长毛对虾海水养殖环境以及虾肠道微生物群落结构研究

为了研究长毛对虾养殖环境以及对虾肠道微生物种群结构的特征,实验分别采集养殖区进水口水体、养殖池底泥、养殖池水体以及长毛对虾肠道样品,采用构建16S rRNA基因克隆文库的方法对不同样品间的微生物群落组成进行了研究。结果表明,4组样品中共获得621条序列,操作分类单元(OTU)总数达212个,表明养殖环境微生物群落结构具有高度的多样性。从遗传进化树分析发现,进水口水体中细菌优势种群为蓝细菌(53.97%)、α-变形杆菌(13.76%)和γ-变形杆菌(10.58%);养殖池水体细菌优势种群为蓝细菌(33.55%)、γ-变形杆菌(14.84%)、厚壁菌(14.19%)、拟杆菌(12.26%)和α-变形杆菌(9.68%);养殖池底泥细菌优势种群大部分属于厚壁细菌(79.12%);对虾肠道细菌优势种群为厚壁细菌(75.79%)、梭杆菌(13.68%)和γ-变形杆菌(10.53%)。在目分类水平上,养殖池底泥、养殖池水体和对虾肠道中芽孢杆菌占有较高的比例,分别占克隆数的69.78%、13.55%和72.63%;进水口水体和养殖池水体中红细菌的比例较高,分别占克隆数的10.05%和9.68%。本研究分析了养殖环境以及对虾肠道微生物的群落结构,揭示微生物从水源到对虾肠道内的演替规律。总体上,本养殖系统微生物群落结构良好,但在养殖池水体和对虾肠道中也检测到黄杆菌类群和少量的弧菌。本研究有助于了解养殖环境对于对虾肠道微生物组成的影响,并为长毛对虾病害的预防提供参考。     

健康和患病凡纳滨对虾幼虾消化道菌群结构的比较

凡纳滨对虾养殖过程中,早期阶段是病害易感阶段,而消化道菌群结构与对虾健康关系密切。因此,探讨幼虾的消化道菌群尤其是弧菌类细菌与对虾发病的关系对病害有效防治具有重要意义。本实验采用Illumina测序研究了凡纳滨对虾健康和患病幼虾的消化道细菌群落结构,并基于纯培养和16S r DNA,rec A和pyr H基因序列比对,分析了幼虾消化道中弧菌的主要种类组成。结果发现,健康幼虾消化道中α-变形菌纲和厚壁菌门丰度较高,而患病幼虾中γ-变形菌纲、拟杆菌门、放线菌门和β-变形菌纲较高,其中放线菌门丰度差异显著。基于科水平的响应比分析,发现患病幼虾消化道中动性球菌科和噬菌弧菌科的丰度显著降低,而弧菌科的丰度显著升高。相似度分析发现,驱动群落变异的OTU主要来源于弧菌属、海洋杆状菌属、冷杆菌属、假交替单胞菌属以及未分类至属的红杆菌科和微杆菌科。健康幼虾消化道弧菌组成以锡那罗亚州弧菌为主,而患病幼虾消化道弧菌组成以坎氏弧菌为主。尽管健康和患病幼虾消化道内细菌群落结构整体差异不显著,但一些重要类群丰度变化显著,其特征与已知的生态功能一致。     

Bacterial Change in the Gut and Hepatopancreas of Tiger Shrimp Reared in Bioaugmented System

The study was conducted in nine 150‐L capacity glass aquaria for 192 h to determine the changes in the microflora in the gut and hepatopancreas of tiger shrimp, Penaeus monodon, after bioaugmenting (water treatment and probiotics). Results showed that luminous bacterial counts were significantly lower in bioaugmented than in nonbioaugmented systems (range 0–5.9 × 10³ cfu/g and 0–3.2 × 10³ cfu/g in gut and hepatopancreas, respectively). Biochemical tests of isolates showed that the bioaugmented systems were dominated by Gram‐positive Bacillus and Streptococcus and the Gram‐negative Vibrio dominated the control. In water‐treated and probiotics‐fed bioaugmented system, the change in bacterial dominance to Bacillus became evident 2 h in the gut and 24 h in the hepatopancreas. Bacterial dominance shifted to Vibrio species after 120 h. Shrimp in the control were observed to become lethargic after 72 h as Vibrio species significantly increased in number. Dissolved oxygen and unionized ammonia levels were significantly lower in bioaugmented system compared with the control.     

