Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

How effective is freezing at killing Anisakis simplex, Pseudoterranova krabbei, and P. decipiens larvae? An experimental evaluation of time-temperature conditions.

The consumption of raw or inadequately cooked marine fish can lead to several disorders caused by the ingestion of viable anisakid nematodes. Although anisakid larvae can be killed by subzero temperatures, making freezing an important control measure for this potential health hazard, these parasites can survive freezing under some conditions. Therefore, the aim of the present study was to experimentally evaluate the time-temperature conditions needed to kill Anisakis simplex and Pseudoterranova spp. The effectiveness of freezing was tested on two species of fish: cod, Gadus morhua from the North Atlantic, and herring, Clupea harengus membras from the southern Baltic Sea. Samples, which comprised skinless fillets of cod (n = 40) with visible parasites and whole herring (n = 240), were separately frozen at - 15, - 18, or - 20 °C for 24 h, or at - 20 °C for 48 h in the single-compressor freezer and at - 20, - 25, or - 35 °C for 24 h in the double-compressor freezer. After thawing, parasites were stained with malachite green and examined under the microscope for viability. All A. simplex and Pseudoterranova spp. larvae in cod fillets died at a temperature of - 15 °C or lower. However, freezing did not kill all the A. simplex larvae in whole herring: spontaneous movement of these parasites was observed in samples stored in the single-compressor freezer at - 15, - 18, and - 20 °C over 24 h. Our results demonstrate that the freezing procedure must consider both the capability of the freezing device and the nature of the fish product to ensure consumer safety.

New host records of three juvenile nematodes in Egypt: Anisakis sp. (Type II), Hysterothylacium patagonense (Anisakidae), and Echinocephalus overstreeti (Gnathostomatidae) from the greater lizard fish Saurida undosquamis of the Red Sea.

Three juvenile nematode parasites were collected naturally from 90 (75 %) out of 120 specimens of the marine greater lizard fish Saurida undosquamis captured from water coasts at Hurghada City along the Red Sea in Egypt during the period from September 2013 to April 2014. Worms were identified on the basis of light and scanning electron microscopy. Two of the recovered worms were isolated from the peritoneal cavity of the infected fish around the wall of the stomach as encapsulated larvae. The anisakid juvenile Anisakis sp. (Type II) was characterized by an anteroventrally triangular mouth, with a boring tooth; its postanal tail was rounded, without a terminal mucron or spine. The gnathostomatid Echinocephalus overstreeti was characterized by the presence of a cephalic bulb armed with six transverse rows of spines which were slightly more compact near the anterior end of bulb with maximal separation near the midbulb; the cephalic bulb terminated at a pseudolabia which situated dorsoventrally and reached its greatest width at the posterior one third of the body, The postanal tail terminated at a pointed mucron. The third juvenile species, Hysterothylacium patagonense (Anisakidae), was isolated from the intestine of the infected fish; they are characterized by a small-sized body with a conical tail provided by a nodulose apex, and the anterior end was equipped with three lips. A dorsal lip slightly smaller than the two subventrals left a deep postlabial groove and prominent lateral flanges in between, and the proximal part of each lip was smooth. The three described species were compared morphologically and morphometrically with some of the previously recorded species of the same genus. From this comparison, the similarity and variations between these species were described and concluded that the present study should be considered as a new host record in Egypt.

Anisakiasis in China: the first clinical case report.

Anisakiasis is a parasitic disease acquired by humans when ingesting raw or undercooked fish infected with L3 larvae of the nematode genus Anisakis or Pseudoterranova. Here we report the first case of human anisakiasis in China. The patient, male, 56 years old, Dalian citizen, was admitted into the hospital with vomiting, peripheral umbilicus and abdominal distension, and frequent mucous diarrhea. The patient was examined using an electronic gastroscope, which displayed a parasite residing in the stomach, and subsequently gastroscope-assisted surgery was implemented. A white round worm was removed from the patient and stained. It was identified as L3 larvae of Anisakis. After the removal of the L3 larvae of Anisakis, the inflammation symptoms disappeared. As the first report of clinical case of Anisakis infection in China, the morphology of L3 Anisakis larvae from the patient is described and discussed. We conclude that anisakiasis should be considered in patients who have a habit of eating raw fish and who display associated symptoms.

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor.

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.

Human anisakiasis in Italy: a report of eleven new cases.

The authors report on eleven new human cases of anisakiasis occurring in Italy, and emphasize the importance of the infection in clinical medicine, histopathology and public health. For ten of these cases, the diagnosis was based on histological findings: an eosinophilic granuloma associated with a larva of Anisakis sp. For one of them, the larva was removed from the stomach by endoscopy. Nine of the subjects were from Apulia and two from Molise (regions of southern and central Italy, respectively). Ten of them were surgically treated, and in one case the endoscopical extraction of the parasite resolved the situation. In two cases, the gastric wall was affected, in three the intestinal wall, in a further three the omentum, in one the spleen, and in the final two the mesentery and the epiploic appendix. In all the cases, the parasite was discovered unexpectedly during surgical treatment of the patients for supposed illnesses, which had originally been misdiagnosed. In three cases, the patients were also affected by cancer. Human anisakiasis must be taken into account in the differential diagnosis of acute, abdominal synddromes in subjects who have ingested raw fish or squid a few hours to a few days before the onset of symptoms. Histopathologists should consider the possibility of this parasitic infection when confronted with an eosinophilic granuloma of the digestive tract, mesentery or peritoneum. The incidence of anisakiasis in Italy is probably higher than reported, as some cases might not be diagnosed and others might heal spontaneously.