morhua) belly flaps and the impact of extensive candling.

The presence of Anisakidae at retail level, after the routine screening via candling, was investigated in cod, the most commonly consumed fish species in Belgium. A total of 780 pre-packed belly flap samples destined for one branch of retail shops were collected from a Belgian wholesale company. To recover all larvae, each sample was first candled and thereafter enzymatically digested. Larvae were morphologically identified to the genus level and a subset was additionally molecularly confirmed by amplification of the ITS fragment and HinfI/HhaI enzyme restriction. The PCR/RFLP profiles of Contracaecum spp. were determined and confirmed with sequencing by the European Reference Laboratory for Parasites (Istituto Superiore di Sanità). The positivity rate of Anisakidae in the individual cod samples was 18% [95%-CI: 15-21%], with a mean intensity of one larva [range: 1-6]. Belly flaps were sold packed primarily by two, with a one-in-three chance of buying an infected package. Pseudoterranova spp. infections (single infections) were most frequently detected (positivity rate 9% [95%-CI: 7-11]), closely followed by Anisakis spp. (7% [95%-CI: 6-9]). Co-infections of Pseudoterranova spp. and Anisakis spp. comprised 8% of the infections, with a positivity rate of 1% [95%-CI: 1-3%]. All belly flaps reportedly were candled prior to our sampling, nonetheless our results indicated that an additional candling screening before packaging would identify an extra third of the infections and larvae. In 19 of the 139 infected samples, all larvae were recovered by the additional candling, thereby removing the infection risk for consumers. In conclusion, this study shows that cod belly flaps infected with zoonotic parasites reach the Belgian consumer. Although a second candling step at retail level could be helpful in reducing the consumer risk, additional measures are needed since 66% of infections would still remain undetected.

Advances in Omic Studies Drive Discoveries in the Biology of Anisakid Nematodes.

Advancements in technologies employed in high-throughput next-generation sequencing (NGS) methods are supporting the spread of studies that, combined with advances in computational biology and bioinformatics, have greatly accelerated discoveries within basic and biomedical research for many parasitic diseases. Here, we review the most updated "omic" studies performed on anisakid nematodes, a family of marine parasites that are causative agents of the fish-borne zoonosis known as anisakiasis or anisakidosis. Few deposited data on Anisakis genomes are so far available, and this still hinders the deep and highly accurate characterization of biological aspects of interest, even as several transcriptomic and proteomic studies are becoming available. These have been aimed at discovering and characterizing molecules specific to peculiar developmental parasitic stages or tissues, as well as transcripts with pathogenic potential as toxins and allergens, with a broad relevance for a better understanding of host-pathogen relationships and for the development of reliable diagnostic tools.

Occurrence of Anisakis and Hysterothylacium larvae in commercial fish from Balearic Sea (Western Mediterranean Sea).

This study investigates the occurrence of anisakids and raphidascarids in commercial fish from Balearic Sea (Western Mediterranean). A total of 335 fish including 19 black anglerfish (Lophius budegassa), 33 white anglerfish (L. piscatorius), 129 European hake (Merluccius merluccius), 30 red mullet (Mullus barbatus), and 124 striped mullet (M. surmuletus) were examined using enzymatic digestion. A total of 948 nematode larvae were isolated (prevalence 52.53%) being the highest prevalence observed in striped mullet. Forty-six larvae were identified using molecular analyses which included PCR and sequencing of the 629-bp fragment of mitochondrial cox2 gene region. Anisakis pegreffii (80.43%), A. physeteris (8.69%), Hysterothylacium fabri (6.52%), and A. simplex (4.35%) were detected based on molecular analyses of larvae. Total nematode prevalence was positively correlated with weight, length, condition factor, and maturity stage of the host and also with fishing ground depth. Statistical differences between total nematode prevalence and geographical sector of capture were observed when fishing hauls were grouped according to the abundance of sperm whales or common bottlenose dolphins. The results also corroborate that fishing water depth may play an important role in anisakid and raphidascarid parasitization.

