Preliminary Morphometrics of Spleen and Kidney Macrophage Aggregates in Clinically Normal Blue Gourami Trichogaster trichopterus and Freshwater Angelfish Pterophyllum scalare

Abstract

Macrophage aggregate (MA) morphometry and pigment composition are believed to be dependent on the species, age, and health status of the fish. The aim of this study was to characterize a “normal” morphometry baseline of spleen and kidney MAs in blue gourami Trichogaster trichopterus and freshwater angelfish Pterophyllum scalare. Three size-classes of clinically normal fish were analyzed. Blue gourami and freshwater angelfish were obtained from three local ornamental fish farms; for each size-class, 10 fish from each farm were analyzed. Hematoxylin- and eosin-stained tissue sections were analyzed by light microscopy at 100× magnification and an image analysis program. The percentage of tissue occupied by MAs, MA size, and MA number were calculated on three arbitrarily selected fields of view from each spleen and kidney. In clinically normal blue gourami, increases in the percentage of tissue occupied by MAs and in MA size were associated with an increase in fish size, but in clinically normal angelfish no correlation was observed. Furthermore, in angelfish, a high variability in MA morphometry was observed, even among fish from the same sample group. In both species, a significant difference in the value of the morphometric parameters was observed among farms. Because iridoviruses inhibit macrophage activity and (possibly) proliferation, MAs in 25 clinical cases of iridovirus-infected blue gourami were analyzed. Preliminary data indicate that in iridovirus-infected blue gourami, there is a decrease in MA size and MA number compared with those of healthy fish.     

Isolation and Classification of a Nocardia species from Diseased Giant Gourami Osphronemus goramy

Abstract

An enzootic caused by bacteria occurred among cultured giant gourami in two freshwater fish farms in Kagoshima, Japan, in 1982. Typical external characteristics of the diseased fish were sloughed scales, exophthalmia, and opaque eyes. Internally, numerous creamy white nodules were found in the gills, spleen, kidney, and liver and a lesser number in the bladder and stomach. Nocardia sp. was isolated from spleen and kidney tissues of all diseased fish examined. To clarify the taxonomic position of the isolates, 164 tests were performed, and results were compared to reference strains of six species of Nocardia and one species of Rhodococcus. The isolates formed a homogenous group in a single taxon at a similarity of 98.7% based on an average linkage analysis and were clearly separated from the reference strains. The isolated strains were more similar to Nocardia kampachi than to the other representative species. However, 12 distinct differences were found between the isolates and N. kampachi, including growth on nutrient agar, sensitivity to 250 and 500 μg hydroxylamine/mL and 0.01% phenol, tolerance of 0.05% salicylate and 0.1% picric acid in brain-heart infusion medium, hydrolysis of starch, nicotinamidase reaction, and acid from fructose, sucrose, and dextrin. Accordingly, the isolates from the diseased giant gourami were classified as a new species of Nocardia.     

Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish

Abstract

Environmental contamination of ground and surface waters by perchlorate, derived from ammonium perchlorate (AP) and other perchlorate salts, is of increasing concern. Exposure to perchlorate can impair the thyroid endocrine system, which is thought to modulate renal and immune function in vertebrates. This study with zebrafish Danio rerio and eastern mosquitofish Gambusia holbrooki examined the histological effects of perchlorate on the trunk kidney, which in teleosts serves excretory and hemopoietic functions and therefore may be a target of perchlorate effects. Adult zebrafish of both sexes were exposed in the laboratory to waterborne, AP-derived perchlorate at measured concentrations of 18 mg/L for 8 weeks. Adult male mosquitofish were exposed to waterborne sodium perchlorate at measured perchlorate concentrations of 1–92 mg/L for 8 weeks. Control fish were kept in untreated water. The region of the body cavity containing the trunk kidney was processed from each fish for histological analysis. Macrophage aggregates (MAs), possible markers of contaminant exposure or immunotoxic effect, were present in the hemopoietic region of the kidney in both species exposed to perchlorate. The estimated percent area of kidney sections occupied by MAs was greater in zebrafish exposed to perchlorate at 18 mg/L (P < 0.05) than in controls. In male mosquitofish, the incidence of renal MAs increased proportionally with sodium perchlorate concentration and was significantly different from that of controls at 92 mg/L (P < 0.05). These observations confirm that in fish the kidney is affected by exposure to perchlorate. The concentrations of perchlorate at which the effects were noted are relatively high but within the range reported in some contaminated habitats.     

