一株解淀粉芽孢杆菌的分离鉴定及其抑菌条件研究

旨在从海洋环境中分离筛选抑制鲈鱼病原鳗弧菌的拮抗菌,进行菌种鉴定,并对其抑菌条件及药物敏感性进行研究。采用十字交叉划线法和平板点种法筛选鲈鱼病原鳗弧菌的拮抗菌,采用形态学、生理生化反应及16S r DNA基因序列的系统发育分析方法对分离拮抗菌进行鉴定,采用单因子变量法对拮抗菌WTD的最佳抑菌条件进行研究,采用平板药敏纸片法对其药物敏感性进行研究。结果显示,从海洋环境样品中分离出126株细菌,筛选出19株拮抗菌。其中菌株WTD的拮抗效果最强,细菌鉴定结果表明,该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。最佳抑菌条件研究表明,该菌在温度35℃、p H7、盐度为1%Na Cl的时候,拮抗效果最好。药敏实验结果表明,该菌对麦迪霉素等29种抗生素敏感,而对多粘菌素具有抗性。拮抗菌解淀粉芽孢杆菌WTD对鲈鱼病原菌鳗弧菌有较强的抑制作用,具有潜在的应用价值。     

滴水湖沉积物中可培养优势微生物种群初探

于滴水湖湖心采集底泥样品,对底泥中可培养优势菌种进行分离、纯化,并利用Biolog微生物自动分析系统进行鉴定。结果显示,滴水湖沉积物中菌落总数为2.43×104CFU/g,分离纯化后的8株优势菌种中,革兰氏阴性菌占87.5%,其中7株为GN-NENT(革兰氏阴性非肠道菌)、1株为GP-ROD SB(革兰氏阳性芽孢杆菌)。鉴定结果显示,8株菌种分别为:鳗鱼气单孢菌(Aeromonas encheleia)、乙酸钙不动杆菌/基因型1(Acinetobacter calcoaceticus/genospecies1)、舒氏气单胞菌(Aeromonas schubertiiDNA group12)、腐败希瓦氏菌B(Shewanella putrefaciens B)、维罗纳/温和气单胞菌(Aeromonas veronii/sobria DNA group8)、坎氏弧菌(Vibrio campbelli)、蕈状芽孢杆菌(Bacillus mycoides)和梅氏弧菌(Vibrio metschnikovii)。     

两株珍珠龙趸病原性哈维弧菌(Vibrio harveyi)的分离与鉴定

自海南陵水和广东深圳养殖场患病珍珠龙趸(pearl gentian)分离到2 株优势菌(X12XC30 和X13SZ03), 经回归感染实验证实2 株菌是珍珠龙趸的病原菌, 对珍珠龙趸的半致死浓度(LD₅₀)分别是6.58×10⁶ CFU·g^-1 和9.83×10⁵ CFU·g^-1。对菌株进行形态、颜色和生长条件等常规生理生化实验, 并进行16S rDNA, rctB 和toxR 等管家基因的序列分析综合鉴定。结果显示: 2 株病原菌均为短杆状革兰氏阴性菌, TCBS 生长为黄色, 对O/129(150 ug·片^-1)敏感, 生理生化指标与哈维弧菌标准株一致; 16SrDNA, rctB 和toxR 序列在Genbank 上检索与哈维弧菌的同源性最高, 根据管家基因的序列构建系统进化树, 自然地与哈维弧菌分支聚类为一支, 确定这2 株菌均为哈维弧菌。药敏实验发现2 株哈维弧菌均耐呋喃唑酮, 而对诺氟沙星、恩诺沙星、环丙沙星和氯霉素等药物敏感。哈维弧菌具有较高的致死率, 在珍珠龙趸养殖业中存在潜在的威胁。     

对虾肠道中T-2毒素降解菌的分离纯化与鉴定

从新鲜健康的凡纳滨对虾肠道中分离筛选和鉴定降解T-2毒素微生物,为日后进一步研究T-2毒素(T-2)降解菌株的相关特性及其分布规律提供参考。取新鲜健康的凡纳滨对虾肠道,通过对该肠道菌群进行定性定量分析;从T-2与对虾肠道共培养液分离纯化得到6株优势菌株;将6株优势菌株分别和T-2在胰蛋白胨大豆(TSB)肉汤中进行共培养,采用LC-MS/MS法检测该培养液前后T-2含量,筛选得到T-2降解菌株,并用微生物鉴定仪VITEK2和16S rRNA鉴定目标菌株。TSB肉汤中检测T-2的标准曲线线性良好(R=0.999 5),T-2加标回收率为91.5%~108.4%,检测限为0.01 ng/m L,相对标准偏差为0.0%~8.4%和精密度为94.5%~107%。对虾肠道中的蜡状芽胞杆菌(Bacillus cereus)和阴沟肠杆菌(Enterobacter cloacae)对T-2毒素降解率最高,分别达到91.8%和78.8%。     

