The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Increasing intensities of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae with weight and sea age in returning adult Atlantic salmon, Salmo salar L., of coastal waters of Norway.

Ninety wild Atlantic salmon, Salmo salar L., (1.5-10.3 kg) were caught in the Namsen Fjord near the mouth of River Namsen, mid-Norway, and examined for the presence and distribution of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) larvae by digestion of the viscera and muscles in a pepsin/HCl solution. All salmon were migrating spawners after 1-4 years of feeding in the Atlantic Ocean. All 90 Atlantic salmon had A. simplex larvae in the viscera, and all, except two, had A. simplex larvae in the musculature. The number of A. simplex larvae in each fish varied between 3 and 181, and the total mean number of nematode larvae was 44.5. The intensity of A. simplex larvae was positively correlated with increasing weight and sea age of the host. However, the proportion of larvae in the muscle fillets decreased with increasing host weight and sea age. Atlantic salmon females had more A. simplex larvae than males. In all the fish examined, 70.2% of the A. simplex larvae were found in the viscera and 29.8% in the musculature. The majority (93%) of the larvae in the musculature occurred in the hypaxial sections anterior to the anus. As A. simplex larvae commonly occur in the musculature of wild Atlantic salmon, consumption of unfrozen, raw or semi-raw musculature represents a risk for humans developing anisakiasis.

Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters.

The third-stage larvae (L3) of the Anisakidae family are parasitic nematodes with zoonotic impact and are frequently encountered in the organs and musculature of various fish intended for human consumption. Since Anisakis simplex (s.s.) and A. pegreffii are the major aetiological agents of human disease, this study aims to combine the morphological and molecular data on the recovered anisakid larvae to contribute to a simplified morphological distinction of those species and conducted a survey of anisakid larvae infection in horse mackerel (Trachurus trachurus). Here, 116 horse mackerel caught in Portuguese waters were analysed for the presence of L3 of anisakids, and 3148 larvae were collected, of which only 30% were retrieved during visual inspection. As such, visual inspection does not appear to be very effective in anisakid detection. A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were 32.1 and 27.1 parasites per fish, respectively. The morphological and molecular analyses of 196 L3 randomly chosen from the total sample of parasites demonstrated the presence of L3 of mostly Anisakis spp., with only one L3 of Hysterothylacium aduncum. Relative frequencies of 62.9% for A. pegreffii and 37.1% for A. simplex (s.s.) were obtained. The morphometry differences between these two sibling species were evaluated, and the results demonstrated significant differences between the length of the ventriculus and the length of the oesophagus. Precisely, A. simplex (s.s.) has a longer oesophagus and ventriculus than A. pegreffii. As such, these differences may be used to distinguish the two species through morphological analysis.

A Case Report of Adhesional Small Bowel Obstruction Caused by Extraintestinal Anisakiasis.

BACKGROUND: Small bowel obstruction (SBO) is a common diagnosis made in the emergency department (ED). We present a case with an unusual underlying cause of SBO: extraintestinal infection with an Anisakis roundworm. CASE REPORT: A healthy young woman with no prior abdominal surgery presented with epigastric abdominal pain, nausea, and anorexia 1 day after eating a raw oyster. Laboratory studies were significant for 14% eosinophilia. Initial abdominal computed tomography (CT) showed small bowel inflammation and small-volume ascites. After discharge home, she returned on day 14 of illness with a closed-loop SBO, to which she was predisposed by an adhesion formed in association with an eosinophilic abscess containing an Anisakis roundworm. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Anisakiasis is an uncommon cause of common symptoms with which patients may present to EDs. The diagnosis should be considered in patients presenting with abdominal pain and recent ingestion of raw seafood, with suspicion raised further by the presence of focal gastric or small bowel inflammation and ascites on abdominal CT. Extraintestinal anisakiasis can cause inflammation leading to intraabdominal adhesions, a sequela of which is small bowel obstruction. If suspicion for gastric or intestinal anisakiasis is high, treatment with endoscopic removal or albendazole may be initiated.

Effectiveness of five flavored Tunisian olive oils on Anisakis larvae type 1: application of cinnamon and rosemary oil in industrial anchovy marinating process.

