辽宁省海鱼异尖线虫感染调查及分子鉴定

目的了解辽宁省市售海洋鱼类异尖线虫感染情况,并对异尖线虫Ⅲ期幼虫进行分子鉴定,确定感染异尖线虫虫种。方法采用直接剖检法从市售海鱼中检出异尖线虫Ⅲ期幼虫后,在显微镜下观察形态进行初步鉴定;提取异尖线虫虫体总DNA,采用通用引物扩增异尖线虫核糖体DNA内转录间隔区(ITS)序列,并对其ITS1和ITS2序列进行序列比对和进化分析。结果采集222份市售海鱼样品进行剖检,共有70份检出异尖线虫幼虫,检出率为31.53%,其中小黄花鱼和带鱼的检出率较高,感染度最高为233条/份。测序结果经序列比对和进化分析,结果显示检出的异尖线虫幼虫分别为异尖线虫属和宫脂线虫属的5种异尖线虫,包括简单异尖线虫(Anisakis simplex)、派氏异尖线虫(Anisakis pegreffii)、典型异尖线虫(Anisakis typical)、内弯宫脂线虫(Hysterothylcaium aduncum)和厦门宫脂线虫(Hysterothylacium amoyense)。结论辽宁省市售海洋鱼类异尖线虫感染情况较严重,且感染的异尖线虫种类多样,其感染优势种为派氏异尖线虫。     

福建省5地区海鱼体Ⅲ期异尖线虫幼虫风险监测

目的 调查福建省5地区海鱼感染Ⅲ期异尖线虫（Anisakis spp.）幼虫的情况。方法 2021年10月至2022年5月期间,采购各海鲜市场不同品种海鱼,直接剖检海鱼或人工消化内脏和肌肉查找、分离Ⅲ期异尖线虫幼虫,经光学显微镜镜检虫种。统计分析福建省5地区和不同鱼种中异尖线虫幼虫的感染情况。结果 共剖检18种133尾海鱼,其中12种68尾海鱼感染异尖线虫幼虫,检获虫体2 214条,海鱼异尖线虫幼虫品种检出率、总感染率、感染度分别为66.7%（12/18）、51.1%（68/133）、32.6条/尾（2 214/68）,海鱼品种中单尾带鱼的感染情况最严重,为416条/尾;未检出异尖线虫幼虫的海鱼品种有黄翅鱼、黄瓜鱼、东星斑、墨鱼、白鲑、鳊鲈。从地区分布看,长乐地区感染率最高（66.7%）,各地区不同品种海鱼检出率芗城最高（71.4%）。结论 福建省5地区海鱼体Ⅲ期异尖线虫幼虫感染严重,海鱼中异尖线虫幼虫的分布密度高,各地区异尖线虫幼虫检出率较高,福建省5地区居民具有高度感染异尖线虫的风险。     

黄海海域鲐鱼感染异尖线虫三期幼虫初步调查

目的 了解异尖线虫三期幼虫在黄海海域鲐鱼体内的感染情况.方法 2010年5月-2011年1月抽取产自黄海海域的鲜活鲐鱼(Pneumatophorus japonicus)113尾(15 ～ 20尾/月),分别检查、记录每个个体的体腔,内脏器官,背部、腹部、尾部鱼肉等感染线虫的情况,将所获线虫经乳酸酚透明液透明后于光镜下进行常规的形态学鉴定,并随机取192条(30 ～35条/月)线虫全基因组进行聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析鉴定.结果 有98尾鲐鱼感染异尖属线虫三期幼虫,感染率为86.7％;共检出4 770条异尖线虫,感染强度为5 ～194条/尾不等,平均感染强度为42.2条/尾;体腔和内脏器官感染虫数占鱼体总感染虫数的88.7％(4 229/4770),腹部肌肉感染虫数占鱼全身肌肉总感染虫数的87.2％ (470/539);感染强度随鱼体重增加而增大;所有检出的异尖线虫依据其形态鉴定均为异尖属线虫I型幼虫;192条异尖属线虫分别为派氏异尖线虫191条、典型异尖线虫1条.结论 黄海海域鲐鱼中异尖属线虫三期幼虫的感染情况比较严重,且感染程度与季节、鲐鱼体重、身体部位有一定关系,检出的异尖属线虫三期幼虫经RFLP分析以派氏异尖线虫为主,这对今后卫生检疫与渔业生产加工出口有一定的提示和指导意义.     

