用PCR和PCR-RFLP方法检测和鉴定进口海鱼中的异尖线虫幼虫

应用PCR和PCR-RFLP方法对厦门口岸进口的各种海鱼中的异尖线虫幼虫进行检测鉴定。结果在带鱼、竹荚鱼、金线鱼、大眼鲷和白姑鱼等海鱼中发现了简单异尖线虫、典型异尖线虫、对盲囊线虫和针回线虫等4种异尖线虫。带鱼和竹荚鱼的异尖线虫感染率达100%,竹荚鱼异尖线虫感染强度最高,白姑鱼感染率和感染强度相对较低。少数海鱼同时感染2种以上异尖线虫,从带鱼中同时检出简单异尖线虫和针蛔线虫,从竹荚鱼中检出简单异尖线虫、对盲囊线虫和典型异尖线虫。内切酶实验结果表明,应用限制性内切酶HinfI有效鉴别上述4种异尖线虫。     

用PCR-RFLP法在海鱼中检出简单异尖线虫幼虫

2008年底至2010年初从进境的竹荚鱼、带鱼等海鱼中多次检出体色米黄色、长16～19mm的一种线虫。用异尖线虫的通用引物和特异性引物对提取的核酸进行PCR扩增，并对扩增产物进行测序和酶切。确定该线虫为简单异尖线虫。     

连云港市售海鱼异尖线虫感染情况的初步调查

异尖线虫病是一种危害严重的食源性人兽共患寄生虫病,人类由于生食或半生食含有异尖线虫Ⅲ期幼虫的海鱼而引起感染,严重威胁人类健康。据报道我国沿海地区海鱼仍有异尖线虫感染,为了解我国黄海海域连云港市售海鱼异尖线虫的寄生情况,于2017年3―10月对该地市售海鱼的感染情况进行了形态学观察与PCR鉴定。随机选取本地海鲜市场采购的不同种类海鱼,剖检鱼体、分离其内脏和肌肉中Ⅲ期幼虫;应用多重PCR方法进行分子生物学鉴定。结果显示,19种海鱼中有11种被检出有异尖线虫感染(57.89%),海鱼的总检出率为37.89%,带鱼、小黄花鱼、梭鱼、黑鱼检出率最高为100.00%,鲳鱼、鲈鱼、鮟鱇鱼、海鲫鱼、沙丁鱼、黄姑鱼、白姑鱼、巴浪鱼均未检出异尖线虫;阳性海鱼平均感染强度为5.24条/尾,总体平均感染强度为1.99条/尾,带鱼感染强度最高为10.60条/尾;感染部位以肠和肠系膜最为严重,占虫体数67.55%。多重PCR鉴定本次获得的虫体为简单异尖线虫、派氏异尖线虫、伪地新线虫和内湾宫脂线虫,其中简单异尖线虫为优势种。结果表明,连云港市售海鱼异尖线虫具有较高的感染率和感染强度,本次调查为连云港市人群预防和控制该病提供了理论依据,对异尖线虫病的流行病学研究具有重要意义。     

进境海鱼异尖线虫幼虫感染情况调查

对来自15个国家和地区的78批次、31种、共255尾海鱼进行了异尖线虫幼虫的检验,100尾海鱼检出异尖线虫幼虫感染,总感染率为39.2%(100/255).16种海鱼检出异尖线虫幼虫,占所检鱼种的51.6%(16/31).共检获异尖线虫幼虫1 554条,平均感染强度为15.5条/尾.在体重≤100 g的海鱼中感染率最高...     

舟山口岸进出境海鱼异尖线虫幼虫感染调查

为了解舟山口岸进出境鱼类自然感染异尖线虫情况,取进出境各种鱼类1 152尾,解剖鱼体进行检查。检获虫体清洗后,用70%酒精固定,在显微镜下鉴定。结果显示检出645尾感染异尖线虫幼虫,总感染率为55.99%(645/1 152),发现检出10 065条异尖线虫幼虫,总感染强度为15.60(10 065/645)。调查结果表明,进出境鱼类异尖线虫的感染率较高,生食海鱼时,应注意对异尖线虫病的预防。     

