致麻鸭输卵管炎的鸭疫里默氏菌的分离鉴定及致病性观察

在对鸭疫里默氏菌进行研究过程中,研究对象主要是具有明显输卵管炎的病死鸭中挑选的有疑似鸭疫里默氏菌的死鸭。并且使用细菌分离的方法从死鸭脑中分离出3株细菌。经过对这3株细菌进行形态鉴定、生化试验、血清型鉴定以及细菌16S rRNA的PCR的鉴定后,基本可以证明被分离出的3株细菌是血清型1型鸭疫里默氏菌,这种病菌具有较高的致病性,然后根据不同的形态特征和致病性对这3种病菌进行分类,经过进一步的检测试验发现,这3株病菌对先锋霉素、氧哌嗪青霉素以及美满霉素有较高的敏感反应。对致麻鸭输卵管炎的鸭疫里默氏菌进行分离鉴定和实验观察,有利于完善对鸭疫里默氏菌的研究资料,有助于鸭养殖业的良性发展。     

杂交鲍稚鲍"漫爬病"潜在病原菌胞外产物的分析及种类鉴定

从发病的杂交鲍(皱纹盘鲍×盘鲍)稚鲍中共分离27株菌株,并对其分泌胞外产物(包括酪蛋白酶、明胶酶、淀粉酶、脂肪酶、磷脂酶和溶血素)的能力进行了分析。试验结果表明,具有分泌胞外蛋白酶、明胶酶、淀粉酶、脂肪酶、磷脂酶和溶血素能力的菌株分别占总菌株数的81.5%(22/27)、74.1%(20/27)、18.5%(5/27)、0%(0/27)、11.1%(3/27)、29.6%(8/27)。其中,1#、14#菌株具有很强的产胞外蛋白酶能力,而明胶酶的最大分泌者为2#、7#、9#、17#菌株,淀粉酶的为11#、14#菌株,磷脂酶的为11#、15#菌株,溶血素的为菌株10#、21#。此外,10#、11#、15#菌株可同时分泌4种胞外产物,而12#、14#、18#、21#、23#、24#菌株可同时分泌3种胞外产物。综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,初步确认11株菌为潜在的致病菌:1#、2#、7#、9#、10#、11#、12#、14#、15#、17#、21#。用API条带法对它们进行了种类鉴定,11株菌中巴斯德氏菌和假单胞菌所占的比例分别为45.5%、27.3%。     

广东汕尾患病黑稚鲍微生物胞外产物的分析

从广东汕尾一鲍鱼养殖厂的患病黑稚鲍中分离筛选到56株菌,并对之进行致病毒力因子(胞外酶及溶血作用)的分析.结果表明,在56株菌中有16株具有较强的分泌胞外蛋白酶、明胶酶、磷脂酶、脂肪酶和淀粉酶的能力,占总菌株数的28.6%;而有溶血现象的仅有6株,占总菌株数的10.7%,且溶血能力不强.其中,8株具有很强的产胞外蛋白酶的能力,3株具有很强的产胞外明胶酶的能力,21株具有产胞外磷脂酶的能力,5株具有产溶血素的能力.此外,同时可分泌4种以上胞外产物的菌株为22株,占39.3%;同时分泌6种胞外产物的菌株为3株.综合考虑每种胞外产物的潜在致病作用以及菌株分泌多种胞外产物的能力,初步确认16株菌为潜在的致病菌.     

鸭传染性浆膜炎的诊断及防治

<正>鸭传染性浆膜炎又称鸭疫里默氏杆菌病,是鸭疫里默氏杆菌引起的以危害幼龄鸭为主的一种急性或慢性、接触性、细菌性传染病。本病一年四季均可发生,不同品种的鸭均可感染,发病率与死亡率都很高,感染率可达90%以上、死亡率达10%～75%左右,急性型病例l～2d内死亡,病程较长者8～10d死亡。在一般情况下,主要发生于1～8周龄的雏鸭。尤以2～3周龄雏鸭最易感,8周龄以上的鸭很少发病。成年鸭罕见发病,但可带菌,成为传染源。     

国内主要鸭养殖区传染性浆膜炎流行血清学调查实验报告

项目研究的目的是调查目前鸭传染性浆膜炎主要流行血清型。对来自全国不同地方送检的临床样品进行细菌分离并进行鉴定,对鉴定为鸭里默氏杆菌进行血清学分型。实验共分离出1151株细菌,实验结果表明,目前国内该病原的血清型主要集中在1-8型,其中1型为主要分离血清型,分离菌株占总菌株的36.5%(420株/1151株)。实验项目将有助于鸭传染性浆膜炎疫苗的制备以用于预防与控制疾病对鸭群的危害,对鸭养殖业具有重大意义。     

雏鸭混合感染鸭疫里默氏杆菌和大肠杆菌的诊断和防治

不少鸭场和农户散养的鸭群,常在2～3周龄发病,死亡率较高.我们从养殖户送检的病鸭和死鸭的肝脏、心血、脑等部位分离出了两种可疑细菌.经病理剖检、接种专用培养基、染色镜检、生化鉴定,确诊为鸭疫里默氏杆菌和大肠杆菌的混合感染.经动物试验证实了分离菌的致病性,并对分离菌进行药物敏感性试验,现将结果报道如下.     