高温热处理对鲭鱼异尖线虫基因和蛋白质的影响

为探讨热处理后鲭鱼中异尖线虫幼虫能否被检测、幼虫残留体蛋白质是否被破坏及其对于消费者的致敏风险。选用高温灭菌和油炸两种高温加工方法处理异尖线虫体蛋白质,利用聚合酶链式反应和实时荧光定量聚合酶链式反应检测其基因变化,采用圆二色谱及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳观察异尖线虫全虫体蛋白质变化情况,借用蛋白免疫印迹技术测定热处理过敏蛋白抗原性变化。结果表明,高温热处理后,异尖线虫幼虫DNA和蛋白质被破坏,抗原性降低。高温灭菌处理60 min过敏蛋白抗原性下降98.2%。实时荧光定量PCR用于检测热加工食品中异尖线虫幼虫残留非常有效。油炸处理对异尖线虫体蛋白质的破坏作用比高温灭菌方法更为显著。实时荧光定量PCR能够快速检测出鲭鱼体内异尖线虫幼虫残留,高温灭菌和油炸热加工方法都能有效破坏残留的异尖线虫体蛋白质,从而降低过敏风险。     

简单异尖线虫重组酶聚合酶等温扩增检测方法的建立

为建立基于重组酶聚合酶等温扩增（RPA）技术的简单异尖线虫检测方法，本研究根据简单异尖线虫rDNA的ITS2基因序列设计特异引物，建立了简单异尖线虫的RPA检测方法。通过对反应体系的优化确定最优反应温度为40℃和最优反应时间为25 min。特异性检测结果显示，本研究建立的简单异尖线虫RPA检测方法与典型异尖线虫、抹香鲸异尖线虫、宫脂线虫、伪地新线虫和对盲囊线虫无交叉反应；该方法对简单异尖线虫基因组DNA最低检出限为10 pg/μL；人工污染检测结果表明，该方法能在100 g鲈鱼肉中检出单条简单异尖虫的混入污染，应用效果良好。因此，本研究所建立的简单异尖线虫RPA检测方法特异性强、灵敏度高、反应快速、无需昂贵设备，可应用于基层单位和现场快速检测。     

进境水产品中典型异尖线虫恒温实时荧光检测法的建立与应用

本研究建立了一套检测进境水产品中典型异尖线虫DNA的恒温实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,根据LAMP方法原理,针对典型异尖线虫ITS2区域设计三套引物,特异性识别靶标基因。对比验证了典型异尖线虫、简单异尖线虫、短棘异尖线虫、抹香鲸异尖线虫、Anisakis nascettii、宫脂线虫、对盲囊线虫和颚口线虫的引物特异性;研究了模板浓度1 ng/μL至10 ag/μL的LAMP反应灵敏度,比传统的PCR方法高100倍;并在最低检测限1 fg/μL的质粒模板进行15次重复试验;对来自不同热带国家和地区的进境的41个实际样品进行测试,与传统PCR方法进行比较,假阳性率为0。恒温实时荧光快速检测方法适用于特异性检测典型异尖线虫。     

恒温实时荧光法快速检测简单异尖线虫方法的建立

为了快速鉴定简单异尖线虫,本研究建立了一套检测简单异尖线虫DNA的恒温实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,根据LAMP方法原理,针对简单异尖线虫ITS2区域设计引物特异性识别靶标基因。进行了特异性、灵敏度、重复性和实际样品的测试,并与传统的PCR方法进行比较。结果表明,该方法能够特异性扩增简单异尖线虫DNA,对含有简单异尖线虫ITS2目的基因片段的质粒DNA检测限为1 fg/μL,灵敏度比传统的PCR方法高100倍,重复性良好,对实际样品进行检测,与传统的PCR测序方法结果相符。本研究建立的恒温实时荧光快速检测方法适用于特异性检测简单异尖线虫。     

