Infection by intracellular bacterium in red claw crayfish, Cherax quadricarinatus (Von Martens), in Ecuador

An intracellular bacterium was detected on several occasions in commercial farms in Ecuador culturing red claw crayfish, Cherax quadricarinatus (Von Martens). Cases A, B and C presented mortality rates of 80%, 45% and 80%, respectively. Each incident occurred in grow-out ponds. Moribund crayfish were observed at the edge of the ponds in some cases. Affected crayfish presented no external differences from healthy ones, except their smaller size. Histological examination of affected red claw crayfish revealed that the cytoplasm of cells in the hepatopancreas, cuticular epithelium, and connective tissue of all organs had been replaced by massive granular and basophilic material. In the hepatopancreas, the tubules were intact but the intertubular connective tissue was replaced by Gram-negative microcolonies. Columnar cells in the cuticular epithelium also contained Gram-negative microcolonies and were necrotic. The heart and nerve cords also contained basophilic material. Transmission electron microscopy showed that the microcolonies consisted of a rod-shaped, obligately intracellular bacterium. The bacterium was delimited by a cell membrane that was composed of two electron-dense zones. Cells contained a single, compact, cytoplasmic condensation that was circumscribed by an electron-lucent halo. The bacterium replicated within the cytoplasm of host cells, which is pathognomic of rickettsiales infections. No host cell nuclei were infected.     

Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador

A new viral agent was found associated with the endoplasmic reticulum of epithelial cells of the Pacific white shrimp Litopenaeus vannamei (Boone) sampled during mass mortalities. A 40% mortality rate affected nursery and grow‐out ponds during the first 50–60 days of culture, and peak mortality in ponds occurred when shrimp reached 2–4 g. Histopathological changes of affected shrimp showed different grades of necrosis in epithelial cells and, in some cases, other tissues were affected. Transmission electron microscopy (TEM) of columnar cells of the cuticular epithelium showed the accumulation of viral particles, either dispersed in the cytoplasm or in a string‐like or paracrystalline array. These arrays of virions were within membrane‐bound vesicles formed from the endoplasmic reticulum (ER), in orderly arrays on the outer nuclear membrane or along the ER. The virus particles had apparently proliferated in the ER. The virions had an opaque area with an approximate diameter of 20 nm and an electron‐lucent surface layer. The approximate diameter of the non‐enveloped virions was 25 nm. The cytological changes observed are similar to those associated with the Picornaviridae and Nodaviridae families. The histopathology and ultrastructure of a new disease in L. vannamei is associated with the presence of a putative new virus. Until further isolation and characterization is performed, it is recommended to refer to the agent as Litopenaeus vannamei viral‐like particles (LvVLPs).     

Epitheliocystis in Atlantic salmon,Salmo salar L., farmed in fresh water in Ireland is associated with ‘Candidatus Clavochlamydia salmonicola’ infection

Intracellular inclusions containing chlamydia‐like organisms are frequently observed in the gill epithelial cells of Atlantic salmon, Salmo salar L., cultured in fresh water in Ireland. In this study, the causative agent was identified in four separate freshwater sites, using 16s rRNA sequencing, as ‘Candidatus Clavochlamydia salmonicola’. Histopathology and real‐time (RT) PCR were used to further assess infections. The prevalence of infection ranged from 75–100% between sites and infection intensity was highly variable. No significant lesions were associated with these infections. As a diagnostic tool, RT‐PCR proved marginally more sensitive than histopathology. The fate of ‘Candidatus Clavochlamydia salmonicola’ in Atlantic salmon post‐seawater transfer was investigated in a 12‐week marine longitudinal study. Both RT‐PCR and histopathological examination indicate that the organism disappears from the gills 4–6 weeks post‐transfer.     

Studies of the ultrastructure and morphogenesis of fish pathogenic viruses grown in cell culture