乌鲁木齐市售海鱼中首次检出异尖线虫

为配合检疫部门检查,2017年8月对乌鲁木齐市市售海鱼随机采样,收集寄生虫并通过形态学和分子生物学进行鉴定。从鳕鱼块中发现5只线虫,虫体无胃盲囊和肠盲囊,黑色圆柱形的胃与"Y"型的肠管以斜线相连,尾短且有一圆塔形的尾突。PCR扩增ITS基因片段获得924 bp的基因序列,测序后经BLAST比对分析显示,与简单异尖线虫(Anisakis simplex)(GenBank登录号:KY275505.1)相似性达99%,确定分离出来的虫体为简单异尖线虫。本研究在远离海洋的乌鲁木齐市发现随海鱼运输携带到新疆的异尖线虫,系异尖属线虫在新疆的首次发现和报道。     

辽宁西部海域主要食用鱼类寄生虫感染情况初报

采用鱼类全身性寄生虫学检查法,在辽宁西部海域随机抽检主要食用鱼类19种共计769尾,其中养殖鱼类4种202尾、野生鱼类15种567尾,发现该海域19种食用鱼类感染体内外寄生虫15种,分别隶属于6纲12科15属。在统计感染率和感染强度的基础上,确定优势虫种为异尖线虫科的3属幼虫:宫脂线虫属幼虫、对盲囊线虫属幼虫、异尖线虫属幼虫,野生和养殖鱼类均有较高的感染率,而针对4种养殖鱼类而言,尤其是牙鲆和大菱鲆,其优势虫种为指状拟舟虫和刺激隐核虫,在各个鱼类养殖场中均有较高的感染率和发病率。     

PCR-RFLP和特异PCR技术用于鲁道夫对盲囊线虫鉴定的研究

本研究运用新建立的Contracaecum rudolphii姊妹种的PCR-RFLP和特异PCR鉴定方法对来自我国青海湖的鲁道夫对盲囊线虫进行分子鉴定。经PCR-RFLP分析，来自我国青海湖的鲁道夫对盲囊线虫的两个样品的带型与C．rudolphiiB的酶切带型一致。经特异PCR扩增，C．rudolphii B的特异引物能从我国鲁道夫对盲囊线虫中扩增出目的片段，与对照C．rudolphii B的代表样品扩增片段大小相同。该两种方法试验结果都说明来自我国青海湖的对盲囊线虫为C．rudolphii B，与PCR-SSCP方法鉴定结果一致。本次研究结果及所建立的分子分类学方法为我国异尖线虫的进一步研究奠定了基础。     

在进境船舶中检出鱼异尖线虫

异尖线虫是寄生于海水鱼类的一种寄生虫,可引起人畜共患传染病-异尖线虫病,该病属于我国禁止进境的动物二类寄生虫病.本病为海洋自然疫源性疾病,周而复始地在海洋中的哺乳类、甲壳类、鱼类中发生.人类主要是生食海鱼,或将鱼的内脏用于喂养畜禽以及其他经济动物,而导致食入异尖线虫引起感染发病.本病的发生与寄生异尖科线虫的鱼类宿主分布及人们的饮食习惯有关,本病呈世界性分布,主要集中于北太平洋和北大西洋沿岸及其岛屿.而与我国相邻的日本、韩国、俄罗斯等国均有发生.1992年6月8日我国农业部根据《中华人民共和国进出境动植物检疫法》第18条规定,制定公布了《进境动物一、二类传染病、寄生虫病名录》,基于异尖线虫病对我国的公共卫生有着极其重要影响,首次将海水鱼类中的异尖线虫病列入我国二类传染病、寄生虫病名录,这也是其中唯一的寄生虫病.自依据《中华人民共和国进出境动植物检疫法》实施检疫以来,许多口岸纷纷从进口的或进境国际航行船舶食品舱中的海水鱼类体内检出异尖线虫,充分说明了必须加强对海水鱼类异尖线虫的检疫,防止本病传入我国,危害人体健康和水产养殖业的发展.连云港口岸于1998年5月首次从塞浦路斯籍进境国际航行船舶中的鳕鱼体内检出异尖线虫,之后在掌握了异尖线虫的正确检疫方法后,大大地提高了本病的检出率,到1999年11月30日止共检出异尖线虫139艘次,占我局一、二类、三类检疫疫情总数的54.3%.下面将1998～1999年连云港口岸进境船舶鱼异尖线虫的检出情况作一简要分析.     