河南省烟草根结线虫种类的PCR鉴定与种群分析

根结线虫病是河南烟草生产上的重要病害,了解根结线虫病病原种类是培育和选用抗病品种的基础。为了弄清河南省烟田根结线虫种群分布及比例,2012年采集了河南省烟叶主产区的8个县共12份烟草根结线虫样本。通过对线虫样本的PCR分子检测和雌成虫会阴花纹的形态鉴定,结果表明,检出烟草根结线虫4种,其中南方根结线虫（Meloidogyne incognita）检出率为55.83% ,花生根结线虫（Meloidogyne arenaria）23.33% ,北方根结线虫（Meloidogyne hapla）17.50% ;爪哇根结线虫（Meloidogyne javanica）3.33%。种群分析表明河南地区仍以南方根结线虫为主,同时其他种类根结线虫的发生危害比例在上升。所有样本均为混合侵染,南方根结线虫（Meloidogyne incognita）、花生根结线虫（Meloidogyne arenaria）和北方根结线虫（Meloidogyne hapla）在河南广泛分布、具有普遍性,爪哇根结线虫（Meloidogyne javanica）在河南分布没有规律性。本研究结果为河南烟区制订根结线虫防治规划和抗线虫病育种提供了依据。      

烟草根结线虫病不同防控措施的田间筛选

为筛选防控烟草根结线虫病的措施,通过田间试验研究了5种植物、1种昆虫病原线虫、2种生防真菌对烤烟农艺性状、产量、产值及烟草根结线虫病防治效果的影响。结果表明,烤烟间作菽麻、间作猪屎豆,施用异小杆线虫和施用淡紫拟青霉在烤烟农艺性状、产量、产值及整个生育期对烟草根结线虫防治效果综合表现较好。除药剂对照外,整个生育期内施用淡紫拟青霉防效最好,平均防效61.2%;间作菽麻次之,平均防效60.4%;其次为1.46×10⁸Js/hm²的异小杆线虫,平均防效53.3%;最差为间作猪屎豆,平均防效为46.2%。     

烟草根结线虫的综合防治

主要介绍了巴氏杆菌的生物学特性、在武隆县的分布情况 ,以及利用巴氏杆菌防治根结线虫病试验结果。同时 ,还介绍了利用与水稻、玉米轮作等措施防治烟草根结线虫病。示范田试验结果表明 ,采用巴氏杆菌和轮作等措施较好地控制了根结线虫对烟草的为害     

Anisakis Infection and Allergy in Humans

Compared with other well-studied parasitic diseases, fish-borne parasitic zoonoses do not get enough attention, especially because these zoonoses have been limited for the most part to populations living in low- and middle-income countries in Europe. Human fishery product-borne parasitic diseases caused by nematodes are the results of infection following ingestion of viable parasites, or as allergic reactions against parasite antigens. With the globalization of the seafood industry, the risk of humans acquiring anisakiasis in developed countries appears to be underestimated. For allergy, the only implicated parasite in fishery products is the nematode Anisakis simplex.     

Compounds of parasitic roundworm absorbing in the visible region: target molecules for detection of roundworm in Atlantic cod

The objective of this study was to contribute to the development of technology that will be able to replace manual operations in processing of fish fillets. Removal of parasites, black lining, remnants of skin, and bloodstains are costly and time-consuming operations to the fish processing industry. The presence of parasites in fish products tends to spoil consumers' appetites. Recent reports questioning the safety of eating cod infected with parasites might lower consumer acceptance of seafood. Presently, parasites are detected and removed manually. An average efficiency of about 75% under commercial conditions has been reported. In this study, we focused on biochemical differences between cod muscle and the prevalent anisakine nematode species (Anisakis simplex and Pseudoterranova decipiens) infecting Atlantic cod (Gadus morhua). Using reversed phase high-performance liquid chromatography equipped with a photodiode array detector, substances absorbing in the range 300 to 600 nm were identified in extracts from parasite material. These substances were not detected in extracts from cod tissue. Significant biochemical differences between cod muscle and parasite material have thus been demonstrated.     