BACKGROUND: Anisakidosis is caused by the ingestion of raw or undercooked fish or cephalopods containing viable Anisakis larvae. Several natural extracts, oils, essential oils, and their compounds have been tested against Anisakis. In this study the effectiveness of Tunisian olive oil with different spices or plants (cardamom, cinnamon, ginger, laurel, and rosemary) was tested against Anisakis larvae type 1. RESULTS: For the in vitro test, larvae were submerged separately in the oils mentioned above and observed to check viability. Cinnamon oil was the most effective against parasites with lethal time (LT) scores being LT50 = 1.5 days and LT100 = 3 days, followed by rosemary. Laurel, cardamom, and ginger oils were less effective. For the ex vivo experiment, cinnamon, and rosemary oils were tested in anchovy fillets, previously artificially parasitized. Cinnamon was the most effective against parasites (dead after 4 days) as compared to rosemary (7 days). CONCLUSION: The use of cinnamon and rosemary-flavored olive oil in the industrial marinating process can be considered as an efficient alternative to the freezing process required by European Regulation EC No 853/2004 to devitalize Anisakis. © 2019 Society of Chemical Industry.

Anisakid Larvae from Anchovies in the South Coast of Korea.

Anisakiasis (anisakidosis) refers to a foodborne zoonosis caused by ingesting raw or undercooked marine fish or cephalopods infected with anisakid larvae. The present study was performed to investigate the prevalence of anisakid larvae in anchovies (Engraulis japonica) purchased from 2 local markets in Gyeongsangnam-do, the Republic of Korea (=Korea), during 2018-2019. Anchovies were transported to our laboratory and examined by pepsin-HCl artificial digestion technique followed by microscopic observations and molecular analyses. The overall prevalence of anisakid larvae was 19.5% (39/200), from which a total of 51 larvae (av. 1.3 larvae/infected anchovy) were recovered. Sequencing of the larvae targeting the ITS region, including ITS1, 5.8S rRNA, and ITS2 genes confirmed the species of larvae as Anisakis pegreffii (54.9%; 28/51), Hysterothylacium sinense (23.5%; 12/51), and Hysterothylacium aduncum (21.5%; 11/51). The results suggested that anchovies could be a potential source of human anisakiasis in Korea.

A scanning electron microscopy study of Anisakis physeteris molecularly identified: from third stage larvae from fish to fourth stage larvae obtained in vitro.

The development of the fourth larval stage (L4) of Anisakis physeteris was studied using scanning electron microscopy (SEM), comparing it with third larval stage (L3) recently obtained from the host fish, blue whiting (Micromesistius poutassou), from the western Mediterranean Sea (east coast of Spain, zone FAO 37.1.1). After molting to L4, samples of the parasite were examined at different times in order to observe their development. Following collection of the L4, a small portion was taken from the middle of the larva for molecular identification, confirming in all cases that it was A. physeteris. The anterior and posterior sections of the larvae were prepared for morphological study by SEM. The development of a row of denticles on each of the three prominent lips, almost reaching the buccal commisures, was observed in the L4. Pores of unknown function were found in the upper external part of each lip. Clearly developed cephalic papillae, amphids, and deirids were also observed in L4, while, although present in L3, these were beneath the cuticle. Phasmids were detected in L4 but not in L3. The L4 tail finished in a conical lobe with a blunt point, absent in L3. In the oldest L4, some preanal papillae were observed beneath the cuticle in males, while, in females, the vulva could be seen by light microscopy, apparently still covered by the cuticle.

Evaluation of different Mediterranean essential oils as prophylactic agents in anisakidosis.