草鱼消化道碱性蛋白酶的分析鉴定

试验结果表明,草鱼肠道粗提取物在pH 10.3有最高酶活力。SDS-底物-PAGE电泳表明,草鱼肠道粗酶中有4种碱性蛋白酶,其中3种是丝氨酸蛋白酶(胰蛋白酶),1种是非丝氨酸蛋白酶,它们的相对分子质量分别为:26 400、30 750、43 000、约105 000。     

鸭疫里默氏菌的分离和鉴定

为明确规模化鸭场病因,开展了鸭疫里默氏菌的分离和鉴定。通过采集病料进行实验室诊断,经细菌分离鉴定及PCR特异性扩增、产物测序鉴定方法,明确从规模化鸭场分离得到的细菌为鸭疫里默氏菌。检测结果为鸭场制定科学的预防和治疗方案提供了参考,对及时治疗病鸭、防止疾病传播、减少养鸭场的经济损失具有重要意义。     

养殖大黄鱼溃疡病的病原菌及其防治药物研究

从症状典型的网箱养殖患病大黄鱼体内分离到1株细菌824-1,经人工感染试验证实为是引起该病的病原菌。经鉴定,菌株824-1为溶藻弧菌(Vibrio alginolyticus)。利用杯碟法研究了其胞外产物的性质,结果表明:其胞外产物具有淀粉酶、明胶酶、酪蛋白酶、卵磷脂酶、脂酶和几丁质酶活性以及溶血活性,其中以淀粉酶、明胶酶和酪蛋白酶的活性最强,但没有脲酶活性。研究了20种化学药物和15种中草药对病原菌的抑菌作用,结果表明:复方新诺明、磺胺 TMP和庆大霉素等3种化学药物和石榴皮、地榆、五味子、大黄等4种中草药的抑菌能力最强,可作为防治该病的有效药物。     

患病九孔鲍苗中异养菌产胞外酶的分析

为探寻南方九孔鲍(Haliotis diversicolor supersicolor supertexta)苗大规模掉板死亡病因,从鲍鱼场患病掉板九孔鲍苗中分离到28株异养菌,并对其分泌胞外酶的能力进行分析.结果表明,分离得到的异养菌中有46.4%能分泌(酪)蛋白酶,60.7%能分泌明胶酶,3.57%分泌卵磷脂酶,17.86%分泌溶血素,但均不能分泌脂肪酶和淀粉酶.推测胞外蛋白酶为鲍苗患病的主要致病因子之一.综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,初步确认有5株异养菌为鲍苗掉板的潜在致病菌.     

Heterogeneity of extracellular proteases produced by different isolates of Aeromonas hydrophila and A. sobria pathogenic for fish

Abstract. Ten isolates of Aeromonas hydrophila and two of A. sobria were investigated with regard to elastase and caseinase production, and the pI, heat and EDTA sensitivity of extracellular caseinase enzymes. An extreme degree of heterogeneity amongst the strains was observed. In isoelectricfocusing, the ECP of the various strains possessed between 19 and 31 proteins, and the number of extracellular proteases ranged from one to 12. Similarities between strains using one criterion of comparison (e.g. IEF patterns) did not hold true by other criteria (e.g. heat or EDTA sensitivity of proteases).     

Extracellular protease production of bacteriolytic bacteria isolated from marine environments

ABSTRACT:   Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas . The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.     

Partial purification and characterization of extracellular metalloproteases with caseinolytic, aminopeptidolytic and collagenolytic activities from Vibrio anguillarum

Abstract. Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the aminopeptidase substrate L-Leu-pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypcs of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35kDa as determined by its mobility on SDS-polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However, the enzyme was strongly inhibited by metal chelators like EDTA and 1, 10-phenanthroline. Also, normal salmon scrum and purified α₂-macroglobulin from salmon serum strongly inhibited the caseinolytic activity of the enzyme.     

Enzyme producing bacterial flora isolated from fish digestive tracts

Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried out.Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media. The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability. Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities. Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities. Maximumactivity of lipase was exhibited by a strain isolated from silver carp. Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut. The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics.     