A birnavirus (infectious pancreatic necrosis virus, IPNV), three rhabdoviruses (viral haemorrhagic septicaemia virus, VHSV; infectious haematopoietic necrosis virus, IHNV; and spring viraemia of carp virus, SVCV) and an iridovirus (isolate from a sheatfish) were investigated with regard to their morphogenetic interactions with cells in culture. In cells infected with birnavirus, a granular viromatrix, single virions randomly distributed in the cytoplasm, viral particles aggregated in pseudocrystals and cytoplasmic tubuli similar in diameter to that of the virus were found. Rhabdoviruses entered the cells by viropexis and replicated within the cytoplasm. Maturation occurred predominantly at the cell membrane and sporadically at membranes of the Golgi cisternae. Inclusion bodies were found partially consisting of viral nucleocapsids. After budding, new virions were found adsorbed to the cell membrane. Viral haemorrhagic septicaemia virus, known to exhibit an atypical shape because of preparative procedures, could be identified by immunostaining using two monoclonal antibodies directed against G- and N-proteins and colloidal gold. Iridoviruses entered the cells by viropexis. Viral particles were found in coated vesicles. Subsequently, vesicles without a clathrin coat were detected. Replication occurred within prominent cytoplasmic inclusion bodies. Isometric viral nucleocapsids were transported in an unknown manner to the cell membrane and matured by budding.     

“红肉病”文蛤中寄生类立克次体的超微结构与细胞病理学

报道了患“红肉病”文蛤的消化盲囊上皮中所发现的类立克次体以及所引起的受感染细胞的病理学变化。该病原包涵体呈球形或椭球形,有强嗜碱性和嗜酸性两种,电镜下观察到上皮细胞内充满了大量的类立克次体,其形态结构及大小表现了极大的多样性,受感染的细胞具有明显的病理学变化,内质网膨大为潴泡状,核糖体脱落;线粒体溶解或固缩,核膜肿胀,染色质固缩并出现液泡结构,高度感染时,细胞核与大部分的细胞器消失,在上皮细胞中,观察到类立克次体以二分裂和出芽方式进行繁殖。     

Identification and characterization of a phospholipase A1 activity type three secreted protein,PP_ExoU from Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea)

Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in farmed Larimichthys crocea in China, encodes a predicted type three effector PP_ExoU, a homolog of the cytotoxin ExoU of Pseudomonas aeruginosa. In this study, secretion of PP_ExoU was tested in various broth, the protein was expressed with the pET30a prokaryotic system, the phospholipase A (PLA) activity of the recombinant protein was determined with fluorogenic phospholipid substrates, fusion expression with green fluorescent protein in transfected HeLa cells was investigated, and the lactate dehydrogenase (LDH) level was measured. The results showed the protein was type three secreted in several media; the recombinant protein displayed significant PLA1 activity with ubiquitin. Fluorescence was observed on the cell membrane and scattered in the cytoplasm of HeLa cells expressing catalytic wild‐type PP_ExoU, blebbing and stretching developed in the cell membranes indicating of membrane damage. Fluorescence scattered in the cytoplasm of cells expressing the catalytic inactive protein. A significant LDH level was detected in HeLa cells expressing wild‐type PP_exoU, but not in the Ser/Asp‐mutated protein, suggestion mutation of predicted catalytic residues abolished the PLA activity. This is the first report on the function of a secreted type three protein from P. plecoglossicida.     

Incidence of white muscle disease, a viral like disease associated with mortalities in hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii (De Man) from the south-east coast of India

Incidence of post‐larval mortalities of 30–100% was reported from commercial freshwater prawn, Macrobrachium rosenbergii (De Man) hatcheries in Andhra Pradesh and Tamil Nadu (south‐eastern states of India) since 2001. Infected postlarvae (PL) exhibited clinical symptoms with lethargy, anorexia and whitening of abdominal muscles and the disease was identified as white muscle disease (WMD). The waterborne infection of WMD was induced in the laboratory by exposing uninfected and healthy M. rosenbergii PL to the filtered muscle homogenates of the naturally infected PL, resulting in mortality that reached 99% within 10 days post infection. Histopathological examination of the infected animals revealed highly necrotic musculature. Degenerated muscle areas showed aggregations of melanized nuclei, many of which looked like inclusion bodies. Bacteriological examination of affected PL showed the presence of Staphylococcus spp. as a predominant organism, while laboratory challenge of healthy PL with this bacterial isolate did not reproduce WMD.     

A light and electron microscope study of epitheliocystis in juvenile steelhead trout, Salmo gairdneri Richardson

Abstract. Ultrastructural analysis of epitheliocystis organisms from gills of anadromous juvenile steelhead trout, Salmo gairdneri Richardson, revealed the presence of several distinct forms. Two oval infectious organisms resembled previously described agents and had chlamydia-like characteristics. One form had a distinct oval head region from which a tail-like structure projected. These prokaryotic forms have an ultrastructural appearance which has not been described in previous reports of epitheliocystis. Morphological analyses of gill epithelial cells of S. gairdneri suggest that each cyst remains contained within the cytoplasm of a single host cell.     

