基于gyrB基因建立快速检测沙雷菌的PCR方法

选择gyr B基因作为靶基因设计特异性引物用于沙雷菌属的PCR检测,该特异性引物扩增产物的片段大小为279 bp。研究所选用的14株沙雷菌分别代表沙雷菌属的7个不同种。此外,2种沙门菌、2种克雷伯菌以及其他5种肠道菌用于进行引物特异性的验证。结果表明:扩增的条带与预期的扩增产物片段大小一致,而非沙雷菌属的其他9个种属均没有扩增条带;该方法检测沙雷菌的最低检测限为9.785 pg。通过对4株从蜜蜂中分离所得的黏质沙雷菌和3株从进口鱼粉中分离所得的居泉沙雷菌进行检测,结果均为阳性。研究表明,基于gyr B基因建立的PCR方法在沙雷菌属的检测中具有特异性强、快速和灵敏度高等特点。     

动物源大肠埃希菌分离与鉴定技术研究

建立动物源大肠埃希菌分离与鉴定方法，对监测大肠埃希菌的耐药情况具有重要意义。样品拭子通过麦康凯显色培养基分离纯化，采用生化或质谱的方法鉴定。用65株已知菌进行方法验证。结果显示：确认大肠埃希菌56株，准确率100%。该方法能准确检测动物源大肠埃希菌。     

进口冻猪肚中鼠伤寒沙门氏菌的分离与鉴定

从欧洲进口的冻猪肚中检出一株细菌，经细菌分离培养，生化反应，梅里奥微生物自动分析仪检测以及血清凝集试验鉴定为鼠伤寒沙门氏菌，抗原模式为4，5，12：i:1,2。该菌株致接种的小白鼠全部死亡；分离菌对先锋V，氟哌酸，环丙沙星，氯霉素高敏，对复方新诺明，青霉素，红霉素耐药。     

进境鱼粉中沙门氏菌的分离与鉴定

近两年来，在厦门口岸进口鱼粉的检测中，多次检出沙门氏菌。本文通过血清学鉴定，确定该菌为甲型副伤寒沙门氏菌和鸭沙门氏菌。动物回归试验说明其致病性很强，药敏试验发现菌必治、先锋霉素V、环丙沙星和氟哌酸对该菌高度敏感．     

进口鱼粉中沙门菌监测与分析

为了解进口鱼粉中沙门菌的污染状况,给制定相关的检验检疫措施提供必要的理论依据,对2011年到2014年期间来自18个国家的543份鱼粉样品进行沙门菌检测。鱼粉样品经过增菌、沙门茵平板分离、VITEKⅡ生化试验和血清学鉴定后,共分离鉴定到27株沙门菌,分属于B、C、E 3个群。沙门茵的总检出率为4.97%,且来自不同国家的鱼粉阳性率不同。结果显示,进口鱼粉中存在着不同程度的沙门菌污染,应加强进口鱼粉的检疫和监管,减少沙门菌病的发生。     

华南地区肉鸽源沙门氏菌的鉴定及遗传进化分析

本试验从华南地区发病肉鸽肠道和肝脏中用BS培养基分离、纯化沙门氏菌,并进行多项生化试验和血清型鉴定试验,结果显示各项生化检测指标均符合沙门氏菌的特点;血清型鉴定试验结果表明该分离菌属沙门氏菌属A-F群。进一步对该分离菌的16S rRNA片段测序及遗传进化分析,确定该分离菌为沙门氏菌菌株,与鼠伤寒沙门氏菌同源性为100%。对该分离菌进行动物回归试验,结果表明该沙门氏菌菌株为华南地区肉鸽的致病菌株。该结果为进一步研究华南地区肉鸽沙门氏菌的致病机理和防控奠定了基础。     

应用HFMa培养基分离猪传染性萎缩性鼻炎菌

从进口的806头猪(在美国出口前作AR菌分离均为阴性)的102头(占总数16.8％)中分离出AR菌。安际检疫证明,用血红素呋喃唑酮改良麦康凯培养基(HFMa)分离AR菌明显优于改良的麦康凯琼脂培养基。     

鸡白痢沙门菌等位基因特异性PCR检测方法的建立

根据鸡白痢沙门菌与鸡伤寒沙门菌的rfbS基因在第237和第598位碱基的不同，设计和合成了等位基因特异性PCR引物，建立了快速检测鸡白痢沙门菌的PCR方法，并应用该方法对鸡白痢沙门菌临床分离样品进行了PCR鉴定。结果显示，该PCR方法能够特异性地鉴定鸡白痢沙门菌，检测灵敏度达100PgDNA。对35个经常规方法鉴定的鸡白痢沙门菌分离株应用等位基因特异性PCR方法进行鉴定，鉴定出33株鸡白痢沙门菌，符合率为94．3％。表明，建立的等位基因特异性PCR方法能够准确而快速地鉴定鸡白痢沙门菌。     

在进口冻鸡脚中检出奇异变形菌

在进口冻鸡脚中检出奇异变形菌邓建新郑雅卡陈铭（中华人民共和国梧州动植物检疫局，广西５４３００２）１９９５年１２月，某公司进口某国一批冷冻鸡脚共１５０吨，抽样后，经细菌分离培养、柒色镜检和生化试验，检出奇异变形菌。奇异变形菌是人兽共患条件致病菌，人食了...     

猪胸膜肺炎放线杆菌的分离鉴定及药敏试验

对从全国30多个猪场送检的疑似猪传染性胸膜肺炎的病猪中，用PPLO培养基进行了胸膜肺炎放线杆菌的分离，对分离菌进行了PCR鉴定和生化鉴定，并测定了该菌的培养特性、致病性和对药物的敏感性。结果成功分离到了15株胸膜肺炎放线杆菌，该菌对先锋霉素类和氯霉素较敏感。     

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.     

