中国养殖斑石鲷(Oplegnathus puncatus)上皮囊肿病的发现及显微观察

2015年,山东省某养殖场工厂化养殖的斑石鲷(Oplegnathus puncatus)幼鱼(全长为15 cm左右)因病陆续死亡,15d内累积死亡率达40％以上.现场调查发现,发病池水温为21℃,盐度为30.患病鱼群散开、不聚集.病鱼身体侧偏,活力差,常贴底或者贴壁,严重者随着水流漂流.病鱼呼吸困难,口部持续张开,鳃盖开合频繁,对投喂的食物无反应.但病鱼反应灵敏,难以捕捉.临床检查和剖检可见病鱼鳃表面覆盖着大量黏液,鳃丝有损伤,肠道无食物.取病鱼鳃丝制成水浸片,在光学显微镜下观察,鳃丝上可见到许多直径约为30-70 μm的囊肿物,外观圆形或卵圆形,呈浅黄棕色.在苏木精-伊红染色的石蜡切片中,病鱼次级鳃丝末端粘连,许多上皮细胞膨大呈囊肿状.囊肿嗜碱性,内部均质化.在扫描电子显微镜下观察,病鱼鳃丝呈棍棒化,鳃小片被大量黏液覆盖,表面光滑的囊肿细胞镶嵌其间.通过上述疾病现场调查、病鱼的临床检查、鳃组织的光学显微镜和扫描电子显微镜病理观察,可以初步确定该病为斑石鲷上皮囊肿病.这是上皮囊肿病在中国养殖斑石鲷中首次被发现和记载.     

Ultrastructural study of epitheliocystis organisms from gill epithelium of the fish Sparus aurata (L.) and Liza ramada (Risso) and their relation to the host cell

Abstract. Ultrastructural study of epitheliocystis organisms from gills of Sparus aurata (L.) and Liza ramada (Risso) revealed in each fish a new form of the organism which differed from epitheliocystis organisms previously described. In their size range, the presence of a plasma membrane, a trilaminated cell wall and a central nucleoid, these organisms resembled chlamydial organisms (PLT). However they differed from them in the following respects: in both species several morphological forms were recognized; hyphae or mycelium-like forms and individual units, not comparable to any of the successive units described for Chlamydia organisms. Both organisms were contained within an inclusion in the host cell, but in epitlieliocysts from S. aM7-ata apparently intracytoplasmic forms were also observed. Ultrastructural studies of the cytoplasmic wall of the epitheliocystis inclusion in S. aurata suggest that the epitheliocystis cyst remains contained in a single unruptured hypertrophied cell, while in i. ramada the final cyst is formed through the coalescence of several epithelial cells.     

Candidatus Actinochlamydia pangasiae sp. nov. (Chlamydiales,Actinochlamydiaceae), a bacterium associated with epitheliocystis in Pangasianodon hypophthalmus

Chlamydial infections are recognised as causative agent of epitheliocystis, reported from over 90 fish species. In the present study, the farmed striped catfish Pangasianodon hypophthalmus (14–15 cm, 70–90 g) with a history of cumulative mortality of about 23% during June and July 2015, were brought to the laboratory. The histopathological examination of gills from the affected fish revealed presence of granular basophilic intracellular inclusions, mostly at the base of the interlamellar region and in gill filaments. A concurrent infection with Trichodina spp., Ichthyobodo spp. and Dactylogyrus spp. was observed in the gills. The presence of chlamydial DNA in the gills of affected fish was confirmed by amplification and sequencing of 16S rRNA gene. BLAST‐n analysis of these amplicons revealed maximum similarity (96%) with Candidatus Actinochlamydia clariae. On the basis of phylogenetic analysis, it was inferred that the epitheliocystis agents from striped catfish were novel and belonged to the taxon Ca. Actinochlamydia. It is proposed that epitheliocystis agents from striped catfish will be named as Ca. Actinochlamydia pangasiae. The 16S rRNA gene amplicons from novel chlamydiae were labelled and linked to inclusions by in situ hybridisation. This is the first report of epitheliocystis from India in a new fish host P. hypophthalmus.     

The developmental cycle of epitheliocystis in carp, Cyprinus carpio L.

