Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术，研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现，采用细菌16S rRNA基因保守区特异性引物，以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象，可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上，检出上述所有细菌，并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为，该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

Evaluation of the link between gyrodactylosis and streptococcosis of Nile tilapia, Oreochromis niloticus (L.)

Streptococcus iniae and Gyrodactylus niloticus are two common pathogens of cultured Nile tilapia, Oreochromis niloticus. We studied concurrent infection of tilapia by G. niloticus and S. iniae and evaluated whether parasitism in tilapia with Gyrodactylus increased susceptibility and mortality following immersion infection with S. iniae. Results showed that death mainly occurred in fish with G. niloticus and challenged with S. iniae (G-S group). The accumulative mortality (42.2%) was significantly higher in the G-S group than in fish not infected by the parasite (6.7%), but exposed to S. iniae. Bacteriological examination revealed S. iniae from > or =92% of dead or moribund fish challenged with S. iniae. Gyrodactylus not only damaged fish epithelium and provided entry for invasive bacteria but also was found to harbour viable cells of S. iniae for 24 and 72 h. Streptococcus iniae was isolated from 60% and 40% of G. niloticus collected from fish infected by intraperitoneal injection or immersion, respectively, at 24 h post-challenge. The present study confirms that parasitism of tilapia by G. niloticus increased host mortality following exposure to the bacterial pathogen S. iniae.     

广东省养殖罗非鱼、海鲈、尖吻鲈海豚链球菌感染调查

利用细菌分离培养方法结合特异PCR技术,对广东省珠三角地区养殖罗非鱼(Oreochromis spp.)、海鲈(Lateolabrax japonicus)及尖吻鲈(Lates calcarifer)的海豚链球菌(Streptococcus iniae)感染情况进行了周年调查。每月固定时间在特定养殖区域采集目标鱼的脑、肝、脾、肾和肌肉等组织,并对其进行海豚链球菌的细菌分离培养鉴定。仅从已经患病的尖吻鲈中分离到3株链球菌,经生理生化鉴定和16S rDNA测序确定为海豚链球菌。利用海豚链球菌特异PCR技术对上述养殖鱼类不同组织进行检测,发现罗非鱼、海鲈、尖吻鲈的海豚链球菌感染率分别为30.21%、23.53%、14.55%,其中罗非鱼脑和肌肉的感染率明显较其他组织高(P<0.05),分别为20.65%和23.75%;海鲈的脑部和肌肉感染率也较其他组织高(P<0.05),分别为12.1%和10%;而尖吻鲈各组织感染率没有较大差异(P>0.05)。另外,研究结果还表明采集样本的海豚链球菌感染率随着其体长的增加而呈现下降趋势。     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Development of a multiplex polymerase chain reaction to detect five common Gram-negative bacteria of aquatic animals

A multiplex polymerase chain reaction (m-PCR) technique was developed as a rapid and accurate diagnostic tool for identifying five major Gram-negative bacilli -Vibrio vulnificus, V. parahaemolyticus, Aeromonas hydrophila, Chryseobacterium meningosepticum and Edwardsiella tarda- that cause major diseases in cultured aquatic animals in Taiwan. The expected amplicons for V. vulnificus, V. parahaemolyticus, A. hydrophila, C. meningosepticum and E. tarda were 410, 368, 685, 180 and 230bp, respectively. The assay was shown to be specific for the target pathogens. The sensitivities of detection were estimated to be 20.5fg～200pg of genomic DNA or 10(2) ～10(4) colony-forming units (cfu) of bacterial isolates when adopted as PCR templates. The m-PCR was capable of simultaneously amplifying target fragments from bacterial genome DNA mixed with the DNA extracted from viscera and tissues taken from fish without affecting the performance of the method.     

Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study.     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.     

Passive immunization of tilapia, Oreochromis niloticus (L.), with anti-Streptococcus iniae whole sera

Passive immunization of tilapia, Oreochromis niloticus, was conducted to determine whether anti- Streptococcus iniae whole sera (ASI), heat inactivated anti- S . iniae whole sera (HIASI) and normal whole sera (NWS) were protective when intraperitoneally (i.p.) injected into tilapia. The ASI was produced in tilapia actively immunized (challenged) with virulent S. iniae by i.p. injection. An antibody response against S. iniae was demonstrated by enzyme linked immunosorbent assay (ELISA) and 18% of the immunized fish died because of the S. iniae infection. The actively immunized tilapia demonstrated a secondary antibody response and immunity to S. iniae after challenge with S. iniae by i.p. injection. Survival was 100% in the actively immunized fish. The NWS was obtained from tilapia free of ASI antibody and susceptible to S. iniae infection (40% mortality). In two separate experiments, significantly higher mortality was noted in tilapia passively immunized with NWS (33 and 53%) and phosphate buffered saline (PBS) (30 and 60%), in comparison with mortalities of 0 and 10% or 3.3 and 6.7% in the fish passively immunized with ASI or HIASI 14 days after S . iniae infection by i.p. injection ( P  = 0.0003 and 0.0023). Results suggest that immunity provided by ASI and HIASI was because of antibody against S. iniae . Inactivation of complement in the HIASI treatment further suggests that ASI antibody plays a primary role in immunity against S. iniae infection.     

