Lectin histochemical studies on Sphaerospora sp. (Myxosporea) from Italian brown trout, Salmo trutta L.

A Sphaerospora sp. (Myxosporea) infection (presumably S. truttae ) was identified on a trout farm in northeastern Italy. Parasites were detected in kidneys from infected brown trout, Salmo trutta L., over a 2-year period. Extrasporogonic, sporogonic stages and mature spores were simultaneously detected in the same fish. Traditional diagnostic methods for Sphaerospora spp. rely on the detection of the myxosporean developmental stages in Giemsa-stained kidney smears or haematoxylin-eosin stained tissue sections. A histochemical method was employed where 10 biotinylated lectins (Con-A, DBA, SBA, GS-I, PHA-P, LEA, PWM, RCA₁, WGA and UEA-I) and the avidin-biotin-peroxidase complex (ABC) were used on Sphaerospora -infected brown trout renal tissues and kidney imprints. Five monoclonal antibodies against PKX (Mab12, MabA3, MabC5, MabD4 and MabB4) were also tested. A lectin glycoconjugate binding pattern for Sphaerospora spp. is presented. This staining method shows that SBA lectin ( Glycine max agglutinin) is a useful tool for the detection of the Sphaerospora spp. Only MabB4 bound some of the most mature sporogonic stages. In contrast Mabs12, A3, C5 and D4, and GS-I lectin ( Griffonia simplicifolia agglutinin) did not stain any of the Sphaerospora spp. stages, but did bind very specifically to the sporogonic and extrasporogonic stages of PKX, the causative agent of proliferative kidney disease (PKD).     

Localization of carbohydrate terminals on Tetracapsuloides bryosalmonae using lectin histochemistry and immunogold electron microscopy

Tetracapsuloides bryosalmonae is the myxozoan parasite that causes the commercially important proliferative kidney disease (PKD) in salmonid aquaculture. Previous studies on the binding of lectins to T. bryosalmonae identified Griffonia simplificola agglutinin I (GS I) as useful for parasite identification. This lectin was also implicated as recognizing antigenic structures on the parasite. Here, we examine the histochemical staining and ultrastructural localization of a panel of 21 lectins on the extrasporogonic stage of T. bryosalmonae. The histochemical staining studies indicated that the majority of lectins bound to the renal stages of T. bryosalmonae, however not all of these lectins could be successfully localized using immunogold electron microscopy. Of the lectins that were localized many, including GS I, bound to membranes associated with the lysosomal pathway within the extrasporogonic primary cell of the parasite, indicating that these organelles are rich in glycoconjugates. The histochemical staining of Erythrina cristagalli ECL was unique and highlighted a different distribution of glycoconjugates in the periphery of some extrasporogonic parasites within the renal sinuses when compared with stages in the interstitium, suggesting the presence of distinct blood forms of T. bryosalmonae.     

Vibriosis in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. An epizootic due to Vibrio anguillarum was observed in channel catfish, Ictalurus punctatus (Rafinesque). Lesions in infected fish included ulceration and petechiae on body surfaces, vent and caudal peduncle. Internally, haemorrhages in liver and kidney were present and the intestinal tract was filled with a clear viscous fluid. Using an isolant recovered during the epizootic, laboratory studies were conducted to determine (a) bacterial dynamics of the agent in blood, kidney and liver and (b) clinical haematologic and biochemical parameters in infected fish. Under conditions of the study, vibrios were apparently sequestered in kidney and liver during initial stages of infection (8–12 h after exposure). Later, bacterial numbers in blood were comparable to those in kidney and liver. Clinical parameters of infected catfish were suggestive of cellular and tissue destruction and renal dysfunction. Based upon data of the present study and those of others, vibriosis appears to be a disease in which the agent is localized in select tissues. Secondary septicaemia may be incidental to factors which compromise host defences.     

Eimeria branchiphila sp.nov. sporulating in the gill filaments of roach, Rutilus rutilus L.

Abstract. Eimeria branchiphila sp. nov. is described from the roach, Rutilus rutilus L., collected in Bulgaria. Sporulated oocysts were found in gill secondary lamellae and less frequently were associated with melano-macrophage centres in the spleen and in kidney interstitial tissue. In the gills, parasite cells identified as zygotes and sporulation stages were also encountered. This unusual site of sporulation represents a mechanism of sporocyst dispersal previously not recorded in fish coccidia.     

A new species of Goussia (Apicomplexa, Coccidia) in the kidney tubules of the cod, Gadus morhua L.