Excretory/secretory products of anisakid nematodes: biological and pathological roles.

Parasites from the family Anisakidae are widely distributed in marine fish populations worldwide and mainly nematodes of the three genera Anisakis, Pseudoterranova and Contracaecum have attracted attention due to their pathogenicity in humans. Their life cycles include invertebrates and fish as intermediate or transport hosts and mammals or birds as final hosts. Human consumption of raw or underprocessed seafood containing third stage larvae of anisakid parasites may elicit a gastrointestinal disease (anisakidosis) and allergic responses. Excretory and secretory (ES) compounds produced by the parasites are assumed to be key players in clinical manifestation of the disease in humans, but the molecules are likely to play a general biological role in invertebrates and lower vertebrates as well. ES products have several functions during infection, e.g. penetration of host tissues and evasion of host immune responses, but are at the same time known to elicit immune responses (including antibody production) both in fish and mammals. ES proteins from anisakid nematodes, in particular Anisakis simplex, are currently applied for diagnostic purposes but recent evidence suggests that they also may have a therapeutic potential in immune-related diseases.

Prevalence and Mean Intensity of Anisakidae Parasite in Seafood Caught in the Mediterranean Sea Focusing on Fish Species at Risk of Being Raw-consumed. A Meta Analysis and Systematic Review.

OBJECTIVE: To assess the prevalence and mean intensity of anisakids in seafood caught in the Mediterranean Sea, focusing on fish species at risk of being raw-consumed. DESIGN: Systematic review and meta-analysis of studies published from 1960-2012. STUDY SELECTION: Main criteria for the inclusion of studies were as follows: Findings of anisakid larvae, in both muscles and viscera; fish species for human consumption caught in the Mediterranean Sea; prevalence and mean intensity data for each species; and sample size equal to or more than 40 fishes. RESULTS: Twelve studies were identified. Among these, four studies considered the following three fish species that are often consumed raw or preserved lightly, or not cooked thoroughly: anchovy, pilchard, and Atlantic mackerel. DATA SYNTHESIS: All pooled analyses were based on the random-effect model. Anisakids prevalence in fish muscle was 0.64% (P < 0.0001), in viscera it was 1.34% (P < 0.0001), and overall prevalence was 0.95% (P < 0.0001). Mean intensity in muscle was 2.31 (P = 0.0083), in viscera it was 1.55 (P = 0.0174), and overall it was 1.81 (P < 0.0005). Heterogeneity indices (I(2)) were significantly high with the exception of viscera mean intensity. CONCLUSIONS: Anchovy, pilchard, and Atlantic mackerel have a low prevalence and mean intensity of anisakidae larvae in both viscera and muscles. Mean Intensity was also low.

Temporal stability of parasite distribution and genetic variability values of Contracaecum osculatum sp. D and C. osculatum sp. E (Nematoda: Anisakidae) from fish of the Ross Sea (Antarctica).

The Ross Sea, Eastern Antarctica, is considered a "pristine ecosystem" and a biodiversity "hotspot" scarcely impacted by humans. The sibling species Contracaecum osculatum sp. D and C. osculatum sp. E are anisakid parasites embedded in the natural Antarctic marine ecosystem. Aims of this study were to: identify the larvae of C. osculatum (s.l.) recovered in fish hosts during the XXVII Italian Expedition to Antarctica (2011-2012); perform a comparative analysis of the contemporary parasitic load and genetic variability estimates of C. osculatum sp. D and C. osculatum sp. E with respect to samples collected during the expedition of 1993-1994; to provide ecological data on these parasites. 200 fish specimens (Chionodraco hamatus, Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi) were analysed for Contracaecum sp. larvae, identified at species level by allozyme diagnostic markers and sequences analysis of the mtDNA cox2 gene. Statistically significant differences were found between the occurrence of C. osculatum sp. D and C. osculatum sp. E in different fish species. C. osculatum sp. E was more prevalent in T. bernacchii; while, a higher percentage of C. osculatum sp. D occurred in Ch. hamatus and T. hansoni. The two species also showed differences in the host infection site: C. osculatum sp. D showed higher percentage of infection in the fish liver. High genetic variability values at both nuclear and mitochondrial level were found in the two species in both sampling periods. The parasitic infection levels by C. osculatum sp. D and sp. E and their estimates of genetic variability showed no statistically significant variation over a temporal scale (2012 versus 1994). This suggests that the low habitat disturbance of the Antarctic region permits the maintenance of stable ecosystem trophic webs, which contributes to the maintenance of a large populations of anisakid nematodes with high genetic variability.

