Effects of anisakid nematodes Anisakis simplex (s.l.), Pseudoterranova decipiens (s.l.) and Contracaecum osculatum (s.l.) on fish and consumer Health.

AbstractThe anisakid nematodes Anisakis simplex (Rudolphi, 1809), Pseudoterranova decipiens (Krabbe, 1878) and Contracaecum osculatum (Rudolphi, 1802) occur as third-stage larvae in marine fish products and may infect consumers ingesting raw or under-cooked fish products. Clinical symptoms associated with the infection, termed anisakidosis, vary from irritation of the oesophagus and stomach, via nausea, vomiting and diarrhoea to severe epigastric and abdominal pain. Third-stage larvae of A. simplex are found in the body cavity, musculature and various organs, P. decipiens occur mainly in the fish musculature (fillet) and C. osculatum larvae reside predominantly in the liver, body cavity, mesenteries and pyloric caeca. Preventive measures, including mechanical removal of worms, heat treatment or freezing to kill worms, are needed in order to reduce the risk of human infections. The anisakid life cycle involves several hosts. A. simplex nematodes use cetaceans (whales) as final hosts whereas P. decipiens and C. osculatum have their adult stage in pinnipeds (seals). Eggs released by worms in these hosts pass with feces to seawater where free-living third-stage larvae hatch from the eggs. Various invertebrates – including euphausiids, copepods and amphipods – feed on these larvae, become infected and serve as intermediate hosts. A range of fish species may serve as transport hosts following ingestion of infected invertebrates and the final stage develops after two additional moults in the stomach of marine mammals which consumed infected fish. Control measures may be implemented to reduce infections of fish stocks and thereby risk of human infections

Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio) Using a GFP-Tagged Yersinia ruckeri

Immersion-vaccines (bacterins) are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr). During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP) and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant) received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC). In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb) Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species

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

Abstract 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

Immunomodulatory effects of excretory/secretory compounds from <i>Contracaecum osculatum</i> larvae in a zebrafish inflammation model

<div><p>Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode <i>Contracaecum osculatum</i> parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of <i>C</i>. <i>osculatum</i> ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.</p></div

Enzyme activity of excretory/secretory proteins produced by <i>C</i>. <i>osculatum</i> third-stage larvae.

<p>Enzyme activity of excretory/secretory proteins produced by <i>C</i>. <i>osculatum</i> third-stage larvae.</p

Evidence of IgE-Mediated Cross-Reactions between <i>Anisakis simplex</i> and <i>Contracaecum osculatum</i> Proteins

Fish consumers may develop allergic reactions following the ingestion of fish products containing nematode larvae within the genus Anisakis. Sensitized patients may cross-react with proteins from insects, mites and mollusks, leading to allergic reactions even in the absence of the offending food. Potential cross-reactivity in Anisakis-allergic patients with larval proteins from other zoonotic parasites present in freshwater and sea fish should be investigated due to an increasing occurrence in certain fish stocks, particularly Contracaecum osculatum. In this work, we evaluated IgE-cross reactions by in vivo (skin prick tests with parasites extracts) and in vitro methods (IgE-ELISA and IgE-immunoblot). In vivo skin prick tests (SPT) proved the reactivity of Anisakis-sensitized patients when exposed to C. osculatum antigens. Sera from Anisakis-sensitized patients confirmed the reaction with somatic antigens (SA) and excretory/secretory proteins (ES) from C. osculatum. Only anecdotal responses were obtained from other freshwater worm parasites. Consequently, it is suggested that Anisakis-sensitized humans, especially patients with high levels of specific anti-Anisakis antibodies, may react to C. osculatum proteins, possibly due to IgE-mediated cross-reactivity

SDS-PAGE of the isolated ES proteins from <i>C</i>. <i>osculatum</i> third-stage larvae; reducing condition, silver staining; Left column: Marker Precision Plus Protein^TM Dual Color Standards; Right column: <i>C</i>. <i>osculatum</i> ES proteins with different molecular weights.

<p>SDS-PAGE of the isolated ES proteins from <i>C</i>. <i>osculatum</i> third-stage larvae; reducing condition, silver staining; Left column: Marker Precision Plus Protein^TM Dual Color Standards; Right column: <i>C</i>. <i>osculatum</i> ES proteins with different molecular weights.</p