Abstract. Segments of the kidney tubules of about 16% of cod caught in the approaches to Halifax Harbour were found to contain a coccidian parasite. Macrogametes developed in an epicellular position, and microgamonts, unsporulated and sporulating oocysts, and free sporocysts, were found in the lumen of the kidney tubule. The epithelial cells of the kidney tubules were damaged and granulomas often formed around infected tubules. The presence of a suture dividing the sporocyst wall into two valves could only be determined using electron microscopy. Other coccidians which have been placed in the genus Eimeria because sutures could not be seen using light microscopy, although no Stieda body was present, may also belong to the genus Goussia. No other coccidians have been reported in the kidney tubules of marine fish, and the name Goussia spraguei sp. nov., is proposed.     

Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L

Juvenile Atlantic cod (10 g) were infected with infectious pancreatic necrosis virus (IPNV) by intraperitoneal injection and cohabitation. Fish showed no signs of disease but IPNV could be re-isolated from kidney tissue for up to 12 weeks. On weeks 2, 5, 8, 10, 11 and 12 following infection, kidney leucocytes were fractionated on Percoll gradients, and cells separated into plastic adherent and non-adherent cell populations after overnight incubation. IPNV was detectable in lysates of both cell populations and in supernatants by culture in CHSE-214 cells. Wells containing 10(5)-10(6) macrophages had an IPNV TCID(50) of about 10(3)/well and in serially diluted macrophages the minimum number of cells required to detect virus ranged from 10(1) to 10(4). These data indicate that about one in 10(4) macrophages were infected and the mean number of virus/infected cell was about 10. Replication of IPNV in the macrophages was low as the titre of the virus in macrophage lysates did not increase between days 1 and 3 of culturing the macrophages, but virus was released into the supernatant over this time.     

The histopathology of Stephanochasmus baccatus Nicoll, 1907 in the digestive gland of Buccinum undatum (L.)

Abstract. Samples of Buccinum undatum (L.), a marine carnivorous prosobranch, from the Loch Ewe area of the West Coast of Scotland were found to be infected with the larval stages of Stephanochasmus baccatus Nicoll, 1907. The digestive glands of infected and uninfected molluscs were examined histologically and the pathogenesis of the larval stages in the digestive gland are described. Two types of lesion were found to occur. In Type I lesions degenerative changes were confined to individual tubules and neighbouring tubules remained unaffected. The Type I lesion was the typical response to the presence of the parasite in the digestive gland. The Type II lesion was less common and was associated with very heavy infections in the early part of the molluscan cycle of infection. In these molluscs large areas of the digestive gland were destroyed, and there was a marked cellular infiltrate of putatively inflammatory cells.     

淡水石斑鱼血细胞发生的观察

通过对淡水石斑鱼血涂片及头肾、肾、脾和肝脏器印片的光镜观察,发现血细胞的发育大致经过3个阶段,即原始阶段、幼稚阶段、成熟阶段。试验对脏器中不同发育阶段的红血细胞、淋巴细胞、单核细胞、粒细胞进行了观察和测量,并进行了分类统计。试验结果表明头肾、肾和脾是淡水石斑的主要造血器官,肝组织印片上未观察到原始阶段血细胞,提示肝脏不是淡水石斑鱼的造血器官。     

Development of Myxobolus bramae (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex

The development of Myxobolus bramae Reuss 1906, a myxosporean parasite of the gills of common bream Abramis brama L., was studied in experimentally infected oligochaetes. In five experiments, uninfected Tubifex tubifex (Müller) and Limnodrilus hoffmeisteri Claparéde were exposed to mature myxospores of M. bramae . In four experiments triactinomyxon type actinospores developed in Tubifex specimens but no infection was found in Limnodrilus . Actinospores were released from oligochaetes 70–81 days after initial exposure. At that time pansporocysts containing eight actinospores were located in the gut epithelium of experimental oligochaetes, but free actinosporean stages were also found in their gut lumen. Each actinospore had three pyriform polar capsules and a barrel-shaped sporoplasm with 32 secondary cells. The spore body joined the three caudal projections with a stout style. The total length of the actinospore was 139 μm on the average.     

Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

Development of proliferative kidney disease in rainbow trout, Oncorhynchus mykiss (Walbaum), following short-term exposure to Tetracapsula bryosalmonae infected bryozoans

The initial site of infection in the fish host for Tetracapsula bryosalmonae , causative agent of proliferative kidney disease (PKD) is poorly understood. Following the recent recognition that freshwater bryozoans harbour the infective stages to salmonid fish, experimental transmission studies were undertaken to investigate (1) the route of entry of the parasite into the fish host and (2) the minimum exposure time required to induce clinical signs of PKD. In-situ hybridization (ISH) studies were carried out on naïve rainbow trout exposed to the naturally infected bryozoan Fredericella sultana for up to 90 min. The sporoplasm of T. bryosalmonae was detected entering the fish via mucous cells in the skin epithelium within the first minute of exposure. In addition, T. bryosalmonae cells were infrequently detected in the skeletal musculature of exposed experimental fish up to 72 h post-exposure. The route of migration through the fish to the kidney and spleen was not determined. All fish exposed to infected, disrupted bryozoans for 10, 30 and 90 min and maintained for up to 8 weeks developed clinical PKD.     