Granulomatous enteritis in rainbow trout (Oncorhynchus mykiss) associated with soya bean meal regardless of water dissolved oxygen level

This study investigated morphological changes associated with soya bean meal‐induced enteritis (SBMIE) in distal intestine (DI) of rainbow trout (Oncorhynchus mykiss) fed a soya bean meal (SBM)‐based diet and exposed to normoxia or hypoxia created by optimal and low water flow rates, respectively. A 28‐day adaption period was followed by a 42‐day challenge period where 600 fish were subjected to dietary challenge and/or hypoxia. Twelve tanks each containing 50 juvenile trout were assigned randomly in triplicate to each treatment. Histopathological and immunohistochemical evaluation revealed pathological features that have not previously been described in association with SBMIE. Vacuolar degeneration of epithelial cells mainly at the base of mucosal folds, epithelial cysts, epithelial dysplasia, necrosis, shedding of necrotic cells, and granulomatous inflammation including infiltration of enlarged, sometimes finely vacuolated or “foamy” macrophages, multinucleated giant cells and increased proliferation of fibroblasts were observed. Acid‐fast bacteria were not detected in enlarged macrophages; however, these cells contained AB‐PAS‐ and sometimes cytokeratin‐positive material, which was interpreted to be of epithelial/goblet cell origin. Hypoxia did not affect the morphological changes in DI. These results suggest that SBM was associated with a granulomatous form of enteritis in DI of rainbow trout regardless of water oxygen level.     

栉孔扇贝急性病毒性坏死症(AVND)病理学观察与分析

2000~2002年,应用光镜和电子显微技术对AVND发生期间栉孔扇贝(Chlamys farreri)各主要组织器官病理变化进行了连续观察。光镜观察显示,除生殖腺、闭壳肌外,濒死栉孔扇贝的外套膜、鳃、肾、消化腺和肠组织都有不同程度的组织病理变化。病灶主要出现在上述各器官的结缔组织以及上皮组织,病理变化表现为细胞核肿大、固缩、破裂,核染色质边缘化或空泡化,受感染细胞崩解、脱落,留下大片均质无结构的空白区域,形成凝固性坏死,结缔组织细胞质中有嗜碱性包涵体样颗粒存在。电镜观察显示,在病灶出现的组织细胞内,细胞核染色质异常凝集和边缘化、核膜周隙扩张或溶解。细胞器病理变化明显,内质网扩张,核糖体脱落,有髓鞘样小体出现;线粒体肿大、嵴融解,整个细胞出现解体现象。病变的结缔组织细胞与间质细胞细胞质中有大量直径为130~170nm、具有囊膜的球形病毒粒子存在,负染电镜观察表明,病毒囊膜表面覆有长20nm放射状纤突。病毒的发生基质在细胞质内,并被一膜性结构所包围。病毒在宿主细胞中的繁殖分为3个阶段,即病毒发生期、病毒装配期和病毒释放期。病理学观察证明,病毒感染与病理变化具有明显的相关性。     

Case report: concurrent herpesviral and presumptive iridoviral infection associated with disease in cultured shortnose sturgeon,Acipenser brevirostrum (L.), from the Atlantic coast of Canada

Approximately 8 weeks after a chlorine insult associated with the city water supply, shortnose sturgeon, Acipenser brevirostrum (L.), from one group presented with small (3–4 mm) irregular foci of cutaneous pallor that involved the dorsocranial integument with progressive ulceration of the nascent lesions. Various bacterial organisms were isolated from the cutaneous lesions, but not from the internal viscera. Histologically, the nuclei of the intralesional and perilesional epidermal cells often exhibited margination of the chromatin that resulted in a homogenous, pale, amphophilic, tinctorial quality of the nucleoplasm consistent with a herpesvirus infection. In addition, rare lamellar epithelial cells were prominently enlarged due to an abundant, dense, basophilic cytoplasm characteristic of an iridovirus infection. Inoculation of cutaneous lesion and kidney, spleen, liver sample pools from affected shortnose sturgeon onto white sturgeon spleen (WSS‐2) cell line induced cytopathic effect characterized by syncytia formation. Ultrastructural analysis of infected WSS‐2 cells revealed viral particles with a characteristic herpesvirus morphology. Intranuclear hexagonal capsids had a diameter of 95–108 nm, and enveloped particles present in the cytoplasm of infected cells had a diameter of 176–196 nm. This is the first report of a herpesvirus and a possible iridovirus‐like infection in shortnose sturgeon.     