PCR-RFLP技术鉴别简单异尖线虫姊妹种的研究

为建立简单异尖线虫姊妹种分子生物学检测体系,以实现对其快速准确的鉴定,本实验对简单异尖线虫单条线虫总DNA进行PCR扩增,获得其rDNA-ITS序列,测序后与基因库中DNA序列比较,并根据rDNA-ITS序列比较结果鉴定线虫的种类,最后通过酶切技术获得线虫的PCR-RFLP图谱并以此作为线虫鉴定的分子标签。     

Parasitism between Anisakis simplex (Nematoda: Anisakidae) third-stage larvae and the spotted mackerel Scomber australasicus with regard to the application of stock identification

The nematode fauna of 369 spotted mackerel of the species Scomber australasicus, collected off the northeastern Taiwanese coast of the northwestern Pacific, was investigated monthly from April 2004 to March 2005. The following nematode species were recorded: Anisakis simplex complex, Hysterothylacium aduncum, Porrocaecum decipiens and Raphidascaris trichiuri. The seasonal variation in the infection with A. simplex third stage larva (L3) was studied throughout the 12 months. The prevalence of A. simplex L3 recorded for total fish samples was 93.6%, varying between 86.7 and 100%. There was an increase in the abundance of this nematode in spring, with the peak occurring in April. To reveal whether intrinsic factors of the spotted mackerel host contributed to infection with this nematode, fish were grouped according to their body weight, age and gonad development (reported as gonadoosomatic index, GSI), respectively, and infection parameters (i.e., prevalence, abundance and intensity) were analyzed. Results showed that abundance was significantly higher in both larger (>450 g) and older (>3 years old) fish. The gonad development of the host fish was not correlated with the intensity of the larval infection in both female and male fish. Two distinct Anisakis species were identified by PCR-RFLP, namely A. pegreffii and a recombinant genotype of A. pegreffii and A. simplex sensu stricto. These species occurred with frequencies of 97% and 3%, respectively. The usefulness of using parasites as biomarkers for spotted mackerel stock identification around Taiwanese waters was confirmed herein. A second group of 58 spotted mackerel were obtained from the coastal waters off southwestern Taiwan. In addition to the two species, A. pegreffii and the recombinant one, which were found with frequencies of 63% and 9%, respectively, an additional Anisakis species A. typica was identified with a frequency of 28% from these fish. Two spotted mackerel stocks could thus be identified based on their infrastructure of Anisakis species community and their frequency. To the best of our knowledge, this is the first report of stock identification of spotted mackerel using endoparasite biomarkers.     

Anisakidae larval infection in fish fillets sold in Belgium.

Fish fillets (n = 1760) sold on the Belgium market were examined for Anisakidae larval infection. Seven sea fish species were examined by means of the candling technique over a period of 4 months, from November 1996 to February 1997. Third-stage larvae of Anisakis simplex and Pseudoterranova decipiens were identified. A. simplex was the most abundant species with a prevalence of 84.5%, and was found in 192 of the examined fillets (10.9%). Pollock (Pollachius pollachius) was the most heavily infected fish species (82.9%). The prevalence in cod (Gadus morhua), saithe (Pollachius virens), and whiting (Merlangius merlangus) was 34.7%, 26.7%, and 15.4% respectively. The prevalence in the three other fish species examined was low, namely 8.9%, 6.6%, and 3.8% for ling (Molva molva), catfish (Anarchias lupus), and Northeast red fish (Sebastes marinus), respectively. Pollock was the fish species with the largest number of Anisakis larvae (7.8 larvae/kg fish fillet).     

Interactions between ecto- and endoparasites in trout Salmo trutta.

Trout fry were experimentally infected with endoparasites (Anisakis sp.) and/or ectoparasites (Gyrodactylus derjavini). During the following 3 weeks the infection level of G. derjavini was significantly lowered in fish infected with live anisakids compared to fish injected with physiological saline, a corresponding amount of Anisakis protein sonicate or to untreated fish. Subsequent scanning electron microscopy (SEM) of recovered nematodes from the trout showed an extensive colonization of the worms with host macrophages. It is indicated that the activation of the fish immune system by infection with live anisakids influences the skin response of the host with a subsequent negative effect on the ectoparasites G. derjavini.     

Molecular identification of Anisakis spp. from fishes collected in the Tyrrhenian Sea (NW Mediterranean)

The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status.     