A Review of Probiotics in Shrimp Aquaculture

Development of shrimp aquaculture has been associated with increases in infectious diseases and environmental degradation. An effective alternative to chemicals and antibiotics used for the prevention of these problems is to administer probiotics into the rearing system. Three bacterial genera, Bacillus, Vibrio, and Pseudomonas, are commonly administered as probiotics in shrimp aquaculture. Candidate probiotics are species specific and need to be tested for their effectiveness for certain species in in vitro and in vivo. Supplementation into feed is more effective in conveying probiotics into animals compared to direct application into rearing systems. Overdosage or prolonged administration of probiotics can induce immunosuppression. A cell-density of 10⁵ colony-forming units (CFU) per ml is widely recommended. A combination of probiotics results in better outcomes for the host than individual probiotics. Probiotics improve water quality while reducing pathogenic bacteria. Probiotics show positive effects through an improvement in the physiological and immune responses of shrimps. Probiotics are increasingly becoming important and more common in any organic shrimp farming.     

Development of primers and a procedure for specific identification of the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.     

Effects of a probiotic (Bacillus subtilis FY99‐01) on the bacterial community structure and composition of shrimp (Litopenaeus vannamei,Boone) culture water assessed by denaturing gradient gel electrophoresis and high‐throughput sequencing

In China, many commercial probiotics are added as water additives into shrimp culture ponds with no sediment. In these ponds, probiotics should have great effects on the bacterial community of culture water at different culture stages. However, the specific effects have not been clarified yet. In this study, the effects of applying the probiotic Bacillus subtilis FY99‐01 on the bacterial community of shrimp (Litopenaeus vannamei) culture water at different culture stages were determined by denaturing gradient gel electrophoresis and high‐throughput sequencing. The water quality parameters, shrimp production, survival rate and feed conversion ratio (FCR) were also analysed. The results showed that application of probiotics improved the water quality by reducing the levels of pH, nitrite and soluble reactive phosphorus, whereas there was no significant effect on shrimp production, survival rate and FCR. Application of probiotics brought no obvious effect on bacterial α diversity while had effect on bacterial β diversity. In addition, probiotics brought different effects on the bacterial community structure at different culture stages, and the effects are stronger in the early and middle phase than in the late phase. Sequencing results demonstrated that probiotics could increase the abundance of Flavobacteria, whereas decrease the abundance of α‐Proteobacteria in the late phase of culture. Besides, principle component analysis results indicated that using probiotics also could increase the abundance of beneficial microalgae (Bacillariophyta and Chlorophyta) and decrease the abundance of Vibrionaceae.     

Enhancement of growth performance, digestive enzyme activities and disease resistance in black tiger shrimp (Penaeus monodon) postlarvae by potential probiotics

Outbreaks of multidrug-resistant pathogens have been constraint on aquaculture production in Thailand, thereby controlling shrimp pathogens by preventive probiotics being importance to sustain the aquaculture system. In this study, the effect of potential probiotics, Bacillus subtilis and Enterococcus sp. was related to growth, digestive enzyme activities (trypsin and chymotrypsin) and pathogenic resistance by postlarval black tiger shrimp (Penaeus monodon). The experiment was divided into two treatments, in triplicate, with and without supplementation of probiotics as a food additive. Shrimp fed with probiotics for a culture period of 84 days showed a significant increase (P < 0.05) in weight (2.03 ± 0.29 g) and survival (71.91 ± 3.15 %) in comparison with non-treated shrimp (1.53 ± 0.28 g and 65.20 ± 5.68 %, respectively). Trypsin activity of treated shrimp was significantly higher (P < 0.05) than that of control shrimp whereas chymotrypsin activities of the two treatments were not significantly different (P > 0.05). After challenging P. monodon postlarvae with a shrimp pathogen, Vibrio harveyi for 10 days, percentage of mortality of P. monodon postlarvae fed with probiotics was 46.67 ± 1.44 %, significantly lower (P < 0.05) than that of non-treated shrimp (61.67 ± 6.29 %). This study showed that potential probiotics was appropriate for application in P. monodon postlarvae cultivation under laboratory condition due to improvement of shrimp weight and survival, enhancement of trypsin activity and reduced mortality causing by pathogenic V. harveyi. This is the first publication reported the effect of probiotics on trypsin and chymotrypsin activities of P. monodon postlarvae.     