CONTEXT: Anisakis Dujardin 1845 (Anisakidae) nematodes can cause gastrointestinal and allergic diseases when humans eat raw or undercooked seafood containing larvae. There is currently no drug available in the market against this parasitic disease, and the study of plant-derived molecules could be useful in the discovery of effective compounds. OBJECTIVE: This research assesses the biocidal activity of a range of essential oils (EOs) from some Mediterranean plants against larvae found in the musculature of fresh fish. MATERIALS AND METHODS: EOs composition was analyzed by gas chromatography-mass spectroscopy. All the EOs were diluted at 5% v/v in olive oil to cover the fish with the solutions for 24 h. The larvae that abandoned the muscle and the larvae obtained from the artificial digestion of the fish were collected. Controls were carried out in parallel. Furthermore, Wistar rats were infected with the live larvae collected from the in vitro trials in order to find any larvae that may have penetrated the gastrointestinal wall. RESULTS: Between 60.8% and 87.6% of parasites treated with EOs abandoned the fish muscle, and the highest in vitro mortality rate was achieved with oregano EO (53.9%). Rats previously treated with oregano, cumin and Spanish lavender EOs showed no detectable lesions in the digestive tract due to the infection with larvae. CONCLUSIONS: Oregano (Origanum vulgare L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae) and Spanish lavender (Lavender stoechas L. Lamiaceae) EOs could be used as promising ingredients in the development of products for the control of anisakiasis.

Experimental anisakid infections in mice.

Anisakidosis is a human parasitic disease caused by infections with members of the Anisakidae family. Accidental infection after fish intake affects the gastrointestinal tract as a consequence of mechanical damage caused by migrating larvae. Infections can also trigger allergies, hives, severe asthma or anaphylaxis with angioedema. Although mouse models of intraperitoneal antigenic stimulation exist, enabling immunological studies, few models using gastric introduction of live larvae are available for the study of immunological and gastrointestinal damage in mice. This study was designed to characterize serum reactivity against Anisakis spp. and Contracaecum spp. in Balb/c mice following orogastric inoculation and to assess gastrointestinal damage. These anisakid species were classified at the Universidade Federal Fluminense (UFF) School of Veterinary Medicine and materials for live larval inoculation were developed at the UFF Immunobiology laboratory. Live larvae were inoculated following injection with a NaCl solution. Blood samples were collected and sera screened for immunoglobulin (Ig)E and IgG anti-larva responses to both nematodes, specific for somatic and excretory/secretory antigens, by enzyme-linked immunosorbent assay (ELISA). The means of the optical densities were analysed using analysis of variance (ANOVA) with Tukey's post-hoc test and the general linear model. This analysis identified the presence of anti-IgG seroreactivity to both somatic and excretory/secretory Anisakis antigens in inoculated animals compared with controls (P< 0.001), and no gastric or intestinal damage was observed. These experiments demonstrated that introduction of live Contracaecum spp. into the gastrointestinal tract did not elicit serum sensitization in animals.

First record of anisakid juveniles (Nematoda) in the European seabass Dicentrarchus labrax (family: Moronidae), and their role as bio-indicators of heavy metal pollution.

This study assessed the anisakid nematode distribution pattern in the fish collected from coasts of Mediterranean Sea, Egypt, during the period September 2010-April 2011. Two hundred thirty out of 300 (76.7%) Dicentrarchus labrax (European seabass) marine fishes belonging to family Moronidae were dissected and found to be infected with larva three nematodes. The larvae had been studied by light and scanning electron microscopy. The present work represents the first record of the presence of the parasite in this fish in the Mediterranean Sea. The concentrations of some heavy metals (Pb, Zn, Fe, Cd, Cu, Mn, Ni) in parasites as well as in tissues of fish were measured. The presented results showed that the nematode parasites are able to accumulate heavy metals in their tissues and in some cases that they are able to accumulate large amounts of heavy metals in a higher amount than host tissues. This demonstrated their sustainability as bioindicators of environmental pollution by removing heavy metals and help in the survival of fish.

Anisakis simplex sensu stricto and Anisakis pegreffii: biological characteristics and pathogenetic potential in human anisakiasis.