Cell types and structures involved in tench,Tinca tinca (L.), defence mechanisms against a systemic digenean infection

Histopathological and ultrastructural investigations were conducted on 36 tench, Tinca tinca (L.), from Lake Trasimeno (Italy). The gills, intestine, liver, spleen, kidney and heart of 21 individuals were found to harbour an extensive infection of larvae of an unidentified digenean trematode. The eyes, gonads, swim bladder and muscles were uninfected. The parasites in each tissue type were embedded in a granulomatous proliferation of tissue, forming a reactive fibroconnective capsule around each larva. Most of the encysted larvae were metacercariae, in a degenerative state, but on occasion some cercariae were found. Many of the granulomas were either necrotic or had a calcified core. Within the granuloma of each, the occurrence of granulocytes, macrophages, rodlet cells and pigment‐bearing macrophage aggregates was observed. Hearts bore the highest parasitic infection. Whilst the presence of metacercariae within the intestine was found positioned between the submucosa and muscle layers, metacercariae in the liver were commonly found encysted on its surface where the hepatocytes in close contact with the granuloma were observed to have electron‐lucent vesicles within their cytoplasm. Metacercariae encysting adjacent to the cartilaginous rods of gill filaments were seen to elicit a proliferation of the cartilage from the perichondrium. Rodlet cells, neutrophils and mast cells were frequently observed in close proximity to, and within, infected gill capillaries. Given the degenerated state of most granulomas, a morphology‐based identification of the enclosed digeneans was not possible.     

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD‐GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related genes (Bax, Bid, Bad and Bcl‐2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase‐3 was significantly activated. The mRNA expression of the Bad gene showed significant up‐regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up‐regulated expression at 72 and 96 h.p.t. and anti‐apoptotic gene (Bcl‐2) was down‐regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.     

褶纹冠蚌珍珠囊发育的超微结构观察

本文报道了用透射电子显微镜对不同发育阶段褶纹冠蚌(Cristaria plicataLeach)珍珠囊的超微结构进行观察的结果。观察表明,珍珠囊上皮细胞在发育的各阶段都具有发达的微绒毛、基底迷路结构和胞间空间;线粒体、核糖核蛋白体十分丰富,高尔基体非常活跃。在发育的早期,上皮细胞及核都呈柱状,常见细胞器的自溶现象以及板层小体的形成;核糖核蛋白体大多呈游离解聚状态。在发育的晚期,上皮细胞呈立方形或扁平状,核的形状不规则;大量的、梅花状的多聚核糖核蛋白体形成。珍珠囊附近的结缔组织主要由成纤维细胞、类结缔组织钙细胞、肌纤维、胞间微纤维、无核细胞样结构和细胞碎片组成。     

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.     

Histopathological survey of diseases and pathogens present in redclaw crayfish, Cherax quadricarinatus (Von Martens), cultured in Ecuador

A histopathological survey of commercial farms in Ecuador culturing redclaw crayfish, Cherax quadricarinatus (Von Martens), revealed the presence of different viral, bacterial and non‐infectious pathologies. A total of 536 animals from 27 different farms were analysed during a 3‐year period; 25, 154 and 357 specimens in 1995, 1996 and 1997, respectively. From the total sampled, a virus referred to as Cherax quadricarinatus bacilliform virus (CqBV) had a prevalence of 44% in animals examined during 1996 and 33% in 1997. A rickettsia‐like organism (RLO) was detected in five specimens sampled from three different farms in 1995. This RLO was detected in 36 and 12% of samples analysed in 1996 and 1997, respectively, and was the infectious agent most frequently associated with mortalities above 60%. Co‐infection by CqBV and the RLO was observed in 18 and 3% of the crayfish examined in 1997 and 1998, respectively. Five specimens out of 25 examined presented no sign of an infectious agent during 1995. The proportion of specimens testing negative by histopathology to known or suspected infectious agents was 25 and 32% in 1996 and 1997, respectively. Although different infectious agents were present in cultured crayfish, farms in general had overall survivals higher than 50% in most ponds. Other conditions observed included an intracellular prokaryotic organism, iron granules in the R and F cells of the hepatopancreas, different species of epibionts on the surface of the gills and body, and two cases of haemocytic enteritis. Two cases of serious mortality in different farms were associated with the presence of Cowdry type A inclusions, pyknosis, karyorrhexis and haemocytic infiltration in the stomach hypodermis and the antennal gland.     