Preliminary investigations of hydrogen peroxide treatment of selected ornamental fishes and efficacy against external bacteria and parasites in green swordtails

The objectives of these preliminary studies were to evaluate the use of hydrogen peroxide (H2O2) for the treatment of selected species of ornamental fishes and its efficacy in treating external bacteria and parasites. In the first part of the study, fish of five species (serpae tetra Hyphessobrycon eques (also known as Serpa tetra H. serpae), tiger barb Puntius tetrazona, blue gourami Trichogaster trichopterus, suckermouth catfish Hypostomus plecostomus, and green swordtail Xiphophorus hellerii) were exposed to H2O2 for 1 h at concentrations between 6 and 34 mg/L or for 24 h at concentrations between 1 and 6 mg/L. The results were species specific: green swordtails tolerated all of the treatments, serpae tetras and tiger barbs were sensitive only to the highest concentration, and mortalities of suckermouth catfish and blue gourami were recorded in every treatment. In the second part of the study, clinically healthy green swordtails and fish infested with external motile rod-shaped bacteria (i.e., Ichthyobodo spp., Trichodina spp., and Gyrodactylus spp.) were treated with several concentrations of H2O2. A single H2O2 treatment of 3.1 mg/L or more for 1 h effectively eliminated external bacteria, concentrations of 6.5 mg/L or more appeared to effectively kill Ichthyobodo spp., and none of the treatments tested was effective against Trichodina spp. or Gyrodactylus spp. These preliminary findings suggest that H2O2 is effective for treating certain external bacterial infections and flagellate infestations in some species of ornamental fish at the dosages tested. Other treatment regimens may need to be tested for effectiveness against Trichodina spp. and Dactylogyrus spp.     

清远麻鸡禽白血病病理组织学观察和PCR诊断

本试验选择临床症状明显、经禽白血病抗原检测为阳性的15只祖代清远麻鸡,采集肝脏、脾脏、肾脏等器官进行病理组织学观察和PCR诊断。结果显示,从病理组织切片中观察到肝脏、肾脏、脾脏、骨髓、腺胃、心脏、肺脏、胰腺和卵巢等组织中有大量的淋巴母细胞呈灶状增生或散在分布,以及少量的成髓细胞增生。禽白血病病毒主要侵害祖代清远麻鸡机体的内脏器官和骨髓,对大脑等神经组织损害较轻;PCR扩增结果表明,所采集的15份样品均能扩增出545 bp左右的特异性片段,证明15份组织样品中均存在J亚型禽白血病病毒。     

Histology of some major immune system organs in Lake Van fish Alburnus tarichi (Güldenstädt, 1814) (Cyprinidae)

The teleostean immune system is variable between and within taxa in terms of morphology. Accordingly, in the current study, the histology of some immune organs (thymus, kidney and spleen) of Lake Van fish (Alburnus tarichi) was investigated. For this purpose, the tissues from eight mature fish were fixed and embedded in paraffin wax, and then the sections were stained with haematoxylin and eosin, Mallory’s triple, periodic acid-Schiff and Alcian blue (pH of 2.5) for histologic examinations. In the thymus, no clear discrimination of the cortex and medulla was observed. Lymphoid cells within a reticulo-epithelial network were the predominant cell type in the thymus. The other components in the thymus were macrophages and myoid, mast-like, plasma-like, cystic cells, Hassall’s corpuscles, and single or multiple epithelial cystic structures. In the kidney, the head kidney, a major haematopoietic site, consisted of lymphoid and non-lymphoid zones within a reticular network. Cells exhibiting mitotic figures were also detected in the haematopoietic tissue of the head kidney. Haematopoietic tissue was also found in the trunk kidney dispersed amongst the excretory components. The spleen was composed of red and white pulp. The red pulp comprised abundant erythrocytes, whilst the white pulp contained leucocytes with a reticular network. Ellipsoids were also determined in the white pulp. Melanomacrophage centres were found in all of the examined lymphoid tissues of the fish. These findings, which were reported, herein, for the first time will provide reference knowledge for future studies of this anadromous fish.     

Prevalence of Flavobacterium psychrophilum bacterial cells in farmed rainbow trout: Characterization of metallothionein A and interleukin1-β genes as markers overexpressed in spleen and kidney of diseased fish

The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10⁴ to 6 × 10⁷ bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10⁶ to 10⁷ bacteria/g of tissue in normally appearing fish vs 10¹¹ to 10¹² bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.     