Anisakiasis is one of the most common fishborne helminthic diseases in Japan, which is contracted by ingesting the larvae of the nematode Anisakis spp. carried by marine fish. Anisakis simplex sensu stricto (s.s.) and A. pegreffii are the dominant species in fish caught offshore Japan. The present study aimed to identify the anisakid species infecting Japanese patients and determine whether there is any difference in the pathogenetic potential of A. simplex (s.s.) and A. pegreffii. In total, 41 and 301 Anisakis larvae were isolated from Japanese patients and chub mackerel (Scomber japonicus), respectively; these were subjected to molecular identification using polymerase chain reaction targeted at a ribosomal DNA internal transcribed spacer region. Chub mackerel larvae were further examined for survival in artificial gastric juice (pH 1.8) for 7 days and for invasiveness on 0.75% solid agar over a 24-h interval. All clinical isolates, including those of asymptomatic, acute, and chronic infections as well as those from the stomach, small intestine, colon, and stool, were identified as A. simplex (s.s.). Chub mackerel harbored A. simplex (s.s.) and A. pegreffii larvae, together with a few larvae of other anisakid species. A. simplex (s.s.) larvae from chub mackerel tolerated the artificial gastric juice better than A. pegreffii, with 50% mortality in 2.6 and 1.4 days, respectively. In addition, A. simplex (s.s.) penetrated the agar at significantly higher rates than A. pegreffii. These results show that A. simplex (s.s.) larvae have the potential to survive acidic gastric juice to some extent and penetrate the stomach, small intestine, or colon in infected humans.

Stereo and scanning electron microscopic studies of the third stage larvae of Anisakis simplex.

This study was to demonstrate the surface anatomy of the third stage larvae of Anisakis simplex in marine fish using stereo and scanning electron microscopes (SEM). The round worm is slender, elongated and of cylindrically shaped. The head of this worm is a globular structure. The mouth is triangularly shaped and surrounded by three lips. A boring tooth projects dorsally at the anterior end. There are four pairs of tactoreceptors, the labial papillae, enclosing the lips. The tail end is blunt and acquires a distinct slender process, the mucron. Stereomicroscopy revealed the esophagus is elongated, bulbous and club shaped, subdivided into an anterior muscular part and a posterior glandular part or ventriculus. The intestine is a long straight tube where the digestion and absorption occur. Waste pass through the intestine and is stored in the rectum until excreted via the anus. A SEM is a powerful tool in distinguishing worm species, as was seen when examining that the mouth of Anisakis simplex, which is triangular shaped and enclosed by three lips with one boring tooth; other species are different. The mucron projection at the distal end is another distinctive structure revealed by SEM.

Anisakis simplex larvae: infection status in marine fish and cephalopods purchased from the Cooperative Fish Market in Busan, Korea.

The infection status of marine fish and cephalopods with Anisakis simplex third stage larva (L3) was studied over a period of 1 year. A total of 2,537 specimens, which consisted of 40 species of fish and 3 species of cephalopods, were purchased from the Cooperative Fish Market in Busan, Korea, from August 2006 to July 2007. They were examined for A. simplex L3 from the whole body cavity, viscera, and muscles. A. simplex L3 were confirmed by light microscopy. The overall infection rate reached 34.3%, and average 17.1 larvae were parasitized per infected fish. Fish that recorded the highest infection rate was Lophiomus setigerus (100%), followed by Liparis tessellates (90%), Pleurogrammus azonus (90%), and Scomber japonicus (88.7%). The intensity of infection was the highest in Gadus macrocephalus (117.7 larvae per fish), followed by S. japonicus (103.9 larvae) and L. setigerus (54.2 larvae). Although abundance of A. simplex L3 was not seasonal in most of the fish species, 10 of the 16 selected species showed the highest abundance in February and April. A positive correlation between the intensity of L3 infection and the fish length was obvious in S. japonicus and G. macrocephalus. It was likely that A. simplex L3 are more frequently infected during the spring season in some species of fish. Our study revealed that eating raw or undercooked fish or cephalopods could still be a source of human infection with A. simplex L3 in Korea.

Expression and functionality of allergenic genes regulated by simulated gastric juice in Anisakis pegreffii.