凡纳滨对虾红体病病原

2001年7月辽宁省盘锦市大洼对虾养殖厂凡纳滨对虾大面积暴发红体病。主要表现为全身发红,活动减弱,食欲减退,壳变硬,死亡率高等。本研究从患红体病的凡纳滨对虾肌肉组织中分离到三株优势菌,经回归感染证实其中一株为引发本次凡纳滨对虾红体病的病原菌,编号为0107。对该菌进行了生理生化鉴定和16SrRNA基因序列同源性分析,将其判定为副溶血弧菌(V.parahaemolyticus)。药敏实验结果显示,该菌对头孢哌酮、头孢曲松等多种抗生素敏感,对氨苄西林、青霉素等抗生素不敏感。     

凡纳滨对虾繁育系统菌群结构及优势菌遗传多样性分析

【目的】探讨凡纳滨对虾(Litopenaeus vannamei)工厂化繁育系统内发生细菌性玻化症(shrimp postlarva bacterial vitrified syndrome,BVS)时期可培养微生物的菌群特征以及优势病原菌的遗传多样性。【方法】采用细菌体外培养方法结合基因测序技术对不同育苗阶段的亲虾、受精卵、无节幼体、蚤状幼体、糠虾幼体、仔虾,及其育苗池水和饵料内可培养细菌菌群的组成与结构特征进行研究,并通过多位点序列分析(multilocus sequence analysis,MLSA)方法解析病原菌的遗传多样性。【结果】系统内分离纯化的526株具有典型形态差异和群落优势的细菌分属于4门5纲16目24科38属113种。在纲水平上γ-变形菌纲(Gammaproteobacteria)丰度最高,共453株,占总分离株的86.1%;在属水平上弧菌属(Vibrio)丰度最高,共369株,占总分离株的70.2%;在种水平上,溶藻弧菌(Vibrio alginolyticus)为最优势种,共112株,占总分离株的21.3%,并且分布于整个繁育系统,在饵料中具有最高丰度。多元关联分析表明,随着对虾幼体的发育,饵料对幼体体内可培养细菌的菌群结构影响逐渐增加。对112株潜在溶藻弧菌的MLSA分析表明,其中100株菌株进一步确认为溶藻弧菌。进一步利用MLSA构建系统发育树分析其遗传多样性发现,100株溶藻弧菌分为9个簇,分离自同类样品的菌株广泛分布在不同的簇中。【结论】在BVS发生时期,凡纳滨对虾工厂化繁育系统中具有丰富的可培养微生物种类。对虾幼体发育过程中,饵料对幼体体内可培养细菌的菌群结构具有重要影响。溶藻弧菌为凡纳滨对虾工厂化繁育系统中的优势弧菌,分布于整个繁育系统,且具有丰富的遗传多样性。本研究为解析对虾繁育系统可培养微生物演替规律提供了数据支撑,也为对虾苗期病原防控和健康养殖提供了科学依据。     

凡纳滨对虾繁育系统菌群结构及优势菌遗传多样性分析北大核心

【目的】探讨凡纳滨对虾(Litopenaeus vannamei)工厂化繁育系统内发生细菌性玻化症(shrimp postlarva bacterial vitrified syndrome,BVS)时期可培养微生物的菌群特征以及优势病原菌的遗传多样性。【方法】采用细菌体外培养方法结合基因测序技术对不同育苗阶段的亲虾、受精卵、无节幼体、蚤状幼体、糠虾幼体、仔虾,及其育苗池水和饵料内可培养细菌菌群的组成与结构特征进行研究,并通过多位点序列分析(multilocus sequence analysis,MLSA)方法解析病原菌的遗传多样性。【结果】系统内分离纯化的526株具有典型形态差异和群落优势的细菌分属于4门5纲16目24科38属113种。在纲水平上γ-变形菌纲(Gammaproteobacteria)丰度最高,共453株,占总分离株的86.1%;在属水平上弧菌属(Vibrio)丰度最高,共369株,占总分离株的70.2%;在种水平上,溶藻弧菌(Vibrio alginolyticus)为最优势种,共112株,占总分离株的21.3%,并且分布于整个繁育系统,在饵料中具有最高丰度。多元关联分析表明,随着对虾幼体的发育,饵料对幼体体内可培养细菌的菌群结构影响逐渐增加。对112株潜在溶藻弧菌的MLSA分析表明,其中100株菌株进一步确认为溶藻弧菌。进一步利用MLSA构建系统发育树分析其遗传多样性发现,100株溶藻弧菌分为9个簇,分离自同类样品的菌株广泛分布在不同的簇中。【结论】在BVS发生时期,凡纳滨对虾工厂化繁育系统中具有丰富的可培养微生物种类。对虾幼体发育过程中,饵料对幼体体内可培养细菌的菌群结构具有重要影响。溶藻弧菌为凡纳滨对虾工厂化繁育系统中的优势弧菌,分布于整个繁育系统,且具有丰富的遗传多样性。本研究为解析对虾繁育系统可培养微生物演替规律提供了数据支撑,也为对虾苗期病原防控和健康养殖提供了科学依据。     

