Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae.
ABSTRACT: Anisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ?, 12 ?, 22 ?, 37 ?) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ? for Anisakis spp. larvae and 37 ? for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ?, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ?. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ?. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.
Project description:Pseudoterranovosis is a well-known human disease caused by anisakid larvae belonging to the genus Pseudoterranova. Human infection occurs after consuming infected fish. Hence the presence of Pseudoterranova larvae in the flesh of the fish can cause serious losses and problems for the seafood, fishing and fisheries industries. The accurate identification of Pseudoterranova larvae in fish is important, but challenging because the larval stages of a number of different genera, including Pseudoterranova, Terranova and Pulchrascaris, look similar and cannot be differentiated from each other using morphological criteria, hence they are all referred to as Terranova larval type. Given that Terranova larval types in seafood are not necessarily Pseudoterranova and may not be dangerous, the aim of the present study was to investigate the occurrence of Terranova larval types in Australian marine fish and to determine their specific identity. A total of 137 fish belonging to 45 species were examined. Terranova larval types were found in 13 species, some of which were popular edible fish in Australia. The sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2 respectively) of the Terranova larvae in the present study showed a high degree of similarity suggesting that they all belong to the same species. Due to the lack of a comparable sequence data of a well identified adult in the GenBank database the specific identity of Terranova larval type in the present study remains unknown. The sequence of the ITS regions of the Terranova larval type in the present study and those of Pseudoterranova spp. available in GenBank are significantly different, suggesting that larvae found in the present study do not belong to the genus Pseudoterranova, which is zoonotic. This study does not rule out the presence of Pseudoterranova larvae in Australian fish as Pseudoterranova decipiens E has been reported in adult form from seals in Antarctica and it is known that they have seasonal presence in Australian southern coasts. The genetic distinction of Terranova larval type in the present study from Pseudoterranova spp. along with the presence of more species of elasmobranchs in Australian waters (definitive hosts of Terranova spp. and Pulchrascaris spp.) than seals (definitive hosts of Pseudoterranova spp.) suggest that Terranova larval type in the present study belong to either genus Terranova or Pulchrascaris, which are not known to cause disease in humans. The present study provides essential information that could be helpful to identify Australian Terranova larval types in future studies. Examination and characterisation of further specimens, especially adults of Terranova and Pulchrascaris, is necessary to fully elucidate the identity of these larvae.
Project description:Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.
Project description:Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France.A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient's nose after she had eaten raw sea bream.These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France.
Project description:Anisakidosis is a zoonotic parasitosis induced by members of the family Anisakidae. The anisakid genera includes <i>Anisakis, Pseudoterranova, Hysterothylacium</i> and <i>Contracaecum</i>. The final definitive hosts of these nematodes are marine mammals with a complex life cycle. These nematode parasites use different crustaceans and fish species as intermediate or paratenic hosts and humans are accidental hosts. Human anisakiasis, the infections caused by members of the genus <i>Anisakis,</i> occurs<i>,</i> when seafoods, particularly fish, contaminated with the infective stage (third stage larvae [L3]) of this parasite, are consumed. Pseudoterranovosis, on the other hand is induced by members of the genus <i>Pseudoterranova.</i> These two genera of anisakids have been implicated in human disease globally. There is a rise in reports of gastro-intestinal infections accompanied by allergic reactions caused by <i>Anisakis simplex</i> and <i>Anisakis pegreffii</i>. This review provides an update on current knowledge on <i>Anisakis</i> as a food-borne parasite with special focus on the increasingly reported diversity of fish and crustacean hosts, allergens and immunological cross-reactivity with invertebrate proteins rendering this parasite a significant public health issue.
Project description:Anisakiasis is a zoonotic disease induced by anisakid nematodes, and endoscopic inspection is used for a diagnosis or remedy for it. Anisakis simplex, Anisakis physeteris, and Pseudoterranova decipiens had been reported to be the major species causing human infections, particularly, in Japan. However, in Korea, recent studies strongly suggested that Anisakis pegreffii is the major species of human infections. To support this suggestion, we collected anisakid larvae (n=20) from 20 human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.