To better understand the molecular mechanisms underlying allergens and parasite immunity and discover the stage-enriched gene expression of fish-borne zoonotic nematodes in the stomach, we used RNA-seq to study the transcriptome profiles of Anisakis pegreffii (Nematoda: Anisakidae, AP) in simulated gastric juice. Mobile L3 larvae were incubated in simulated medium at 37 °C in 5% CO2 (AP-GJ) and the control group larvae were collected in PBS under the same conditions (AP-PBS). We found that the sequences of A. pegreffii were highly similar to Toxocara canis sequences. Among the transcripts, there would be 138 up-regulated putative genes and 251 down-regulated putative genes in AP-GJ group. Several lipid binging-related genes were more highly expressed in AP-GJ larvae. Moreover, 17 allergen genes were up-regulated and 29 were down-regulated in AP-GJ larvae. Eleven allergen genes belonged to one or more of the following three categories: biological process, cellular component, and molecular function. According to KEGG analysis, the main pathways that were represented included protein processing in transcription, immune system, cancer, and infectious disease. In particular, the most significant changes in the expression of parasite-derived allergen products occurred in AP-GJ larvae. This study helps us to extend our understanding of the biology of the fish-borne zoonotic parasite A. pegreffii and could be helpful for more precise risk assessment and providing guidelines for allergic consumers.

Shotgun Proteomics for L3 and L4 Anisakis simplex Development Stages.

Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are present in many fish species and cephalopods all over the globe, might be consumed and develop occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and economic concern. In recent years, proteomic methods have gained greater acceptance among scientists involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)-based shotgun proteomics to define differences in proteomic composition between L3 and L4 development stages of A. simplex s.s.

Multiple acute parasitization by Anisakis simplex.

Hypersensitivity to Anisakis is an increasingly prominent medical problem throughout the world, due to a better understanding of diseases induced by parasites and to modern culinary habits of eating raw or undercooked fish. We describe the case of a patient who presented epigastric pain, wheals, erythema, and pruritus 3 hours after the ingestion of fish. More than 200 larvae were obtained by endoscopy. However, the patient only developed an immune response with specific immunoglobulin E and eosinophilia peaking at day 18 and decreasing during the 17-month follow-up. Only eosinophilia reached normal limits.

Antigenicity and viability of Anisakis larvae infesting hake heated at different time-temperature conditions.

Heat treatments (40 to 94 degrees Celsius, 30 s to 60 min) were applied to different batches of Anisakis simplex L3 larvae isolated from hake ovaries and viscera to study the effect of heat on the viability of the larvae measured as mobility, emission of fluorescence under UV light, and changes in color after staining with specific dyes, and on A. simplex antigenic proteins. The aim was to determine the lowest time-temperature conditions needed to kill the larvae to avoid anisakiasis in consumers, and to evaluate whether high temperature modifies the antigenicity of A. simplex extracts. Heating at 60 degrees Celsius for 10 min (recommended by some authors) was considered unsafe, as differences in viability between batches were found, with some larvae presenting spontaneous movements in one batch. At higher temperatures (> or = 70 degrees Celsius for > or = 1 min), no movement of the larvae was observed. Antigenic protein Ani s 4 and A. simplex crude antigens were detected in the larvae heated at 94 + or - 1 degrees Celsius for 3 min. This indicates that allergic symptoms could be provoked in previously sensitized consumers, even if the larvae were killed by heat treatment.

Direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid screening of anisakid larvae in seafood.

BACKGROUND: Anisakid larvae are one of the most important pathogenic parasites in marine products; however, simple and rapid analytical techniques for them are still very limited. In this research, based on specific rabbit polyclonal antibodies which were raised against crude extracts of Anisakis larvae, purified by protein A affinity chromatography and labeled with horseradish peroxidase, a direct competitive enzyme-linked immunosorbent assay (ELISA) was developed and validated for detection of anisakid larvae in seafood. RESULTS: The established method exhibited a broad selectivity to Anisakis larvae and Pseudoterranova larvae, and the lowest detection limit to them was estimated to be about 5 parasites kg(-1) in food matrix. Using Pseudopleuronectes yokohamae, Scomberomorus niphonius and Ommastrephes bartrami as samples and within spiking concentrations from 20 to 100 larvae kg(-1), the determination recovery for Anisakis larvae and Pseudoterranova larvae ranged from 77.8% to 107.0%, with relative standard deviations all less than 20%. CONCLUSION: The results allowed us to suggest the established direct competitive ELISA as an effective analytical tool for fast screening of anisakid larvae in sea foods.

Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity.

Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites ("gastroallergic anisakiasis"), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.