Comparison of histopathology caused by Vibrio anguillarum and Vibrio ordalii in three species of Pacific salmon*

Abstract. The histopathology associated with naturally acquired vibriosis in chum salmon, Oncorhynchus keta (Walbaum), fingerlings caused by Vibrio anguillarum was compared with that caused by infection with Vibrio ordalii. Pathogenesis of the two forms was found to be different. Bacteraemia caused by V. anguillarum occurred in the early stages with pronounced histopathological changes in blood, loose connective tissue, kidney, spleen, gills and posterior gastrointestinal tract. Bacterial cells appeared uniformly dispersed throughout the affected tissues but were most abundant in the blood. With V. ordalii, bacteraemia developed only in late stages of the disease and the concentration of bacterial cells per ml of blood was less than in the V. anguillarum infection by a factor of 10²–10³. Tissues with most pronounced changes were skeletal and cardiac muscle, anterior and posterior gastrointestinal tract and the gills. Vibrio ordalii observed in the tissues was not evenly dispersed but was present within tissue as colonies or aggregates of cells. The differences in pathology observed in naturally infected chum salmon were produced experimentally with each pathogen by waterborne exposure of chum; coho, Oncorhynchus kisutch (Walbaum); and chinook salmon, Oncorhynchus tshawytscha (Walbaum). Severe decreases in circulating leucocytes accompanied bacteraemia caused by either bacterial species.     

苏丹鱼甘露糖受体的基因克隆表达和免疫特性

为了解苏丹鱼MR(Lh MR)的结构特点及其在抗感染免疫反应中的作用,本研究克隆并分析了LhMR基因,采用实时荧光定量PCR(qRT-PCR)技术检测了LhMR在正常及柱状黄杆菌感染苏丹鱼组织中的表达。结果显示,LhMR开放阅读框4296 bp,编码1431个氨基酸(aa)。LhMR的氨基酸序列和分子结构与其他物种高度相似,胞外1个富含蓖麻类β型三叶草的结构域(RICIN)、1个纤连蛋白Ⅱ型结构域(FNⅡ)、8个串连的C型凝集素样结构域(CLECTs)、1个跨膜区和1个胞内区。通过qRT-PCR检测显示,LhMR在脾脏、体肾、心脏、脑、皮肤、肌肉、鳃、肝脏、后肠、前肠和中肠11种组织中均有表达,其中脾脏中表达量最高;经柱状黄杆菌感染苏丹鱼后,脾脏、肠、体肾和鳃中LhMR基因的相对表达量显著升高。通过免疫组织化学(IHC)检测发现,在脾脏、肾脏和鳃中的LhMR蛋白表达水平提高。通过苏木精-伊红染色病理组织切片表明,在体肾、肠、肝脏和鳃中均有不同程度的病变。体肾的肾小管有大量的血细胞浸润,上皮细胞发生坏死;肠壁变薄,组织大量弥散坏死;肝脏组织中有多个空泡;鳃小片肿胀、混乱、坏死和脱落。研究表明,LhMR在苏丹鱼感染柱状黄杆菌免疫反应中发挥重要作用,为苏丹鱼柱形病的防控提供新的参考。     

An assessment of the variation in the prevalence of renal myxosporidiosis and hepatitis in wild brown trout, Salmo trutta L., within and between rivers in South-West England

The prevalence of renal myxosporidiosis in wild brown trout, Salmo trutta, in seven river catchments in South-West England was investigated. Three hundred and twenty-seven fish were sampled from 16 sites, of which 54 (16.5%) were found, by histological examination of the kidney, to be infected with Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease. No T. bryosalmonae infected fish were found in one river catchment, in other catchments the prevalence ranged from 2.5% to 36%. Hepatitis was strongly associated with the presence of T. bryosalmonae (odds ratio = 20.2, P < 0.001). Chloromyxum schurovi was found in 25% of fish and in six of seven river catchments, where the prevalence ranged from 2.4% to 63%. There was a strong negative association between the presence of T. bryosalmonae and C. schurovi (odds ratio = 0.10, P < 0.001). A hierarchical binomal model of the variance indicated that for T. bryosalmonae most of the variance existed at the site level, whereas for C. schurovi most variance existed at the river catchment level, suggesting that prevalence of T. bryosalmonae infection is determined largely by site level factors (e.g. presence of alternate host). The intraclass correlation coefficients (ICC) were 0.2 and 0.4 for T. bryosalmonae and C. schurovi, respectively, indicating the latter has higher effective transmission because of a higher level of infectiousness and/or abundance of alternate oligochaete hosts. These values can be used in future studies to estimate the sample sizes required to generate prevalence estimates with the required precision.     