丹参植株内生菌的分离纯化及其抗病性研究

为探明丹参(Salvia miltiorrhiza Bge)内生菌的种类,筛选拮抗农作物病害菌的生防菌株,从健康丹参植株中进行内生菌分离,依照形态特征以及16SrDNA序列对菌株进行初步分类鉴定,采用琼脂块法进行拮抗菌株筛选,并选取拮抗活性较强的菌株液体发酵进行体外抑菌试验。结果表明:(1)从丹参植株中共分离得到69株内生菌(真菌62株、放线菌7株),其中23株内生真菌属于无孢类群或现有条件不适合产孢,其余真菌菌株中串珠镰孢菌属(Fusarium moniliforme Sheld.)和交链孢菌属(Alternaria sp)为优势菌群;7株内生放线菌均为链霉菌属。(2)病原菌拮抗性实验表明,有44株内生菌对病原菌具有不同程度的拮抗活性,其中12株内生菌对2种及以上靶标病原菌具有拮抗活性,表明丹参内生菌具有一定的广谱抗菌性。(3)放线菌菌株A232的次级代谢产物对白色假丝酵母(Canidia albicans)和苹果腐烂菌(Valsa mali)都表现出较强的拮抗活性,通过形态、培养特征以及16SrDNA序列分析,鉴定其为Streptomyces luteoverticillatus。     

罗非鱼源无乳链球菌肠道拮抗芽孢杆菌的筛选及其生物学特性

【目的】从健康尼罗罗非鱼(Oreochromis niloticus)肠道中筛选一株对罗非鱼源无乳链球菌等病原菌具有拮抗功能的益生菌。【方法】取健康尼罗罗非鱼肠道,匀浆后进行10倍系列梯度稀释,然后涂布BHI平板,培养1–2d,挑取单克隆菌落。采用点种法初步筛选对罗非鱼源无乳链球菌有拮抗作用的菌株,选取其中一株拮抗效果较好的菌株LF01,通过形态学、生理生化特征以及分子生物学分析,对LF01菌株进行鉴定。然后对LF01菌株的生长特性、水解淀粉和酪蛋白能力、药物敏感特性、抗菌谱和生物安全性进行测定和分析。【结果】根据菌落形态和生长时间的差异,从健康尼罗罗非鱼肠道中筛选出64株细菌,通过拮抗试验筛选出6株具有明显拮抗效果的菌株,其中LF01菌株的拮抗效果最好。根据LF01的形态、生理生化特征和gyr A基因的进化分析,确定该菌株为贝莱斯芽孢杆菌(Bacillus velezensis)。LF01菌株的最适生长温度为30°C,最适p H值为7,最适盐度为5‰,而且该菌株具有水解淀粉和酪蛋白的功能。药敏试验结果显示,LF01菌株对多数抗生素敏感,仅对杆菌肽耐药。拮抗试验结果显示LF01株对无乳链球菌、海豚链球菌、迟缓爱德华氏菌、鮰爱德华氏菌、嗜水气单胞菌、舒氏气单胞菌、维氏气单胞菌、简氏气单胞菌、鰤鱼诺卡氏菌等病原菌均具有拮抗作用,其中对鰤鱼诺卡氏菌的拮抗作用最强,平均抑菌圈直径达28.3 mm。生物安全试验表明,LF01菌株对尼罗罗非鱼、斑马鱼(Danio rerio)和乌鳢(Channa argus)等3种鱼均无致病性,具有良好的安全性。【结论】本研究筛选了一株贝莱斯芽孢杆菌LF01株,该菌的生物安全性良好,而且可拮抗常见的水产病原菌,具有防控多种水产经济动物疾病的潜力,应用前景十分广阔。     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon

The role of an extracellular cysteine protease, produced by pathogenic luminous Vibrio harveyi strain 820514 originally isolated from diseased tiger prawn (Penaeus monodon), in the disease process in the prawns was studied. The protease was lethal to P. monodon with an LD50 value of 0.3 microgram protein g-1 prawn. The lethal toxicity of the extracellular products (ECP) of the bacterium was neutralized by pre-incubation of the ECP with rabbit antiserum to the cysteine protease. Pre-incubation of ECP with CuCl2 (an inhibitor of cysteine protease) also inhibited toxicity. This suggests that cysteine protease is the major toxin produced by the bacterium. The present protease is the first toxic cysteine protease to be found in Vibrio species.     

Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon

P.-C. LIU, K.-K. LEE AND S.-N. CHEN. 1996. The pathogenicity of six Vibrio harveyi strains in tiger prawn, Penaeus monodon , was studied, using both live bacteria and extracellular products (ECP). The organisms originally isolated from diseased penaeids were more virulent using both live bacteria and ECP (LD₅₀, 4.87–8.65 times 10⁴colony-forming units (cfu) and 1.20–1.51 μg protein g^-1body weight) than the two reference strains originally isolated from either sea water (ATCC 25919; LD₅₀, 3.18 times 10⁶cfu and 2.70 μg protein g^-1body weight) or diseased Talorchestia sp. (ATCC 14126, 0.418 times 10⁶cfu and 2.34 μg protein g^-1body weight). Each strain was reisolated from the haemolymph and the hepatopancreas of moribund prawns following each bacterial challenge. Both the live bacteria and the ECPs of the penaeid isolates exhibited stronger proteolytic (caseinase), phospholipase and haemolytic activities than those of the reference strains. These results indicate that there are differences between penaeid and non-penaeid isolates of V. harveyi in pathogenicity and reveal that proteases, phospholipases, haemolysins or exotoxins might play leading roles in the pathogenicity of V. harveyi in the tiger prawn, Penaeus monodon .     

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus.     

Effects of extracellular products of Vibrio alginolyticus on penaeid prawn plasma components

The effects of both crude extracellular products (ECP) and a partially purified protease of Vibrio alginolyticus on the plasma components of kuruma prawn ( Penaeus japonicus ) and tiger prawn ( P. monodon ) were studied using crossed immunoelectrophoresis (CIE). A component of the plasma, tentatively identified as coagulogen, apparently disappeared after incubation with the ECP, while the amount of a component tentatively identified as haemocyanin decreased. The coagulogen and an unknown component (component 1) in the penaeid plasma showed an increased migration rate after incubation with a partially purified 33 kDa protease of the bacterium. In contrast, incubation with protease had no detectable effect on the amount of haemocyanin. These events may significantly contribute to the pathogenicity of Vibrio alginolyticus in penaeids.     

Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture

AIMS: The present work aims at finding potential probionts from marine sources as a biocontrol agent against pathogenic Vibrio species in shrimp larval culture. METHODS AND RESULTS: A total of 109 bacterial strains were isolated from seawater, sediment and marine fish-gut samples, and were screened for their antagonistic activity against Vibrio species. Three strains (Q, Q1 and M) isolated from the marine sediment were found antagonistic against Vibrio strains. Based on 16S ribosomal DNA gene sequence analysis, the strain Q was identified as Paenibacillus spp. (EF012164); Q1 as Bacillus cereus (DQ915582); and the M as Paenibacillus polymyxa (DQ915580). Further, the two bacterial species, Paenibacillus spp. and B. cereus were challenged separately at two different concentrations of 10(4) and 10(5) CFU ml(-1) for probiotic activity in the postlarvae of Penaeus monodon against pathogenic Vibrio harveyi and Vibrio spp. CONCLUSIONS: The present study identified the probiotic activity of Paenibacillus spp., B. cereus and Pa. polymyxa against the pathogenic Vibrios in the postlarvae of P. monodon. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo study reveals that the marine bacterial species can be used as probionts against pathogenic Vibrios in shrimp larval culture practices.     

牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。     

Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon).

During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included.     

Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon).

During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included.     

水产动物副溶血性弧菌的拮抗菌研究进展

细菌性疾病的爆发常造成水产养殖业的巨大经济损失,其中副溶血性弧菌(Vibrio parahaemolyticus)引起的细菌性疾病更是引起了人们的关注.拮抗菌在代谢活动中通过分泌抗菌物质直接对病原菌产生抑制或竞争作用来抑制或杀死病原菌,在病害防治中发挥着重大作用.对副溶血性弧菌拮抗菌的种类、产生拮抗物质的种类、筛选拮抗...     

Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon

Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood. In this study, the penetration, fate and the clearing of V. vulnificus were investigated in Penaeus monodon. A bacterial disease isolate from a shrimp farm was identified as V. vulnificus biotype I. Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp. and other gram-negative bacteria. The bacteria were then administered to P. monodon juveniles by injection, immersion and oral intubation. An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp. Bacteria were cleared by a common route, regardless of the method of administration. Observations in immersion challenge were similar to a combination of those for oral and injection challenges. With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae. Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue. They were either phagocytosed by haemocytes, or broken down outside host cells. Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph. HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ. Free bacterial antigens also accumulated in the heart and lymphoid organ. Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas. Bacterial antigens passed through the hepatopancreas into the haemolymph. Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ. Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes.