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为了研究江苏地区感染南美白对虾(Penaeus vannamei)的肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的形态学特点,分析与国外报道的差异性,本研究以江苏地区感染肝肠胞虫的南美白对虾肝胰腺组织为研究材料,通过蔗糖密度梯度离心等方法进行肝肠胞虫初步分离纯化,构建一种肝肠胞虫荧光染色检测方法,显微观察肝肠胞虫的孢子形态结构。结果发现感染江苏地区南美白对虾的肝肠胞虫孢子主要分布在35%~40%浓度的蔗糖层面,可推算其密度为1.15~1.17 g/m L。荧光显微镜下可见孢子椭圆或圆形,呈亮蓝色,孢子微小但可计数,显微观察可见肝肠胞虫孢子大小约为1.7μm×0.9μm,外壁上有大量白色瘤状物,疑似胞壁蛋白,孢子表面布满细小褶皱,孢子具有一个细胞核,极丝4~6圈,细胞壁由一相对薄的电子透亮的孢子内壁和电子致密的孢子外壁组成。结论认为江苏地区南美白对虾肝肠胞虫的形态结构与国外报道的吻合,本研究建立的肝肠胞虫分离纯化、荧光染色方法以及取得的肝肠胞虫形态学资料可为其检测和研究提供参考。 相似文献
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采用差速离心和密度梯度离心,从感染虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)对虾的肝胰腺中,尝试纯化出孢子,并应用透射电镜、荧光桃红染色和血球计数板计数的方法对纯化出的孢子进行了观察和计数。结果表明,从1g的EHP载量为(4.7±2.2)×104 copies/ng DNA的肝胰腺组织中经差速离心和密度梯度离心方法分离到EHP的孢子,电镜观察孢子为大小在0.7～1.2μm的椭圆形,纯化孢子悬液用血球计数板计数显示孢子浓度为5.30×103个/μL,蔗糖密度梯度中的纯化孢子区带含有的孢子总数约1.06×106个。 相似文献
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舟山地区大棚凡纳滨对虾生长缓慢病因的调查分析 总被引：1，自引：0，他引：1
2013年以来,浙江省舟山地区大棚养殖的凡纳滨对虾(Penaeus vannamei)普遍出现生长缓慢、养殖成功率低的现象。为了查明该原因,本研究采用分子生物学和组织病理学等方法对引起对虾生长缓慢的病因开展了调查分析。结果显示,采集的270份病虾样本中,对虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)PCR阳性检出率高达85.19%,传染性皮下及造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)检出率为0;所有采集的病虾样本中也未分离到常见的致病菌;54份正常的对虾样本中EHP和IHHNV均未检出。将病虾PCR扩增产物进行序列测定和比对分析,结果获得的序列片段与Gen Bank中已有EHP相关序列相似性高达99.55%;病虾的肝胰腺组织病理切片观察显示,在虾肝胰腺组织中可观察到处于各个生长发育阶段的EHP。通过上述研究,初步认为EHP是引起舟山地区大棚养殖对虾生长缓慢的一个重要病原。 相似文献
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为全面、系统地了解滨州市地区凡纳滨对虾(Litopenaeus vannamei)苗种携带虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)及爆发情况，对滨州地区37家凡纳滨对虾养殖池塘随机采集对虾苗种样本，根据SC/T 7232-2020 《虾肝肠胞虫病诊断规程》采用套式PCR法进行对虾苗种虾肝肠胞虫的检测，检测结果表明滨州地区虾肝肠胞虫阳性检测率为4.84%,同时监测虾肝肠胞虫病发病池和未发病池的水质，发现发病池的水体中pH值、COD等指标并没有显著差异，说明虾肝肠胞虫病爆发并不是由于养殖池水质导致的。 相似文献
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近年来，虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)流行区域不断扩大，已经成为我国南美白对虾(Litopenaeus vannamei)养殖重要病原之一。本研究根据Gen Bank中虾肝肠胞虫18S r RNA保守区域设计一套特异性引物和荧光探针，通过优化反应条件，建立检测EHP的TaqMan探针实时荧光定量PCR(Real-time quantitative polymerase chain reaction,qPCR)检测方法。建立的方法呈良好的线性关系(R2=0.998)，灵敏度是巢式PCR的10倍。特异性试验表明：检测其他常见病原均为阴性，特异性好；重复性试验显示：批内批间变异系数均小于1.5%，重复性良好；对临床样品检测，与巢式PCR的符合率为97.81%。使用该方法对中山市、江门市和珠海市珠三角部分地区南美白对虾开展EHP流行病学调查，其中虾苗检出率为10.75%，成虾检出率为54.07%，部分区域检出率较高，要防范爆发风险。本研究建立的实时荧光定量PCR灵敏度高、特异性和重复性良好，具有较好的应用前景。 相似文献
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2013年,河北、天津等地区养殖的凡纳滨对虾(Litopenaeus vannamei)育苗期出现死苗、出苗率低的情况,生产上,仔虾个体大小差异较大,造成了严重损失.本研究采用荧光定量PCR方法(Real-time PCR)对天津大港地区采集的108尾凡纳滨对虾仔虾样品进行单尾病原检测.结果显示,传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)均有检出.IHHNV阳性检出率100％,每微克对虾组织DNA的病毒拷贝数为103-107,且个体较大的样品(1.2-2.0 cm)携带病毒拷贝数偏高;EHP阳性检出率为49.1％,每微克对虾组织DNA的拷贝数为103-105,且集中于个体较小样品(0.7-1.1 cm).对IHHNV和EHP阳性凡纳滨对虾样品进行生物学体长与病毒载量指数相关性分析,显示IHHNV载量指数与对虾生长速率呈正相关,虾组织IHHNV平均载量达8.51×104 copies/μg DNA,为较高的感染水平;EHP的载量与对虾生长速率呈负相关关系,与较大个体阳性检出率较低相对应,虾组织EHP平均载量达到2.19× 104 copies/μg DNA,为较高的感染水平.由此,该批凡纳滨对虾仔虾患病为IHHNV和EHP的混合感染所致,本研究数据为IHHNV和EHP病原混合感染流行情况及其对养殖育苗期仔虾生长的影响提供科学依据. 相似文献
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S. Thamizhvanan S. Sivakumar S. Santhosh Kumar D. Vinoth Kumar S. Suryakodi K. Balaji T. Rajkumar S. Vimal S. Abdul Majeed G. Taju A. S. Sahul Hameed 《Journal of fish diseases》2019,42(3):447-454