凡纳滨对虾不同生长阶段肠道可培养细菌耐药性研究

在凡纳滨对虾(Litopenaeus vannamei)养殖过程中多使用微生态制剂来调节水质,为避免破坏池塘菌群结构,很少使用抗生素.为了解凡纳滨对虾肠道细菌耐药性与不同生长阶段的关系,本研究选取江苏地区4种主要养殖模式凡纳滨对虾成虾和虾苗作为研究对象,利用K-B纸片法和qRT-PCR技术,研究对虾样本肠道可培养细菌对...     

健康与患病凡纳滨对虾肠道菌群结构及功能差异研究

为探究病害发生后健康与患病凡纳滨对虾肠道菌群结构和功能的差异,并筛选肠道指示菌群来评估宿主健康状况,评价凡纳滨对虾肠道菌落的功能冗余性。实验采集健康和患病凡纳滨对虾样品,通过Illumina高通量测序技术测定肠道菌群组成,并利用PICRUSt进行功能预测,以此比较健康与患病凡纳滨对虾肠道微生物的群落结构和功能差异,并预测功能与群落组成的相关性。结果显示,病害的发生伴随着肠道菌群结构的显著变化,而多样性无显著差异。与健康凡纳滨对虾肠道细菌组成相比,患病凡纳滨对虾肠道中放线菌门(Actinobacteria)、浮霉菌门(Planctomycetes)和疣微菌门(Verrucomicrobia)相对丰度降低,而γ-变形菌纲(Gammaproteobacteria)和拟杆菌门(Bacteroidetes)增加。同时,筛选出16个指示细菌科,能够很好地指示宿主健康状况。与健康组相比,患病凡纳滨对虾中参与弧菌侵染的过程显著增加,而溶酶体和过氧化物酶等免疫功能代谢过程显著减弱。此外,肠道微生物群落结构与功能组成呈显著正相关,表明凡纳滨对虾肠道菌群组成具有较低的功能冗余性。研究表明,健康与患病凡纳滨对虾肠道菌群结构存在显著差异,并且由细菌介导的功能随之发生改变,能够用指示微生物评估宿主健康状况。     

Integrated application of two different screening strategies to select potential probiotics from the gut of channel catfish Ictalurus punctatus

In this study, 342 bacterial strains were isolated from the gut of channel catfish Ictalurus punctatus. Two different screening strategies, including the production of antagonistic effects against pathogens and the capacity of secreting exoenzyme, were used to select potential probiotics from the bacterial strains. Among them, 6 isolates showed antagonism against the pathogens, 15 isolates could produce high amylase and protease, and 3 isolates (BHI344, BH0142 and BH03124) had both the two characteristics . Then, BHI344, BH0142, and BH03124 were tested for survival rate in channel catfish bile and at low pH, antimicrobial susceptibilities, and pathogenicity in channel catfish. The results of the obtained assays showed that BHI344 possessed the broadest inhibitory potential, a strong ability of secreting extra-cellular amylase and protease, high viability in simulated gastrointestinal conditions, and sensitivity to most of the used antibiotics and non-pathogenic to channel catfish. Therefore, it could be selected as the most promising probiotics candidate. BHI344 was identified as Bacillus subtilis based on the phenotypic characterization and 16S rRNA gene sequencing.     