Abstract. Epitheliocystis infections in the gills of carp from Israel and Portugal occurred in lining epithelial cells as well as mucus and chloride cells. Ultrastructural studies of infected cells revealed several morphological forms of epitheliocystis showing the existence of a pleomorphic developmental cycle. The following stages were observed: chlamydia-like round cells (RC), non-dividing round cells (NRC) and bullet-shaped small cells (SC). All these were identical in shape to RC and SC described from epitheliocystis of other fish, The rickettsia-like cells (PLC and ILC) which occur in epitheliocystis from other fish species and produce the SC were absent in carp epitheliocystis and instead the RC were seen transforming into SC. Transformation to SC, however, occurred at any stage of the developmental process, even before the replication potential of the RC was exhausted, i.e. before reaching the NRC stage.     

Epitheliocystis in silver perch, Bidyanus bidyanus (Mitchell)

Cultured juvenile silver perch, Bidyanus bidyanus (Mitchell), were affected by epitheliocystis, with a prevalence of up to 75%. The condition affected 2–96% of gill filaments. Granular basophilic cysts ranged from 10 to 87 μm and had a round-to-oval shape. The infected cells were positioned centrally within a lamella, often close to its tip, and their identity was difficult to determine, but the involvement of pillar cells could not be precluded. The size of pathogen was within the range previously reported. The pathogen had two morphological forms, which were positioned differently within the cyst. The form located more in the centre was rod-shaped with an electron-dense core and electron-lucent vesicles on the sides of the core. Its mean length was 498.14 nm (SD = 47.68 nm), and its mean width was 145.14 nm (SD = 9.53 nm). The form present in a more peripheral position had an irregular shape, often did not show the electron-dense core and did not have the electron-lucent vesicles. Its mean length was 704 nm (SD = 170.29 nm) and its mean width was 152.4 nm (SD = 16.40 nm). Both forms were enclosed by a double trilaminar membrane. This is the first confirmed report of epitheliocystis in Australian freshwater fish.     

弗氏柠檬酸杆菌感染诱导斑马鱼皮肤免疫相关基因的差异表达

从草鱼肠道中分离到1株细菌,通过形态学观察、生理生化特征和16S rDNA序列分析等鉴定为弗氏柠檬酸杆菌,动物试验结果表明,该菌对斑马鱼有致病性;从感染诱导的斑马鱼皮肤组织中提取总RNA,经Biotin荧光标记与拥有15 617个cDNA片段的斑马鱼基因芯片(affymetrix)杂交筛选分析,获得斑马鱼皮肤免疫相关差异表达基因88个,其中有74个上调表达基因和14个下调表达基因;进一步根据基因功能聚类(GO)分析,初步将88个差异表达基因分为8个生物学功能,其中主要参与补体激活、急性期反应、应激防御反应、细胞凋亡、抗原加工提呈、细胞迁移粘附、凝血因子和血小板激活等免疫应答过程;同时进行信号通路(pathway)分析,结果表明MAPK(hsp70、daxx、nfkbiab)、JAK/STAT(ifn1)和TGF-β(thbs1)等信号通路参与斑马鱼皮肤抗弗氏柠檬酸杆菌感染免疫应答。采用基因芯片方法筛选与鱼类皮肤免疫相关的功能基因,为将来以斑马鱼为模型研究鱼类皮肤局部免疫应答的分子机制提供科学依据。     

养殖裸盖鱼(Anoplopoma fimbria)疥疮病病原菌的分离与鉴定

裸盖鱼(Anoplopoma fimbria)是一种名贵的冷水经济鱼类,已在我国北方地区开展小规模的工厂化繁育和养成.目前,我国关于裸盖鱼的病害研究仍是空白.本研究对山东烟台一养殖场自然发生疥疮病的裸盖鱼进行了病原分析,从发病鱼体内分离得到形态一致的优势菌株,命名为AF-1,并对其进行了致病性检测、菌种鉴定及药物敏感性研究.人工感染实验证明,AF-1对裸盖鱼有致病性,呈现症状与自然发病状态一致;结合形态观察、革兰氏染色、生理生化特征、16S rRNA和gyrB基因序列进化树分析,将AF-1鉴定为杀鲑气单胞菌(Aeromonas salmonicida);药物敏感性实验结果显示,AF-1对青霉素、阿莫西林、头孢噻吩等13种抗生素具有耐药性,对氟苯尼考、氟甲喹等16种抗生素敏感.综上所述,本研究首次报道了我国养殖裸盖鱼感染杀鲑气单胞菌病例,为裸盖鱼养殖过程中的疾病防控和疫苗开发提供了参考.     