White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively. 相似文献
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为探究虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)感染对脊尾白虾(Exopalaemon carinicauda)肠道菌群的影响,本研究基于16S rRNA基因的测序结果,对感染EHP的脊尾白虾肠道菌群进行了分析。结果显示,感染虾与健康虾肠道菌群差异较大,且其肠道菌群结构多样性显著低于健康虾。研究发现,病虾中归属于变形菌门(Proteobacteria)的脱硫弧菌科(Desulfovibrionaceae)、弧菌科(Vibrionaceae)、未分类蓝细菌科(unidentified Cyanobacteria)、支原体科(Mycoplasmataceae)和未分类α变形菌科(unidentified Alphaproteobacteria)为优势菌,而归属于厚壁菌门(Firmicutes)的乳杆菌科(Lactobacillaceae)、双歧杆菌科(Bifidobacteriaceae)、芽孢杆菌科(Bacillaceae)及噬几丁质杆菌科(Chitinophagaceae)细菌在健康虾中占据优势地位。EHP侵蚀导致感染虾肠道内潜在致病菌显著增加(P<0.05),增加了其他疾病的易感性。此外,通过Tax4Fun功能预测,发现感染虾肠道菌群主要用于新陈代谢,从而抵抗EHP侵染,维持机体正常功能;健康虾肠道菌群则多用于个体生长与环境信息处理,进而保证生长与存活。本研究从虾肠道菌群结构方面入手,进一步探究了EHP感染对脊尾白虾肠道菌群的影响,以期为EHP的防治提供帮助。 相似文献
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A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus . Viable spores stained green with FDA and non-viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi , actinosporean spores of M. cultus , stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm–2 for M. artus myxosporean spores, 600 mW s cm–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments. 相似文献
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Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens. 相似文献
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Chin-I Chang Chia-Che Wu Ta Chih Cheng Jyh-Ming Tsai & King-Jung Lin 《Aquaculture Research》2009,40(10):1182-1190
A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens. 相似文献
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Michael L. Kent Diane G. Elliott Joseph M. Groff Ronald P. Hedrick 《Aquaculture (Amsterdam, Netherlands)》1989,80(3-4):211-222
Loma salmonae (Putz et al., 1965) infections were observed in five groups of coho salmon, Oncorhynchus kisutch, reared in seawater net-pens in Washington State, U.S.A. in 1984–1986. Ultrastructural characteristics, size of spores, tissues and host infected, and geographical location identified the microsporidium as Loma salmonae. Preserved spores measured 4.4×2.3 (4–5.6×2–2.4) μm and exhibited 14–17 turns of the polar filament. Infections were evident in the gills of some fish before seawater entry, but few parasites were observed and they caused little tissue damage. Infections observed in fish after transfer to seawater were associated with significant pathological changes in the gills. A mixed inflammatory infiltrate was associated with ruptured microsporidian xenomas within the vessels and interstitium of the primary lamellae. Microsporidian spores were dispersed throughout the lesions and were often seen inside phagocytes. The parasite was also observed in the heart, spleen, kidney and pseudobranchs; however, the inflammatory lesions were common only in the heart.
Monthly examination of fish after transfer to seawater showed peak prevalences (33–65%) of gill infections during the summer. Although moribund fish were often infected with other pathogens, the high prevalence of L. salmonae infections and the severity of the lesions it caused, suggested that this parasite significantly contributed to the recurrent summer mortalities observed at this net-pen site. 相似文献