Project description:In this work a total of 949 fish samples were analysed for the identification of nematode larvae belonging to the Anisakidae family. Biomolecular application for the identification of Anisakidae larvae can be an optimal instrument for the traceability of fish products, described on the Reg. EC 178/2002. Results confirm a correlation between geographical distribution of fishes and presence of specific Anisakid larvae. FAO 37 zone (Mediterranean sea) showed a prevailing distribution of Anisakis pegreffii and a minimal presence of A. simplex s.s. in hybrid form with Anisakis pegreffii. FAO 27 zone showed a prevailing distribution of A. simplex s.s. in fish like Brosme (Brosme brosme) and infestation prevalence of Pseudoterranova krabbei and P. decipiens s.s. in Gadus morhua. Obtained results validate the hypothesis that molecular biology methods for identifying Anisakidae larvae are effective traceability markers of fish products.
Project description:<h4>Background</h4>In the marine environment, transitional zones between major water masses harbour high biodiversity, mostly due to their productivity and by containing representatives of species characteristic of adjacent communities. With the aim of assessing the value of larval Anisakis as zoogeographical indicators in a transitional zone between subtropical and sub-Antarctic marine currents, larvae obtained from Zenopsis conchifer were genetically identified. Larvae from Pagrus pagrus and Merluccius hubbsi from two adjacent zoogeographical provinces were also sequenced.<h4>Results</h4>Four species were genetically identified in the whole sample, including Anisakis typica, A. pegreffii, A. berlandi and a probably new species related to A. paggiae. Anisakis typica and A. pegreffii were identified as indicators of tropical/subtropical and sub-Antarctic waters, respectively, and their presence evidenced the transitional conditions of the region. Multivariate analyses on prevalence and mean abundance of Anisakis spp. of 18 samples represented by 9 fish species caught south of 35°S determined that host trophic level and locality of capture were the main drivers of the distribution of parasites across zoogeographical units in the South-West Atlantic.<h4>Conclusions</h4>Most samples followed a clear zoogeographical pattern, but the sample of Z. conchifer, composed mostly of A. typica, was an exception. This finding suggests that population parameters of A. typica and A. pegreffii could differ enough to be considered as a surrogates of the identity of larvae parasitizing a given host population and, therefore, a step forward the validation of the use of larval Anisakis as biological indicators for studies on host zoogeography.
Project description:BACKGROUND:Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus Anisakis. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of Anisakis are detected. Thus, molecular-based diagnostic approaches are warranted. METHOD:We have developed a PCR method that amplifies the DNA of Anisakis spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA cox2 gene were used and sequence analysis was performed according to the procedures already established for species of Anisakis. RESULTS:The sequence obtained (629 bp) was compared with those of the other species of Anisakis which have so far been genetically characterized and with sequences obtained from larval stages of Anisakis collected from the Mediterranean fish Engraulis encrasicolus. This enabled the genetic identification of the larva in the human tissue as A. pegreffii. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin. CONCLUSION:The case of human anisakiasis presented reinforces the pathological significance of the species A. pegreffii to humans. The molecular/genetic methodological approach based on mtDNA cox2 sequence analysis, described here, can allow easy and rapid identification of Anisakis spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.
Project description:Anisakiasis in humans in South Korea has been considered to be caused exclusively by the larvae of Anisakis simplex sensu stricto and Pseudoterranova decipiens. Recently, however, DNA sequencing of larvae from 15 of 16 anisakiasis patients confirmed the cause to be Anisakis pegreffii infection. Molecular analysis should be performed for all extracted larvae.
Project description:Ascaridoids are one of the main parasitic hazards in commercial fish. Candling is the current industrial screening method whereby visible ascaridoid larvae are detected on a light table and manually removed. The aim of this study was to assess the sensitivity (Se) and negative predictive value (NPV) of this method. To make targeted recommendations to the fish industry, the Se was calculated per fish part, larval genus, and fish species. All fish parts (n = 615) were first candled, and larvae were collected, followed by enzymatic digestion to recover the remaining larvae. A fish part was considered positive if at least one larva was detected using candling and/or enzymatic digestion, with both methods combined as reference standard. The overall Se of candling was 31% (95% CI 23-41%) and NPV was 87% (95% CI 85-90%). The Se increased with higher numbers of larvae/100 g infected muscle. A low NPV was found for the belly flaps, therefore we either advise the removal or proper freezing of this part. Lastly, the Se and larval recovery was the highest for the darker and larger Pseudoterranova spp. larvae. Due to the low overall efficacy of candling, further assessment of its cost-benefit and impact on consumers' health risk should be conducted.