Molecular identification and population dynamic of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) isolated from the European anchovy (Engraulis encrasicolus L.) in the Adriatic Sea

Anchovy Engraulis encrasicolus (L.) is a coastal pelagic and euryhaline species that represents the only European species of the family Engraulidae, with a widespread distribution. In Croatia, it is marketed fresh, frozen, salted or marinated and mainly exported to Italy and Spain, however Anisakis sp. larval infection is frequently the reason for border rejection. Since it is known that the prevalence and intensity of Anisakis infection varies with fish species, fishing area and season, the aim of our study was to identify Anisakis sp. parasitizing European anchovy and infer its population dynamic through a 2.5-year period. Larvae were found coiled and encysted on the external wall of intestine (94%) and reproductive organs (6%), rarely in fillets. Prevalence was 76.1% (95% confidence limits 74.51-77.56%), mean abundance 6.59 (bootstrap 95% confidence limits 5.81-7.26) and mean intensity 8.67 (bootstrap 95% confidence limits 7.82-9.35). The partial CO2 mitochondrial DNA sequence of the isolated anisakids confirmed clustering of the anchovy parasite within A. pegreffii sister group. Parasite population structure showed plasticity inferred by fishing ground, sampling year and fish gender and size. Compared to anisakid prevalence/abundance in other fish, the European anchovy in the Adriatic Sea represents a moderately high-infected paratenic host, although in the Mediterranean and Atlantic waters, anchovies have shown strikingly lesser values of prevalence. Since this host represents one of the most attractive Mediterranean fisheries products traditionally consumed without thermal preparation that in any case would not disrupt larval antigenicity and prevent human allergies, and given the high prevalence of the anisakid within the host, it is necessary to include anchovy into more firm risk assessment frames in order to develop measures that will support the safe alimentary production and consumption of seafood.     

A quantitative sandwich ELISA for the detection of <Emphasis Type="Italic">Anisakis simplex</Emphasis> protein in seafood

There is a growing awareness by consumers and food safety authorities regarding the possible presence of parasites or parasite-related potentially hazardous substances in seafood. Anisakis simplex is among the most frequently occurring parasites in wild-caught marine fish. Except for various visual inspection techniques and PCR-based methods for the detection of more or less intact worms or parasite DNA, respectively, there are at present no validated methods for the quantification of A. simplex proteins in processed fish products. This work describes the development and validation of a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification and analysis of proteins from A. simplex in seafood products. The ELISA is based on a polyclonal rabbit anti-A. simplex antibody for capture and a biotinylated conjugate of the same antibody for detection. The ELISA is specific for A. simplex and does not cross-react with other species. Recoveries ranged from 72–101% in typical food matrixes, while intra- and inter-assay precisions were <11 and <25%, respectively. With a limit of detection of 1.1 μg A. simplex protein/g of sample, the sensitivity of the A. simplex sandwich ELISA appears to be sufficient to detect even low levels in seafood products.     

Anisakis antigens detected in fish muscle infested with Anisakis simplex L3

Anisakis simplex is a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E-mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakis larvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex.     

Anisakis simplex larva killed by high-hydrostatic-pressure processing

Anisakis simplex is a common nematode parasite present in many marine fish, including finfish and squid. It can pose a public health problem if it is not destroyed during food processing. Anisakis larvae were isolated from fish tissue, and their survival of high-pressure treatments in distilled water and physiological isotonic solution was assayed. Treatment at a pressure of 200 MPa for 10 min at a temperature between 0 and 15 degrees C kills all Anisakis larvae, with a lack of motility being used as an indicator of larval death. Lower pressures can be successfully employed down to 140 MPa, but with lower pressures, the treatment time must be increased by up to I h to kill all larvae. Meanwhile, most larvae treated for >10 min at pressures of >120 MPa were dead, with the autofluorescence method being used to determine death. Cycles of compression and decompression increase the destruction of larvae compared with a single pressure treatment for a similar treatment time. Our results indicate that high-pressure treatment is an alternative nonthermal method for killing this nematode. The possible mechanism of death and damage by pressure is discussed, and uses for this treatment in food processing are suggested.     