Reno-, hepato- and splenomegaly of common carp fingerlings (Cyprinus carpio L.) diseased in swimbladder inflammation caused by Sphaerospora renicola Dyková et Lom, 1982

The weight of internal organs (swimbladder, kidney, liver, spleen) in relation to the body weight was studied in common carp fingerlings divided into three groups on the basis of swimbladder appearance and microscopic examination of the kidney. The fish had been collected from different Hungarian fish farms at the time when swimbladder inflammation (SBI) usually occurs (in July and August). The first group comprised fish with severe signs of SBI and massive renal sphaerosporosis, the second group consisted of fish with milder swimbladder changes and/or kidney infection by a low number of Sphaerospora renicola, while the third group was constituted by infection-free common carp fry. Statistical analysis of swimbladder, kidney, liver and spleen weight in relation to the body weight revealed that in the infected groups the internal organs were substantially enlarged. This suggests that in common carp fry with SBI the swimbladder changes are accompanied by reno-, hepato- and splenomegaly.     

Yersinia ruckeri septicaemia in experimentally infected carp (Cyprinus carpio L.) fingerlings.

The presence of Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in salmonids and a few other freshwater fish, has so far been reported from a variety of sources including the intestine of healthy carp. Since there are no data on the pathogenicity of this bacterium for carp, 15 fingerlings were experimentally infected by intraperitoneal injection of about 5 x 10(5) cells. Thirteen injected fish were moribund or died within 4 days with septicaemic lesions. Two survivors were sampled on Day 28 after infection. Yersinia ruckeri was reisolated from the internal organs of all experimental fish. By histopathological examination moribund fish had generalised bacteriaemia with inflammation, degeneration and necrotic foci in kidney, liver and spleen, corresponding to findings described previously in ERM of rainbow trout. Survivors of challenge on Day 28 had a chronic disease characterised by prominent peritonitis and enteritis, exhaustion of the erythroid, granuloid and lymphoid components in haematopoietic kidney tissue as well as focal degeneration and necrosis in organs. These data indicate a high sensitivity of carp to intraperitoneal infection with a relatively low dose of Y. ruckeri.     

Odontogenic hamartomas in cultured angelfish (Pterophyllum scalare)

BackgroundOdontogenic tumors present diverse clinical and histopathological behaviors that range from benign odontoma or hamartoma lesions to malignant tumors. In angelfish Pterophyllum scalare, findings of tumorous masses have been reported in the frontal region of the mouth, encompassing both the maxilla and mandible. Histologically, oral tumors have been characterized as angelfish lip fibroma or ameloblastoma.MethodsIn this report, we describe the occurrence of a compound odontoma/hamartoma in 60 angelfish from a population of 300, all of which were collected from a Mexican ornamental fish farm.ResultsAll fish presented a tumor-like mass on the rostral part of the mouth. Histological analyses revealed the tumor-like mass was covered by a hyperplastic stratified squamous epithelium, with fully differentiated denticles in various states of organization, immersed among normal dental tissues and showing a mild disorganized pattern. Radiograph analysis revealed radiopaque denticles in a radiolucent protuberance. Bacterial and viral isolation procedures and electron microscopy analysis revealed no presence of these microorganisms.Conclusion and clinical relevanceAlthough findings of odontogenic tumors in the mouth of angelfish are reported as sporadic, our study detected odontogenic mouth tumors in 60 angelfish, but the possible cause of alteration is not yet clarified. Further research is needed to clarify pathogenesis and to identify possible genetic abnormalities.     

Communications: Isolation of Several Mycobacterium Species from Fish

Abstract

Several species of marine fish caught in the wild and of freshwater ornamental fish were used in this study. Infected organs (liver, spleen, and kidney) were sampled for mycobacteria. Decontaminated tissue samples were plated onto selective media for mycobacterial recovery. After initial isolation, fluorescent and acid-fast staining techniques identified bacterial colonies to genus. Profiles of biochemical growth characteristics were used to further identify the isolates to species. Five species of Mycobacterium were identified: M. simiae, M. scrofulaceum, M. marinum, M. chelonae, and M. fortuitum. Of these, M. simiae and M. scrofulaceum have not been previously reported from fish. Tissue samples containing focal granulomatous lesions were prepared for electron microscopic examination.     