Bacterial inhibitors affecting proteases produced by Aeromonas salmonicida, and the occurrence of such inhibitors in furunculosis vaccines

Abstract. Cultures of Aeromonas salmonicida , subspecies salmonicida and achromogenes , produced considerable quantities of inhibitors that affected extracellular bacterial proteases (endopeplidases), as well as some animal trypsins (from pig, salmon and trout) included in this study. Electrophoretic separation of these inhibitors revealed a complex of three to four single factors which were similar for the two subspecies. One of the factors only inhibited protease from the homologus subspecies, while another only affected enzyme from the other subspecies (cross-wise inhibition). Both subspecies produced a factor which inhibited animal trypsins only. Subspecies salmonicida also possessed a factor which inhibited most of the examined proteases unspecifically. In young cultures (2–3 days at 15°C), the inhibitors were demonstrable both in the disintegrated cell material and in cell-free culture filtrate. The activity of the factor which only inhibited the protease from subspecies salmonicida could be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram-negative bacteria on the proteases of the A. salmonicida subspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspecies salmonicida and achromogenes were demonstrated in cell-free filtrates of two commercially available vaccines against furunculosis in fish.     

Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum)

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.     

草鱼和银鲫肠道产消化酶细菌的研究

检测了分别从草鱼(Ctenopharyngodon idellus)和银鲫(Carassius auratus gibelio)肠道中分离的180株细菌的蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果显示,两种鱼肠道内可分泌胞外消化酶的细菌包括Aero-monas(气单胞菌属,Aer.)、Vibrio(弧菌属,Vib.)、Bacillus(芽孢杆菌属,Bac.)、Pseudomonas(假单胞菌属,Pse.)四个种属的细菌,Aer.在其中占主要优势,45.71%的Aer.可分泌胞外消化酶。草鱼可分泌上述四种胞外消化酶的菌株共有33株,占肠道菌总数的36.67%;银鲫43株,占47.78%。产酶菌的分布上,草鱼中肠内产消化酶细菌数量显著多于前肠和后肠(P<0.05),前、中、后肠分别是6株、20株和7株;银鲫中肠和后肠数量差异不显著,前肠分布最少。草鱼分泌蛋白酶、脂肪酶、淀粉酶和纤维素酶的菌株分别有21株(23.33%)、10株(11.11%)、30株(33.33%)和16株(17.78%)。银鲫肠道内未检测到可分泌纤维素酶的细菌,蛋白酶、脂肪酶和淀粉酶菌株的数量分别是21株、37株和17株。可见鱼类肠道细菌对食饵消化有重要作用。     

Activity,molecular mass and hydrolysis on baker's yeast protein of extracellular proteases from the putative probiotic bacteria Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8N

The bacteria Microbacterium sp. 8L and Exiguobacterium mexicanum 8N are known to improve the culture of Artemia franciscana using baker's yeast as food. Using spectrophotometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), substrate‐SDS‐PAGE and pH‐stat in vitro‐digestibility assays, the activity, molecular mass and hydrolysis on baker's yeast protein of proteases from extracellular polymeric substances (EPS) of the strains 8L and 8N along with the pathogenic strains Microbacterium sp. 8R and Vibrio parahaemolyticus 588 CECT (Vp) were studied. The EPSs of 8L and 8R showed one activity band, on which the serine inhibitor phenylmethylsulphonyl fluoride (PMSF) had no effect. The EPSs of 8N showed four bands; two were unaffected by PMSF, whereas one was affected, and the other was partially affected. The EPSs of Vp showed two bands, one partially inhibited by PMSF. No inhibitory effects from 1‐chloro‐3‐tosylamido‐7‐amino‐2‐heptanone (trypsin inhibitor) were observed in the protease bands of the studied bacteria. The EPSs of 8L and 8N showed a similar degree of hydrolysis (pH‐stat). The EPSs of 8L had the lowest Dice index of similarity of yeast protein profiles at 1 h of reaction. We conclude that the strain 8L could benefit A. franciscana by providing bacterial proteases for digestion of baker's yeast.     

Genotype and virulence of Streptococcus iniae isolated from diseased olive flounder Paralichthys olivaceus in Korea

In the study, we characterized 29 Streptococcus iniae isolates from diseased olive flounder Paralichthys olivaceus in Korea from 2000 to 2005. Biochemical characteristics of 29 isolates using API 20 strep were identical. Through analysis of repetitive sequence-based PCR (rep-PCR) using BoxA primer and random amplified polymorphic DNA using p14 primer, 29 isolates of S. iniae were divided into two genotypes. The isolates were divided into two clusters by comparison of genetic distance using a sequence of the capsular polysaccharide D gene that was consistent with genotyping by the rep-PCR. The isolates belonging to genotype 1 in rep-PCR analysis showed a high virulence in the flounder, while the isolates belonging to genotype 2 were relatively low in virulence. Therefore, a correlation between the genotype and the virulence of S. iniae isolates has been identified.     

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S‐23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype‐specific biochemical characteristics and genetic characteristics of 16S‐23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.