Anisakidae infection in fish of the Aegean Sea

Nematode worms of the family Anisakidae are the causative agents of infections in humans when fish is consumed raw and of serious allergies up to the death, when fish is consumed raw or cooked by previously sensitized people. From April until November 2009, 462 fish belonging to 26 species, fished in three areas of the Aegean Sea were tested for Anisakidae larvae. Anisakidae larvae were detected in 87 (18.83%) fish of 13 species. These larvae were identified by morphology as the third-stage larvae of the genera Hysterothylacium sp. or Anisakis. Larvae of the genus Anisakis were identified by PCR-RFLP as belonging to A. simplex s.str., A. pegreffii, or as hybrids between A. simplex s.str and A. pegreffii.     

Nematode infection in fish from Cartagena Bay, North of Colombia

Parasitic nematode infection indices were recorded in different fish species collected at Cartagena Bay, North of Colombia. Among 19 studied species, the Mugil genus presented the highest prevalence (83.9-100%), although Sciades herzbergii, Caranx hippos and Centropomus undecimalis were also found infected with nematodes. Parasites were found in the liver, intestinal mesenteries and encysted near the intervertebral joints, with an average parasite abundance of 4.0 ± 0.3 nematodes per fish. Morphological analysis allowed the identification of these nematodes as Contracaecum sp. A small, but positive correlation was found between parasite abundance and length (R=0.294, P<0.001) and weight (R=0.244, P<0.001). In contrast, the correlation between parasite abundance and condition factor was negative (R=-0.191, P<0.001). These results are the first describing the presence of nematodes in several fish species of this ecosystem, and it highlights the need for monitoring parasitism in Mugil species in order to avoid parasite ingestion during fish consumption.     

Anisakidae nematodes and Trypanorhyncha cestodes of hygienic importance infecting the king mackerel Scomberomorus cavalla (Osteichthyes: Scombridae) in Brazil

From February to October 2007, thirty specimens of the king mackerel, Scomberomorus cavalla (Cuvier, 1829) were purchased from markets in the municipalities of Niterói and Rio de Janeiro. The fishes were measured, filleted and further had their organs investigated for helminths. Ten out of the thirty fish specimens were parasitized with anisakid nematodes represented by Anisakis sp. and Contracaecum sp. with prevalence of 1% and 16%, mean intensity of 2 and 3.31 and mean abundance of 0.02 and 0.53, respectively. The infection range with Contracaecum sp. was 1-9. The sites of infection were the stomach serosa and mesentery. Seventeen fish specimens (53%) out of the 30 investigated were parasitized with Trypanorhyncha metacestodes, identified as Callitetrarhynchus gracilis, Pterobothrium crassicole, Callitetrarhynchus speciosus and Tentacularia coryphaenae in the mesentery, with prevalence of 26, 20, 6, 3%, intensity and mean intensity of 3.25, 3.5, 1, 2 and mean abundance of 0.86, 0.7, 0.06 and 0.06, respectively. The infection range due to C. gracilis and P. crassicole were of 1-5 and 1-20, respectively. Anisakis sp., C. speciosus and P. crassicole are reported in S. cavalla for the first time. Considerations on the zoonotic potential of the parasites and their rules in sanitary inspection are presented.     

开放环境实验猕猴肠道寄生虫感染状况调查

为了解开放环境普通级猕猴（Macaca mulatta）肠道寄生虫的感染情况,采用卢戈氏碘液染色法和饱和糖溶液漂浮法对108份粪便样品进行检查。结果共检查出阿米巴（Amoeba sp.）、结肠小袋纤毛虫（Balantidium coli）、福氏类圆线虫（Strongyloides fuelleborni）、鞭虫（Tri...     

Anisakiasis with proventricular perforation in a greater shearwater (Puffinus gravis) off the coast of Georgia, United States

A juvenile male greater shearwater (Puffinus gravis) found off the Georgia coast, United States, was lethargic, hypothermic, dehydrated, and emaciated. It was provided with supportive care but subsequently died. The coelomic cavity contained fibrinous fluid intermixed with hundreds of nematodes, some of which protruded through a perforation in the proventriculus. Nematodes were embedded in the wall of the proventriculus from the lamina propria to the serosal surface and were surrounded by granulocytes, epithelioid macrophages, multinucleate giant cells, lymphocytes, and fibroplasia. A full-thickness tear in the proventriculus was lined by heterophils and multinucleate giant cells. The nematode histomorphology was consistent with Anisakis spp. The definitive hosts of Anisakis spp. are marine mammals, which are infected through ingestion of parasitized fish and crustaceans. Marine birds are aberrant hosts of Anisakis spp., and young, inexperienced, immunocompromised birds might be more susceptible to severe infestations. Fatal anisakiasis is rarely reported in birds.