Epitheliocystis disease in the cultured amberjack, Seriola dumerili Risso (Carangidae)

Epitheliocystis infection affecting the gill and the pseudobranch of the cultured amberjack, Seriola dumerili Risso, is described. In hyperinfected fish, proliferative cell response around the epitheliocystis capsule resulted in gill and pseudobranch lamellar fusion, which led to mass mortalities in the 0+age class. Histopathological and scanning electron microscope observations showed that the target cell was the chloride cell since (1) epitheliocystis organisms were first found within the chloride cell; (2) chloride cells underwent degeneration in the filament epithelium, proliferated along the lamellae, and hypertrophied; and (3) cysts were found only in the trailing edge of the gill filament and in the interlamellar spaces where, in healthy fish, chloride cells are mainly located. In some cases, the pseudobranch of infected fish was much more severely affected than the gill. It is therefore suggested that, for diagnostic purposes, the pseudobranch should also be sampled when epitheliocystis disease is suspected.     

Molecular evidence for the lack of transmission of the monogenean Sparicotyle chrysophrii (Monogenea, Polyopisthocotylea) and isopod Ceratothoa oestroides (Crustacea, Cymothoidae) between wild bogue (Boops boops) and cage-reared sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax)

In the Mediterranean area, wild fish have often been suggested as either the reservoirs of the causative agents or at least the carriers of the pathogens responsible for disease outbreaks in cultured fish. However, no epidemiological investigations on actual pathogen/disease interactions between farmed and wild fish have been conducted even for the most important fish pathogens. Only sporadic isolations and identifications of various pathogens in wild fish have been done and real associations with the pathological conditions that exist within the farm environment and vice versa have not been established. Monogenean ectoparasite Sparicotyle chrysophrii and isopod Ceratothoa oestroides are commonly found in the Mediterranean cage-reared sea bream and sea bass and in the surrounding wild fish population. Both species were recognized as pathogens that seasonally inflict serious losses in fingerlings and juveniles of sea bream and sea bass, being potentially propagated and exchanged during wild and caged fish interaction. In order to evaluate the degree of pathogens transfer between wild and farmed fish, we investigated genetic population structures of these two important parasitic pathogens inferred by mtDNA cytochrome oxidase I locus. Parasites isolated from wild and farmed fish on the two most productive Adriatic fish farms showed genetic heterogeneity, contradicting widely accepted hypothesis of cross-contamination, at least in case of S. chrysophrii and C. oestroides. As far as we know, this is the first report that in a study of pathogen transfer molecular evidence was employed to asses the genetic population structure of shared parasites.     

子二代中华鲟分枝杆菌感染及血液生理生化指标的变化

鱼类分枝杆菌病是由分枝杆菌属的非结核分枝杆菌造成的长期性感染疾病。本研究发现,养殖中华鲟(Acipenser sinensis)子二代可感染该病,典型病理特征是腹水、腹膜肉芽肿、肾脏水肿、肝脏呈灰白色等。采集腹水,通过细菌分离培养获得一株缓慢生长型的抗酸杆菌,进一步通过16S r DNA和IS2404重复序列进行分子生物学鉴定,发现该菌与Mycobacterium liflandii相似性最高。将10尾健康子二代中华鲟3次体检的血液参数与4尾分枝杆菌感染子二代中华鲟12次检查的血液参数进行比较,发现在26项生理生化指标中,有9项指标差异显著(P0.05),即中性粒细胞、淋巴细胞、钠、氯、钙、总蛋白、球蛋白、外周血白细胞总数(TWBC)和谷丙转氨酶(ALT),其中TWBC、中性粒细胞和ALT这3项指标显著升高,其他指标显著下降,说明分枝杆菌感染对鱼体肝脏和肾脏的损伤较大。采用盐酸卡纳霉素、红霉素和利福平三种抗生素联合用药进行治疗,给药时间75 d,治疗后腹水明显减少,RBC、血红蛋白、PCV、MCV和球蛋白这5项血液理化指标均有显著性升高(P0.05),说明药物治疗初期具有一定的效果,但随着病程的加剧,该尾鱼最终未能治愈。以上结果表明,分枝杆菌在中华鲟广泛流行,对中华鲟的危害较大,目前尚未见有效的疫苗和药物,因此对该病的预防、早期诊断和治疗极其重要。     

Francisella sp., an emerging pathogen of tilapia, Oreochromis niloticus (L.), in Costa Rica

Francisella sp. is an emergent bacterial pathogen that causes acute to chronic disease in warm and cold water cultured and wild fish species. During the past 3 years, the bacterium has been detected in tilapia, Oreochromis niloticus , cultured in Costa Rica. Infected fish presented non-specific clinical signs, such as erratic swimming, anorexia, anaemia, exophthalmia and high mortality. Upon macroscopic and microscopic examination, several internal organs (mainly spleen and kidney) were enlarged and contained white nodules. Histological examination revealed the presence of multifocal granulomatous lesions, with the presence of numerous small, pleomorphic, cocco-bacilli. The bacteria were isolated from infected tilapia on selective media and grown on several media with and without antibiotics. Specific PCR primers to the Francisella genus were used to confirm the preliminary diagnoses. In comparison with several bacterial 16S rRNA sequences, our isolate was found to share 99% identity with other Fransicella spp. isolated from fish, and more than 97% identity to the human pathogen Francisella tularensis . Koch's postulates were fulfilled after experimental intraperitoneal and gill exposure challenges.     

两种疾病发生对养殖半滑舌鳎肠道菌群结构的影响分析

为研究疾病发生情况下鱼类肠道菌群的结构变化,运用高通量测序方法分别对感染腹水病和皮肤溃疡病的工厂化养殖半滑舌鳎病鱼及同一养殖系统中健康鱼的肠道菌群结构进行了对比分析。结果发现,养殖半滑舌鳎肠道中的细菌种类要明显少于环境中的细菌种类,仅为养殖池进水和颗粒饲料中细菌种类总数的14.3%~44.7%。在疾病发生后,病鱼肠道中的细菌种类数量仅是同一养殖系统中健康鱼肠道细菌种类数量的45.8%和64.5%,病鱼肠道中细菌多样性下降非常明显。进一步分析肠道菌群结构的组成发现,虽然发病鱼和健康鱼肠道中超过60%的细菌种类不相同,但优势菌群的结构组成差异不明显,丰度最高的前10种细菌种类基本相同,其比例超过肠道相对总菌量的80%。不同疾病的发生对半滑舌鳎肠道菌群结构的影响也不同,腹水病会造成肠道中优势菌株的种类和相对丰度都发生比较明显的改变,而皮肤溃疡病发生前后肠道菌群结构组成几乎没有变化。研究表明,养殖半滑舌鳎的肠道菌群结构相对比较保守,而且并不是所有的疾病种类都会引起肠道菌群结构发生改变。     

Detection of the bacterium Flavobacterium psychrophilum from a natural infection in rainbow trout, Oncorhynchus mykiss (Walbaum), using formalin-fixed, wax-embedded fish tissues

The bacterial pathogen Flavobacterium psychrophilum was successfully identified from formalin-fixed, wax-embedded tissue blocks of infected rainbow trout heart and spleen tissues, using a polymerase chain reaction (PCR)-based assay. Filamentous bacteria were observed in haematoxylin and eosin and Giemsa-stained sections but no bacteria were recovered from the diseased fish using standard bacteriology isolation techniques. All infected fish had histopathological evidence of myocarditis or rainbow trout fry syndrome. Immunohistochemistry was attempted using three different anti-F. psychrophilum sera but the results were inconclusive, and an alternative molecular approach was therefore attempted. This paper describes the use of a PCR-based assay to help identify bacteria present in formalin-fixed, wax-embedded tissue samples. This is the first time that this technique has been used for the detection of fish bacteria from diagnostic samples.     

灿烂弧菌的疏水性和生物被膜形成能力

以仿刺参化皮病病原菌—灿烂弧菌(Vibrio splendidus)AP622为试验菌株,研究了培养材料、培养时间、培养基、葡萄糖浓度对菌株生物被膜形成能力的影响,证实鞭毛和菌毛介导的运动性,同时比较了浮游细菌与形成生物被膜的细菌对抗生素的敏感性,并研究了菌株的疏水性。结果表明,菌株AP622为高疏水性和高生物被膜形成菌株,在聚氯乙烯为培养材料、含葡萄糖浓度为0.5%、LB培养基中形成的生物被膜最多,生物被膜形成周期为24 h。菌株AP622明显表现出鞭毛介导的群集性和Ⅳ型菌毛介导的颤搐等运动力。生物被膜中的菌株AP622对抗生素的抵抗力明显强于浮游细菌,其最小抑菌浓度(MIC)是浮游细菌的32倍。综上所述,菌株AP622具有很强的疏水性和高生物被膜形成能力,判断其具有很强的黏附力,同时具有明显的抗药性。     