Development of a PCR protocol for the detection of Aeromonas salmonicida in fish by amplification of the fstA (ferric siderophore receptor) gene

The aims of the study were to evaluate a new PCR protocol designed to detect Aeromonas salmonicida in fish tissues and to develop a non-destructive method for the diagnosis of furunculosis. A set of primers (Fer3, Fer4), flanking a fragment of the fstA gene (coding for the ferric-siderophore receptor) was designed, showing to be sensitive and specific. When compared to PCR methods previously reported, the new protocol recognized all the 69 A. salmonicida strains evaluated, with no cross-reactions with the other bacterial species analysed. Sensitivity assays were performed in fish tissues seeded with serial dilutions of pure cultures of A. salmonicida and mixed cultures of this bacterium with Vibrio anguillarum and Aeromonas hydrophila. Detection limits obtained were of 60 and 450 bacterial cells 100 mg(-1) of tissue, respectively. Mucus and blood were evaluated in order to develop a non-destructive tool to detect the pathogen. The detection limits in seeded mucus and blood samples were 2.5 x 10(2) and 1 x 10(5) bacterial cells mL(-1), respectively. When the method was used to detect A. salmonicida in asymptomatic wild salmon, four samples of mucus and six of blood were positive, corresponding to 6 out of the 31 fish examined, whereas only one of the samples resulted positive by culture methods. It is concluded that the PCR protocol evaluated is fast, specific and sensitive to detect A. salmonicida in infected and asymptomatic fish, and will be helpful for the control of the disease through the prompt detection of carriers within fish populations.     

Protection against atypical Aeromonas salmonicida infection in carp (Cyprinus carpio L.) by oral administration of humus extract

Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to common carp (Cyprinus carpio L.) induced effective protection against experimental atypical Aeromonas salmonicida infection. Mortality of fish and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with 10%, 5% or 1% humus extract adsorbed on dry feeding pellets. The median surviving days was also greater in fish treated with 10% or 5% humus extract than in untreated fish. Atypical A. salmonicida was isolated from ulcerative lesions of part of dead fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying bacterial population changes during the progression of skin lesions. These results clearly show that treatment of fish with humus extract is effective in preventing A. salmonicida disease.     

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.     

A comparison of total and specific immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in salmon naturally infected with Aeromonas salmonicida subsp. achromogenes.

Healthy Atlantic salmon and salmon with a history of chronic natural Aeromonas salmonicida subsp. achromogenes infection were compared with respect to total serum protein and the concentration and specificity of serum immunoglobulin. The immunoglobulin level was measured using competitive ELISA and the specific antibody activity against Aeromonas salmonicida subsp. achromogenes was measured using double sandwich ELISA. Significant elevation of serum protein and immunoglobulin concentration was observed in the infected salmon compared with the healthy fish. This was accompanied by weak anti-A. salmonicida activity in the infected fish which seemed to contribute to the raised immunoglobulin level to only a limited degree.     

Amoxycillin in the control of furunculosis in Atlantic salmon parr.

The efficacy of amoxycillin in the control of laboratory induced Aeromonas salmonicida infection in Atlantic salmon parr was investigated. When given in the diet at a dose rate of 80 mg per kg bodyweight it was effective against both a moderate and severe challenge (with mortality rates in untreated groups of 75 per cent and 45 per cent). At 40 mg per kg it was effective against the moderate challenge only. The plasma levels in these regimens were 1.25 micrograms per ml and 0.3 to 0.6 micrograms per ml and the minimum inhibitory concentration of the challenge strain of A salmonicida was 0.6 micrograms per ml. The potential of the Charm radiobioassay system in detecting antibiotic residues in fish tissue was studied. The level of amoxycillin in muscle and bone from fish in mid-treatment at 80 mg per kg was 0.32 micrograms per ml. After a 12 day withdrawal period at 18 degrees C no residue was detected within the 0.005 micrograms per ml limit of this test.     

Protection against atypical Aeromonas salmonicida infection in common carp, Cyprinus carpio L., by oral administration of a mixed microbial culture of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.     

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.     

In vitro adherence of two candidate probiotics from Atlantic cod and their interference with the adhesion of two pathogenic bacteria

The potential of two candidate probiotic bacteria (GP21 and GP12), isolated from the gut of Atlantic cod, to adhere to primary cultures of the epithelial cells from the different regions of the intestine and to interfere with the adhesion of two pathogens, Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida were investigated. The intestinal isolates showed clear preference in adhering to the cells from the different intestine segments. GP12 adhered strongly to the fore- and mid intestine cells. The adherence of GP21 was most to the cells from the hind intestine followed by those from the mid-segment. The adhesion of V. anguillarum was affected by both GP21 and GP12; GP12 interfered through competition, but a specific mode of action was not observed for GP21. In the case of A. salmonicida, competition was the principal mechanism by which GP21 interfered with their adhesion, while exclusion mechanism was favoured by GP12. In addition, GP21 was more auto-aggregative than GP12, but the latter was more co-aggregative with both the pathogens. The isolates were also capable of lowering lactate dehydrogenase activity compared to that by the pathogen and they reduced the caspase-3 activity in the epithelial cells from the hind intestine, to which the pathogens adhered the most. Thus it could be concluded that the adhesion of the candidate probiotics is segment-specific and their interference with the adhesion of pathogens is dependent on both source of the epithelial cells and the mechanism adopted by the isolates. This information is novel in the case of fish and the manner in which potential probiotic organisms interfere with the pathogen adhesion provides supportive information for disease control.