Evolution of the Anisakis risk management in the European and Italian context.

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy.

Sobrevivência de larvas de anisakídeos de peixe espada, Trichiurus lepturus (L) submetidos aos processos de salmouragem e cocção / Survival of anisakids larvae of cutlass fish, Trichiurus lepturus (L) submitted to brine and cooking processes

Um total de 40 espécimes de peixe espada, Tríchíurus lepturus, foram analisados para se observar a sobrevivência de larvas de anisakídeos submetidos à salmoura e cocção. Os parasitos coletados pertenciam aos gêneros Contracaecum, Phocanema e Anísakís com prevalências de 95,5%, 3,8% e 0,7%, respectivamente. O efeito da salmoura a 20º Bé (Baumé) na sobrevivência das larvas demonstrou que, ao final de 72 h de exposição, todas encontravam-se mortas. Nos peixes submetidos à cocção a temperatura de 100ºC, 5,9% das larvas permaneceram vivas ao final de 30 min. e todas morreram ao final de 60 min. Estes resultados sugerem que o melhor método para matar larvas de anisakídeos em peixe espada é a cocção.

Fourty specimens of cutlass fish Trichiurus lepturuswere analyzed in arder to observe the survivallevel of the anisakids larvae submitted to brine and cooking processes. The collected parasites were of the genera Contracaecum, Phocanemaand Anisakiswith a prevalence of 95.5%, 3.8% and 0.7%, respectively. Alllarvae died after being exposed to 72 hr of brine at 20º Bé. In cooked fishes at 100ºC, 5.9% of larvae remained alive after 30 min, and ali of them died after 60 min. These results suggest that cooking is the best method to kill anisakids larvae in cutlass fish.

Larvas de anisakideos em Pagrus pagrus (L) e seu controle através de baixas temperaturas / Anisakids larvae in Pagrus pagrus (L) and its control under low temperature

Em 100 exemplares de Pagrus pagrus examinados, pescados no litoral dos Estados do Rio de Janeiro e Espírito Santo, Brasil, 88% achavam-se parasitados por larvas de anisakideos. Nos 16 primeiros peixes necropsiados para evidenciação do parasitismo, observou-se que 100% estavam parasitados pelo gênero Contracaecum, 65% pelo gênero Raphidascaris 44% pelo gênero Phocanema e 44% pelo gênero Terranova. Cinquenta e um peixes foram mantidos à temperatura de refrigeração (0°C) por 120 horas. Ao final deste tempo 68,37% dos parasitos morreram. Trinta e três peixes foram colocados à temperatura de -15°C. Após duas horas de exposição à temperatura de -2,9°C todas as larvas coletadas encontravam-se mortas. A localização dos parasitas estava restrita às serosas das vísceras e serosas abdominais.

A sample of 100 Pagrus pagrus caught in the coast of Rio de Janeiro and Espírito Santo States, Brazil, was examined and larval stages of anisakid nematode were observed affecting 88% of the sample. Post-mortem examinations were carried out in the first 16 fish inorder to identify the genera of the parasites and it was noted that 100% belonged to the genus Contracaecum, 63% of the genus Raphídascaris, 44% to the genus Phocanema and 44% to the genus Terranova. Among 51 fish wich were maintained at 0°C for 120 hours it was observed that 68,37% was dead at the end of this time. Thirty-three fish were kept at -15°C and after two llours at -2,9°C ali previously noted larva were dead. Ali the parasites were found in the vísceral and abdominal serosa.