The eel immune system: present knowledge and the need for research

The European eel, Anguilla anguilla, is one of the most important warm water fish species cultured in southern Europe and the Mediterranean as well as in northern countries including Germany, the Netherlands and Denmark. The Japanese eel, A. japonica, is an important cultured fish in several Asiatic countries including Japan, China and Taiwan. During recent decades, research has been performed to elucidate the immune response of these species against different pathogens (viruses, bacteria or parasites). Nevertheless, there is very limited information in terms of both cellular and humoral immune responses. This review summarizes the present knowledge relating to the eel immune system and includes new data.     

Gill histopathology of wild marine fish in Tasmania: potential interactions with gill health of cultured Atlantic salmon, Salmo salar L

This study surveyed conditions in the gills of wild marine fish in Tasmania to determine potential interactions between wild and cultured fish. Wild marine fish of 12 species were captured from three Atlantic salmon farm sites and three reference sites around Tasmania. The survey concentrated on three species, red cod, Pseudophycis bachus, sand flathead, Platycephalus bassensis, and jack mackerel, Trachurus declivus. Seventy-six per cent of salmon pens contained wild fish species. The number of species found in a pen ranged from one to nine and the number of individuals ranged from one to 23. Trichodinids were prevalent and occurred on seven of the 13 species examined. Trichodina occurred on the gills of all but one specimen of red cod. Monogenean gill flukes were observed on all three major species sampled and were abundant on sand flathead. Other parasites and conditions observed in the survey included metacercariae of digenean trematodes, epitheliocystis and cysts of unknown origin. Infestations of trichodinids on red cod and monogenean gill flukes on sand flathead were significantly more intense at farm sites than at reference sites. Atlantic salmon sampled at the same time from the farms were only affected by amoebic gill disease and isopods.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Aetiology of an ulcerative disease in goldfish Carassius auratus (L.): microbiological examination of diseased fish from seven locations

Abstract. A comparative diagnostic study was conducted on goldfish, Carassius auratus (L.), with a cutaneous ulcerative disease from five locations in the United States and one each in England and Japan. Fish were examined for parasites, viruses and bacteria. Fish from all locations examine d were infested by ectoparasites; no single parasite species was common to all locations. No virus-associated cytopathology was observed in fathead minnow (FHM) or adult goldfish (CAR) monolayer cell cultures inoculated with homogenates of cutaneous lesions, kidneys and livers from diseased fish. The only bacterium cultured from fish from all locations was an atypical, often late-pigmenting strain of Aeromonas salmonicida . This organism was isolated from 64 of 83 (77%) of the total lesions cultured and was most prevalent in early lesions. A second commonly isolated organism was Aeromonas hydrophila , which was cultured from fish at four of the seven locations and from 28 (34%) of the total lesions cultured. A. hydrophila was most prevalent in terminal lesions. From these studies it was concluded that A. salmonicida was the probable cause of ulcers noted In the cases examined an d that A. hydrophila was a secondary invader.     

Evidence of fish and human pathogens associated with doctor fish (Garra rufa,Heckel, 1843) used for cosmetic treatment

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well‐known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.     

革胡子鲶出血性败血症病原菌的分离鉴定

从暴发性死亡的革胡子鲶(Clarias gariepinus)中分离出可疑病原菌株2010111403(简称1403),分别采用细菌全细胞脂肪酸鉴定系统、Biolog微生物自动鉴定系统及细菌16 S rDNA序列分析三种方法对其鉴定,结果表明菌株1403为嗜水气单胞菌(Aeromonas hydrophila)。分离菌株对健康革胡子鲶致病性测试表明其对革胡子鲶的半数致死剂量(LD50)为6.32×106菌落形成单位(CFU);实验感染革胡子鲶出现与自然发病相似症状,表明菌株1403是引起革胡子鲶发生暴发性死亡的主要致病病原。药敏试验表明,庆大霉素、四环素、阿奇霉素、氟哌酸、先锋霉素等9种抗生素对分离菌株有较强的抑制作用,但分离菌株对复方新诺明、克林霉素、罗红霉素等4种药物表现出耐药性。