Isolation and distribution of bacterial flora in farmed rainbow trout from Mexico

Trout farming is a growing aquaculture industry in Mexico, with stock mainly supplied by the importation of eyed eggs. The aim of the present study was to determine the frequency of bacterial isolations in farmed rainbow trout Oncorhynchus mykiss from Mexico. Sixty-five farms distributed among seven states of Mexico were included in the study. Individual samples from gills, liver, spleen, intestine, and kidney were obtained from 563 apparently healthy fish. In total, 371 bacterial isolates were recovered from sampled fish; isolates of the genera Aeromonas, Edwardsiella, Enterobacter, Escherichia, Klebsiella, Plesiomonas, Pseudomonas, and Yersinia were identified. Aeromonads were the most frequently isolated bacteria. Renibacterium salmoninarum was not isolated from any of the sampled fish. Our results showed the presence of bacteria that are potential pathogens of both rainbow trout and humans.     

三聚氰胺对雌性Wistar大鼠的慢性毒性研究

三聚氰胺（melamine,MA）对小鼠的经口LD50为4550mg/（kg.bw）,急性毒性属于低毒物质。为证实和客观评价其慢性毒性,以每千克饲料500、1000和2000mgMA/kg饲料的剂量对6周龄雌性Wistar大鼠进行了6个月慢性毒性试验。结果表明,各剂量组雌性Wistar大鼠肾、肝和膀胱均有不同程度地功能异常和器质性病变;血液ALT、AST、CRE和UA等生化指标均有不同程度地升高,肾、肝和膀胱黏膜均有不同程度地病理变化或损伤;每千克饲料2000mgMA/kg饲料的剂量能够抑制大鼠生长和采食,肾病理变化显著,出现结石;染毒组大鼠脾脏、骨髓等未见病变;MA对大鼠的最大无作用剂量应小于（35.7±15.4）mg/（kg.bw.d）。     

安徽省部分猪场猪圆环病毒2型感染情况调查

本研究应用PCR技术对采集自安徽省3个猪场(FH、CX、SC)30头发病仔猪的脾、肺、肾、淋巴结共计120份样品,进行猪圆环病毒2型(PCV2)感染情况调查。结果显示,FH猪场PCV2感染率最高达70%,SC和CX两个猪场均为20%;脾、淋巴结PCV2的检出率与肺、肾之间差异显著。表明安徽省猪场的猪群中普遍存在PCV2的感染,且有的猪场PCV2感染的现象相当严重;进一步研究表明PCV2主要感染部位为脾和淋巴结。     

Ranking freshwater fish farms for the risk of pathogen introduction and spread

A semi-quantitative model is presented to rank freshwater rainbow trout farms within a country or region with regards to the risk of becoming infected and spreading a specified pathogen. The model was developed to support a risk-based surveillance scheme for notifiable salmonid pathogens. Routes of pathogen introduction and spread were identified through a process of expert consultation in a series of workshops. The routes were combined into themes (e.g. exposure via water, mechanical transmission). Themes were weighted based on expert opinion. Risk factors for each route were scored and combined into a theme score which was adjusted by the weight. The number of sources and consignments were used to assess introduction via live fish movements onto the farm. Biosecurity measures were scored to assess introduction on fomites. Upstream farms, wild fish and processing plants were included in assessing the likelihood of introduction by water. The scores for each theme were combined to give separate risk scores for introduction and spread. A matrix was used to combine these to give an overall risk score. A case study for viral haemorrhagic septicaemia is presented. Nine farms that represent a range of farming practices of rainbow trout farms in England and Wales are used as worked examples of the model. The model is suited to risk rank freshwater salmonid farms which are declared free of the pathogen(s) under consideration. The score allocated to a farm does not equate to a quantitative probability estimate of the farm to become infected or spread infection. Nevertheless, the method provides a transparent approach to ranking farms with regards to pathogen transmission risks. The output of the model at a regional or national level allows the allocation of surveillance effort to be risk based. It also provides fish farms with information on how they can reduce their risk score by improving biosecurity. The framework of the model can be applied to different production systems which may have other routes of disease spread. Further work is recommended to validate the allocated scores. Expert opinion was obtained through workshops, where the outputs from groups were single point estimates for relative weights of risks. More formal expert opinion elicitation methods could be used to capture variation in the experts' estimates and uncertainty and would provide data on which to simulate the model stochastically. The model can be downloaded (in Microsoft(?)-Excel format) from the Internet at: http://www.cefas.defra.gov.uk/6701.aspx.     