Anisakid larvae in the musculature of the Argentinean hake, Merluccius hubbsi

We report the infection levels of third-stage anisakid larva in the muscle of the Argentinean hake, Merluccius hubbsi, in relation to fish size and location in the musculature. The musculature of 42 hake was separated into hypaxial (ventral) and epiaxial (dorsal) parts and surveyed for nematode larvae. Two anisakid species were detected: Anisakis sp. (prevalence, 52.4%; mean +/- SD abundance, 1.2 +/- 1.7) and Pseudoterranova sp. (prevalence, 9.5%; mean +/- SD abundance, 0.2 +/- 0.7). Since the fish were gutted after capture, the occurrence of anisakids in the flesh indicates that the worms had migrated into the muscle before capture. The number of Anisakis sp. in muscle was not correlated with fish length or weight. Therefore, fish size cannot be used as a predictor of parasite loads in the muscle. Only one Anisakis sp. and one Pseudoterranova sp. appeared in the epiaxial musculature. The density of Anisakis sp. in the hypaxial muscles was significantly higher than that in the epiaxial ones. This suggests that removal of the hypaxial musculature can reduce the risk of anisakid-induced allergies and gastrointestinal anisakidoses among consumers.     

Genetic relatedness between Listeria monocytogenes isolates from seafood and humans using PFGE and REP-PCR

Listeria monocytogenes has been isolated from catfish and various non-catfish seafoods. Despite progress that was made to understand the relationship between L. monocytogenes isolated from seafood and humans, no study has emphasized the genetic relatedness between catfish and non-catfish seafood and human isolates. The objectives of this study were to (1) investigate the genetic relationship between L. monocytogenes isolates from catfish, non-catfish seafood and humans using pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic element-based PCR (REP-PCR) linking with serotypes; and (2) compare the distinct capabilities of these methods. The genetic relationship among 36 catfish, 35 non-catfish seafood, and 57 human isolates were analyzed using Bionumerics software. Among a total of 128 isolates, only two subtypes by PFGE with ApaI-digestion, one subtype by PFGE with AscI-digestion and three subtypes by REP-PCR were shared by non-catfish seafood or catfish with human isolates. Although most of the non-catfish seafood and catfish isolates were genetically distinct from human isolates, the human isolates used in this study may not be truly representative of all clinical isolates. Serotype 4b was dominant in human isolates, whereas serotypes 3b, 1/2a, and 1/2b and serotypes 4b, 1/2a and 1/2b were commonly found in catfish and non-catfish seafood, respectively. Furthermore, 97, 87 and 94 subtypes of L. monocytogenes were revealed using the ApaI-digestion, the AscI-digestion, and the REP-PCR, respectively. Their respective discriminatory indexes were 0.994, 0.990 and 0.994. Distinct genetic groups based on the difference of flagella antigens were observed in all three methods. The study suggests that the REP-PCR possesses a similar discriminatory ability as the PFGE for subtyping L. monocytogenes. Therefore, the REP-PCR that is rapid and less expensive could be considered as an alternative method for subtyping L. monocytogenes.     

水产品油脂的研究进展

水产品油脂中含有大量有益于人类健康的药用活性成分,具有降血脂、降血压、抗癌等多种独特功能。目前水产品油脂的研究大多集中在鱼类油脂的开发上,而对其他水产品油脂的研究相对较少。为此,立足于国内外水产品油脂的研究成果,对水产品油脂的研究开发做一个简要的阐述,以适应开发利用海洋资源特别是水产油脂资源的迫切要求。