The response of intestinal mucous cells to the presence of enteric helminths: their distribution,histochemistry and fine structure

Histochemical and ultrastructural investigations were conducted on the mucous cells of the intestine of brown trout, Salmo trutta L., naturally infected with the cestode Cyathocephalus truncatus (Pallas, 1781) and the acanthocephalan Echinorhynchus truttae Shrank, 1788. A subpopulation of 45 S. trutta were examined of which 15 specimens harboured E. truttae, 15 of which were infected with C. truncatus and 15 fish, the control group, were uninfected. In histological sections, hyperplasia and hypertrophy of the mucous cells were evident at the site of parasite infection. Enhanced mucus secretion was also recorded in infected fish. The number of mucous cells close to the site of parasite attachment within the intestine was significantly higher than the number detected in uninfected individuals and in infected individuals at sites 1 cm or greater from the point of parasite attachment. There were no significant differences between the number of mucous cells found at the latter two sites. Alcian blue and periodic acid‐Schiff’s staining of representative histological sections revealed a significant increase in the number of mucous cells staining positively for acid glycoconjugates compared to the number of cells found in the intestines of uninfected S. trutta. In transmission electron microscopy sections, each mucous cell typically possessed an elongated, basally positioned nucleus. The cytoplasm was observed to possess numerous electron dense and lucent vesicles, in addition to well‐developed rough endoplasmic reticulum, Golgi apparatus and a few round mitochondria.     

中华绒螯蟹围食膜结构观察及其主要蛋白质种类的鉴定

为揭示中华绒螯蟹围食膜的分泌、形态和功能,实验采用组织切片技术和电镜技术观察了中华绒螯蟹消化道及围食膜的基本形态,采用几丁质酶溶解法和SDS-PAGE电泳法研究了围食膜的基本组成成分,采用液相色谱电离串联质谱(LC-ESI-MS/MS)分析了围食膜蛋白的种类。显微切片结果显示,中华绒螯蟹整个消化道表面都有围食膜存在,除中肠外围食膜区室化作用明显;围食膜与上皮细胞分离时,柱状细胞变成不规则圆形,核消失。电镜结果显示,围食膜形成时上皮细胞内含有大量线粒体和分泌泡;中肠上皮细胞表面密生微绒毛。围食膜能被几丁质酶溶解,围食膜蛋白质SDS-PAGE图谱显示有7条比较清晰的电泳条带,蛋白质分子量主要分布在40 ku以上。LC-ESI-MS/MS分析结果发现,中华绒螯蟹围食膜蛋白主要包括钠、钾-ATP酶、ATP合成酶、肌动蛋白、微管蛋白、硫氧还原蛋白、加氧酶等。研究表明,中华绒螯蟹的围食膜由上皮细胞分泌形成,可能参与免疫、渗透调节和营养物质转运等生理过程。     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

Nephroblastoma in bester sturgeon,a cultured hybrid of Huso huso × Acipenser ruthenus: Diagnostic imaging,clinical and histopathological study

Abdominal distention occurred at an incidence of 1% (15 from 1500 fish) in the population of 1‐year‐old bester (Huso huso × Acipenser ruthenus). Computed tomography (CT) images and radiographs showed a soft tissue mass compressed the posterior part of the swim bladder. Ultrasonography showed that the masses had different patterns. Internal examination revealed the abdominal cavities to be filled with large masses which appeared to encompass most of the visceral organs, including the swim bladder. The masses originated from the posterior kidney. Histologically, the masses were composed of mixtures of embryonal epithelial (tubules and glomeruli), blastema and mesenchymal tissues. The tubules showed cystic, papillary and tubular patterns. Tubules and glomeruloid structures were surrounded by proliferating blastema cells. The primitive mesenchyme was composed of loose streams and whorls of spindle to stellate cells with elongate nuclei. Histological findings in the skeletal muscles, hypoderm and spleen confirmed the metastatic tumour from the kidney in two cases. Immunohistochemically, neoplastic cells of the tubules and glomeruloid structures were positive for cytokeratin AE1/AE3. Sections stained with Masson's trichrome showed blue staining of the stroma. The histopathologic findings were consistent with nephroblastoma.