虾肝肠胞虫感染对脊尾白虾肠道菌群的影响

为探究虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)感染对脊尾白虾(Exopalaemon carinicauda)肠道菌群的影响,本研究基于16S rRNA基因的测序结果,对感染EHP的脊尾白虾肠道菌群进行了分析。结果显示,感染虾与健康虾肠道菌群差异较大,且其肠道菌群结构多样性显著低于健康虾。研究发现,病虾中归属于变形菌门(Proteobacteria)的脱硫弧菌科(Desulfovibrionaceae)、弧菌科(Vibrionaceae)、未分类蓝细菌科(unidentified Cyanobacteria)、支原体科(Mycoplasmataceae)和未分类α变形菌科(unidentified Alphaproteobacteria)为优势菌,而归属于厚壁菌门(Firmicutes)的乳杆菌科(Lactobacillaceae)、双歧杆菌科(Bifidobacteriaceae)、芽孢杆菌科(Bacillaceae)及噬几丁质杆菌科(Chitinophagaceae)细菌在健康虾中占据优势地位。EHP侵蚀导致感染虾肠道内潜在致病菌显著增加(P<0.05),增加了其他疾病的易感性。此外,通过Tax4Fun功能预测,发现感染虾肠道菌群主要用于新陈代谢,从而抵抗EHP侵染,维持机体正常功能;健康虾肠道菌群则多用于个体生长与环境信息处理,进而保证生长与存活。本研究从虾肠道菌群结构方面入手,进一步探究了EHP感染对脊尾白虾肠道菌群的影响,以期为EHP的防治提供帮助。     

Analysis of the composition of the bacterial community in puffer fish Takifugu obscurus

The food chain has been postulated as one pathway for tetrodotoxin (TTX)-producing bacteria to enter into fish. However, the background composition of the bacterial community in puffer fish is unclear. Using 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) technology, we investigated the composition of bacterial communities in the skin, intestines, and TTX-accumulating organs (ovary, liver) of the striped puffer fish Takifugu obscurus. A total of 38 species of different culture-independent bacteria were isolated and classified according to phylogenetic analysis. Of these species, those belonging to the class γ-Proteobacteria dominated the microbial community in the puffer fish, while others belonged to the group of low DNA G + C content, Cytophaga–Flavobacterium–Bacteroides (CFB) group, α-Proteobacteria, β-Proteobacteria, ε-Proteobacteria and Spirochaetales. Gram positive bacteria of the group of low DNA G + C content were seen as the dominant component in the intestine, while γ-Proteobacteria was the dominant group in the skin or TTX-accumulating organs. The TTX-accumulating organs were found to contain bacteria from all TTX-producing genera reported previously. We also observed large differences in the bacterial assemblages in the intestines of fish fed natural and artificial diets.     

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 10⁷ CFU ml^− 1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 10⁵ to 10⁸ CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD₅₀ to BALB/c mice was found to be 10⁹ CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections.     

Species diversity of the marine diatom genus Skeletonema in Japanese brackish water areas

The genus Skeletonema includes phytoplankton species that are important primary producers in marine food chains. Brackish waters have been reported to be one of the important habitats of some species of Skeletonema. To elucidate the species diversity of Skeletonema in brackish waters, we investigated three Japanese brackish bodies of water: the coastal waters of Toyama Bay, a tidal area of the Chikugo River, and a constructed reservoir in Isahaya Bay. We used molecular analysis based on large subunit rDNA and fine morphological structure to identify species. Skeletonema costatum s.s. (sensu stricto) was isolated at salinities as low as 0.6, but Skeletonema dohrnii, Skeletonema subsalsum, and Skeletonema tropicum were not found at salinities below 11.0. S. costatum s.s. could survive transfer from a medium with a salinity of 15 to a salinity of 2, but S. dohrnii did not survive in the same experiment. Only S. costatum s.s. germinated from the sediment of a reservoir in which the salinity was 0.1–1.4; incubation conditions included temperatures of 10, 15, 20, 25, and 30 °C and salinities of 5 and 30. Skeletonema costatum s.s. was identified as the species most adaptable to low-level salinity variations throughout its lifecycle.     