Effects of metomidate hydrochloride sedation on blood glucose and marketability of transported threespot gourami Trichogaster trichopterus

Our objectives were to determine whether sedation with metomidate hydrochloride (hereafter, "metomidate") during transportation of threespot gourami Trichogaster trichopterus would prevent an increase in blood glucose levels and improve fish marketability (i.e., based on appearance and behavior) in comparison with unsedated controls. Threespot gourami are obligate air-breathers that possess a labyrinth organ, enabling the fish to respire air above the water surface; these fish should be lightly sedated during transport. Fish were transported for approximately 24 h via truck and domestic airline. Blood was sampled at 0, 2, 6, and 12 h posttransport ation, and appearance and behavior were observed at 0, 2, 4, 6, and 12 h and 7 d posttransportation. Metomidate concentrations tested were 0.0 (control), 0.1, 0.2, 0.3, and 0.4 mg/L. At the concentrations tested, metomidate neither inhibited elevations in blood glucose nor improved marketability. Fish that were transported with 0.3-mg/L metomidate were less marketable based on behavioral indices, and fish that were transported with 0.4-mg/L metomidate had higher glucose levels than control fish. Use of metomidate as a transport sedative for threespot gourami should be considered with caution and may be problematic at the concentrations tested; however, further research examining additional indices of stress may clarify metomidate use for this species.     

Evaluation of a point-of-care blood analyzer and determination of reference ranges for blood parameters in rockfish

OBJECTIVE: To compare values of blood parameters in rockfish obtained by use of a point-of-care portable blood analyzer with values determined by a veterinary diagnostic laboratory, calculate reference ranges for various blood parameters in black rockfish, and compare values of blood parameters in clinically normal fish with those of fish with clinical abnormalities. DESIGN: Prospective study. ANIMALS: 41 captive adult black rockfish (Sebastes melanops) and 4 captive adult blue rockfish (Sebastes mystinus). PROCEDURE: Rockfish were anesthetized with tricaine methanesulfonate for collection of blood samples. Heparinized blood samples were immediately analyzed with a point-of-care analyzer. Blood sodium, potassium, chloride, urea nitrogen, and glucose concentrations; Hct; pH; partial pressure of carbon dioxide; total carbon dioxide concentration; bicarbonate concentration; base excess; and hemoglobin concentration were determined. A microhematocrit technique was used to determine PCV, and a refractometer was used to estimate total plasma protein concentration. Paired heparinized blood samples were transported to a veterinary diagnostic laboratory for analyses. RESULTS: Data obtained with the point-of-care analyzer were reproducible; however, values for most blood parameters were significantly different from those obtained by the veterinary diagnostic laboratory. Fish with poor body condition had several blood parameter values that were lower than corresponding values in clinically normal fish. CONCLUSIONS AND CLINICAL RELEVANCE: Point-of-care blood analyses may prove useful in rockfish. Point-of-care data for a large number of clinically normal fish must be obtained for reference ranges to be calculated, and further assessments of clinically abnormal fish are necessary to determine the relevance of the data.     

ULTRASOUND-GUIDED TISSUE-CORE BIOPSY OF LIVER, SPLEEN AND KIDNEY IN NORMAL DOGS

Six normal dogs were subjected to ultrasound-guided biopsy of the liver, spleen and kidney to examine the accuracy of the technique (i.e. the presence of targeted tissue) and the histologic quality of the biopsies. Five consecutive tissue-core biopsies of each organ were taken on one or more occasions. The accuracy of the technique was 77% for hepatic, 90% for splenic, 53.5% for left kidney and 40% for right kidney biopsies. The histologic quality of the liver and kidney samples was sufficient, although for some samples the diagnostic value was limited by their size and in renal samples either cortical or medullary tissue was sometimes lacking. In contrast, the quality of the splenic sections was not good. The effect of reused and resterilized needles on the quality of the specimens was evaluated by histologic inspection of the samples and by the amount of biopsies lacking tissue. All tissue samples, including those taken with reused or resterilized needles had sharp-cut edges. Twenty-two of the total number of 120 biopsies (18%) contained no tissue. Absence of tissue in the samples was observed in biopsies taken with all needle types. The animals were observed for possible complications of the repeated needle biopsy. Apart from one case of hematuria, no complications were encountered.     

堆型艾美耳球虫感染后组织抗氧化功能的变化

通过测定堆型艾美耳球虫(Eimeria acervulina)感染鸡心脏、肝脏、脾脏、肺脏和肾脏组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的水平,研究E.acervulina感染对雏鸡的组织抗氧化功能的影响。结果表明,雏鸡在感染E.acervulina后,早期各组织SOD活性均低于对照组水平,但很快都又恢复到正常对照组水平;各组织中MDA含量均高于对照组水平;心脏、肝脏和脾脏中CAT活性在感染后下降,肺脏和肾脏中CAT活性在感染后升高;各组织GSH-Px活性在感染后均有不同程度的下降。表明病理氧化应激参与了球虫感染过程,损伤了机体的氧化平衡。