罗非鱼湖病毒核蛋白的克隆表达、抗体制备及其组织分布

近年来罗非鱼湖病毒(TiLV)在多个国家流行,对世界罗非鱼养殖业造成严重威胁。中国是罗非鱼第一养殖大国,尽管我国大陆还没有TiLV的正式报道,鉴于吉富罗非鱼是我国重要的罗非鱼养殖品种,其对TiLV的感染特性研究具有重要意义。本实验采用TiLV对吉富罗非鱼进行人工感染,随后在肝脏组织中克隆和测定了TiLV第6片段基因。罗非鱼湖病毒第6片段基因cDNA全长1044 bp,开放读码框(ORF)为954 bp,编码317个氨基酸,预测分子量为36.38 ku;5′非编码区(NCR)为19 bp,3′非编码区(NCR)为972 bp。系统进化树分析表明该蛋白属于TiLV核蛋白(NP)。随后在大肠杆菌中表达和提纯了GST融合NP蛋白,在新西兰大白兔上制备了多克隆抗体。通过酶联免疫吸附实验(ELISA)检测抗体效价为1∶51200,且抗体可特异性识别感染组织中的病毒NP蛋白。对吉富罗非鱼不同组织进行苏木精—伊红(H.E)染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞出现空泡、坏死,含铁血黄素增多,头肾细胞坏死,鳃丝上皮细胞明显解离脱落,鳃小片黏连,脑组织细胞肿大。通过蛋白印迹法(WB)和免疫组化(IHC)对人工感染TiLV的吉富罗非鱼不同组织进行检测,结果显示,NP蛋白在肝脏、脑、体肾和头肾等组织中均有表达,以肝脏组织中表达量最高。为了解吉富罗非鱼对TiLV的免疫反应,通过实时荧光定量PCR(qRT-PCR)测定免疫因子TNF-α和TGF-β在主要免疫器官脾脏和头肾组织中的表达。研究表明,在感染早期(感染后12~24 h),病毒可显著抑制TNF-α和TGF-β在脾脏和头肾中的表达,可能通过抑制宿主这些免疫因子来促进病毒自身早期的复制。本研究将为进一步解读TiLV的致病机理及其高效防控提供理论基础。     

二倍体和三倍体皱纹盘鲍精子发生过程的超微结构

比较了二倍体和三倍体皱纹盘鲍精子发生过程中细胞和细胞器的超微结构变化。结果表明,二倍体皱纹盘鲍的精子发生经历了精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子5个阶段。其形态结构发生了一系列变化,主要包括：核染色质浓缩、线粒体的发达与融合、顶体形成和胞质的减少。三倍体皱纹盘鲍各种生精细胞的直径和核径均大于二倍体,精原细胞结构与二倍体相似;初级、次级精母细胞的胞质中,除溶酶体外,线粒体、内质网等细胞器少于二倍体,线粒体大小与二倍体没有差别,但形态不典型,层状嵴不发达;三倍体皱纹盘鲍的精子发生停滞在精子细胞阶段,表现出各种畸形状态,很多趋于解体,没有发现成熟精子。     

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post-smolts, almost all kidney macrophages ex vivo contain a low level of non-replicating virus

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.     

成年似鲇高原鳅外周血细胞及血细胞发生的观察

通过对成年似鲇高原鳅(Triplophysa siluroides)外周血细胞涂片及中肾、肝脏和脾脏组织的印片进行瑞氏(Wright’s)、过碘酸雪夫氏(PAS)和苏丹黑B(SBB)染色,观察各类血细胞形态、大小、分布及细胞化学特征。结果表明:似鲇高原鳅血细胞可分为红细胞、粒细胞、单核细胞、淋巴细胞和血栓细胞,各类血细胞的发生(除血栓细胞)均可分为原始、幼稚和成熟三个阶段。外周血中红细胞比例最多,为97.56%,白细胞中血栓细胞数量最多占52.86%,居白细胞的首位;三种粒细胞中嗜中性粒细胞数量最多占30.25%。粒细胞的主要发生场所在中肾占45.18%,红细胞的主要发生场所在脾脏,占71.62%,肝脏中未见原始血细胞。在中肾中同时可见嗜酸性、嗜碱性和嗜中性三种粒细胞的发生。各阶段的白细胞PAS染色均显示阳性,但只有各阶段的粒细胞SBB染色显示阳性。