饲料中添加益生菌对凡纳滨对虾抗感染和5种免疫基因表达的影响

为研究饲料中添加益生菌对凡纳滨对虾抗副溶血弧菌感染能力和非特异性免疫基因表达水平的影响,以初体质量为(6.95±1.20)g的凡纳滨对虾为研究对象,在室内养殖箱进行3周的养殖实验和2周的副溶血弧菌人工感染实验;其中,对照组每日投喂普通商品饲料,实验组每日投喂在普通商品饲料中添加地衣芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌/枯草芽孢杆菌(1:1)配制成的3组实验饲料,实验饲料中益生菌的终浓度为10⁷ cfu/g。并采用实时荧光定量RT-PCR方法,对保护率最高的地衣芽孢杆菌/枯草芽孢杆菌实验组进行凡纳滨对虾相关免疫基因表达水平的分析。实验结果表明,在饲料中添加单一益生菌或复合益生菌均可显著提高对虾抗副溶血弧菌感染的能力(P<0.05),且复合益生菌的保护效果更佳,其相对免疫保护率为31.11%。感染副溶血弧菌后,地衣芽孢杆菌+枯草芽孢杆菌实验组凡纳滨对虾血淋巴中的先天免疫缺陷基因(innate immune deficiency gene,IMD)、对虾素3a分子(penaiedin 3a)、酚氧化酶原(prophenoloxidase,proPO)、溶菌酶(Lysozyme,LZM)和甲壳素Crustin的mRNA的相对表达量均显著上调,且分别在18～24 h达到最大值。实验结果提示:饲料中添加芽孢杆菌可有效提高凡纳滨对虾抗副溶血弧菌感染的能力,这种能力的提高可能是通过增加抗病相关基因的表达量实现的。     

Characterization of bacterial community in intestinal and rearing water of Penaeus monodon differing growth performances in outdoor and indoor ponds

Penaeus monodon provides a high‐quality protein source for humanity, and pond cultured shrimp often presents asynchronous growth. Microbial communities are important for the digestion and immunity of shrimp. Therefore, in this study, we investigated the bacterial characteristics of the intestine and rearing water of asynchronously growing P. monodon that were cultured in outdoor and indoor pond respectively. The results showed that the bacterial community of the rearing water was more complex than that of the intestine; the fast‐growing shrimp in the indoor pond had higher intestinal bacterial diversity. Besides, the dominant bacterial composition of the water was more complex than that of the intestinal. Specifically, the abundance of Proteobacteria in the intestine was consistent with the growth performance of shrimp in the outdoor pond, which was exactly the opposite in the indoor pond. At the genus level, two cyanobacteria, Limnothrix and Cyanobium PCC‐6307, were dominant in the indoor and outdoor water respectively. In the outdoor pond, Bacillus was dominant in the slow‐growing shrimp intestines, while Vibrio was dominant in the fast‐growing shrimp. The intestinal microbes of the fast‐growing shrimp had higher proteasome metabolic capacity. These results can provide new insights into microbiome characteristics involved in the asynchronous growth of shrimp.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

Evaluation of microalgae diets for Litopenaeus vannamei larvae using a simple protocol

We evaluated native marine microalgae isolated from NortheasternVenezuela, as food for Litopenaeus vannamei larvae,compared to six microalgae commonly used in aquaculture. We evaluated themicroalgal ingestability calculating the biomass consumed by larvae, later weevaluated three monoalgal diets on protozoea stages of L.vannamei over 72 hr based on the larval biomass,RNA/DNAindex and the proportion of stage retarded larvae. The presence of everymicroalgae species in the protozoea's digestive tract indicated theirconsumption. Chaetoceros spp. showed high ingestabilityandTetraselmis spp. and Skeletonema sp.Ch1 showed low ingestability. The native microalgaeChaetoceros sp. A1 and Chaetocerossp.G1 were evaluated as monoalgal diets compared to Chaetocerosgracilis. Ch. gracilis demonstrated the bestresults when the evaluation was performed with the RNA/DNA index. However, thesurvival rate, larval biomass and percentage of stage retarded larvae fed withChaetoceros sp. G1 were not statistically different fromthose fed with Ch. gracilis. Taking into account thatnative microalgae produced higher biomass in shorter times when they werecultured in field conditions, the results suggest that native microalgae areadequate for large-scale culture conditions, thus reducing costs. Thedetermination of microalgal size and the larval consumption, dry and organiclarvae biomass and specially the RNA/DNA index, were valuable features of thisevaluation. The protocol developed in this study could be used to evaluatemicroalgae quality under different environmental conditions and productionregimes.     

Virulence of different isolates of Aeromonas hydrophila in channel catfish

The virulence of nine Aeromonas hydrophila isolates from diseased fish, diseased Macrobrachium (freshwater shrimp) and from pond water was determined in channel catfish (Ictalurus punctatus) fingerlings. According to the lethal dose ? 50% end point (LD₅₀) of each isolate, the water organisms were significantly (P < 0.05) less virulent than the isolates from diseased fish. All isolates were biochemically similar.