Novel decaplex PCR assay for simultaneous detection of scallop species with species‐specific primers targeting highly variable 5′ end of the 16S rRNA gene

Scallops (family Pectinidae) comprise species of high commercial value, supporting both commercial fisheries and mariculture activities. Accurate and reliable molecular methods for the species level identification are of outstanding utility for taxonomic and food authentication surveys. The mitochondrial 16S rRNA gene has been used to design species‐specific primers for identification of different bivalve species. However, the low interspecific variability at the 3′ end of this gene has limited its utility and only few scallop species have been assessed. In this study, we used the high variable 5′ end of the 16S gene to develop a novel decaplex PCR assay that enabled a fast and accurate identification of eight commercially important scallop species in a single PCR reaction. A total of 285 individuals including fresh and manufactured samples from eight different processed presentations from 11 different scallop species were collected representing diverse locations around the world. Our assay accurately identified all the analysed samples at the species level. Furthermore, to enhance the utility of our assay, the PCR product amplified by the family specific primer set that was utilized as positive control was also used for the identification of unknown (non‐target) scallop species by DNA sequencing analysis. In its present form, our multiplex PCR method can be of great utility for different types of studies involving scallop species and for research institutes and governmental agencies that regulate seafood authentication around the world.     

温和气单胞菌PCR快速诊断方法的建立

根据NCBI公布的温和气单胞菌(Aeromonas sobria)丝氨酸蛋白酶的基因序列,设计并选取一对能够快速而准确地检测温和气单胞菌的PCR引物,建立PCR快速检测体系,并对患病的鱼组织进行检测。研究结果表明,使用设计的引物能够扩增出与预计大小相符合的131 bp的特异性片段,具有较好的检测特异性,对靶标DNA的检测灵敏度为1.0 pg/20μL,对温和气单胞菌的检测灵敏度为50 cfu/20μL。样品的检测结果与实际发病情况一致,表明本研究成功建立了温和气单胞菌常规PCR检测体系,该体系可以用于温和气单胞菌的诊断和样品的检测。     

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real‐time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS‐s/IGS‐a, which targets the 16S‐23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post‐injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post‐injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.     

Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases

Streptococcus iniae is an important bacterial pathogen of fish, causing up to 50% mortality in stocks, which has recently been associated with human infections. To determine whether S. iniae isolates from humans and fish are similar, the present authors examined the biochemical profiles and genetic relatedness of these isolates by random amplified polymorphic DNA (RAPD) analysis and repetitive primer polymerase chain reaction(REP PCR). The biochemical profiles differentiated between the human and fish isolates of S. iniae using pyrrolidonyl arylamidase, arginine dehydrogenase, ribose, β-glucoronidase and glycogen as markers. These biochemical results suggest that the fish and human S. iniae isolates are genetically different. However, RAPD and REP PCR do not have the discriminatory power to differentiate between these streptococcus isolates using five different RAPD primers and BoxA primer.     

Identification and Detection of the Pathogenic Bacteria Responsible for Swollen Abdomen Disease in Cultured Turbot,Scophthalmus maximus,and Flounder,Paralichthys olivaceus

Swollen abdomen disease (SAD) seriously threatens the aquaculture of turbots and flounders. Two dominant bacterial strains, FS1 and FS2, were isolated from the livers and kidneys of fish with diagnosed SAD. Applications of biochemical analyses, sequence analyses of 16S ribosomal RNA gene and heat shock protein 60 gene revealed two distinct pathogenic bacterial species, Edwardsiella tarda and Vibrio alginolyticus. These two hypothesized SAD‐causing pathogens were validated by challenge trials on flounder, Paralichthys olivaceus. Challenges with E. tarda and V. alginolyticus demonstrated lethal dose 50 (LD₅₀) values at 1.51 × 10⁵ colony‐forming units (CFU) and 1.05 × 10⁵ CFU, respectively. To develop a rapid SAD diagnosis method in flounders and turbots, a multiplex polymerase chain reaction (PCR) assay method was developed to simultaneously detect E. tarda and V. alginolyticus. Our multiplex PCR assay successfully detected as low as 10⁵ CFU/mL E. tarda and V. alginolyticus in flounders and turbots. No other common fish pathogens were detected with the multiplex PCR, suggesting a high specificity of this assay. The multiplex PCR assay developed in this study showed a great reliability in detecting SAD‐causing bacterial pathogens. Further optimization of this assay may contribute to the development of a novel SAD diagnosis tool in aquaculture.     

鱼类环境DNA研究中通用引物的筛选验证

为了筛选一个通用性和适用性良好的能够运用于环境DNA(eDNA)研究的鱼类引物,从相关文献中选取了鱼类线粒体基因组部分片段的5对引物,分别对线粒体D-loop区、16S rRNA基因、COI基因以及Cytb基因部分片段进行扩增。对千岛湖48种鱼类基因组DNA进行扩增后比较发现,引物16s和COI均可以取得良好的扩增效果,通用性优于其他几对引物。引物16s的扩增产物经凝胶电泳检测均出现明亮的目的条带,引物COI则有3种鱼的条带经凝胶电泳检测亮度较暗。利用上述引物对环境样品eDNA扩增时发现,只有16s和COI的引物具有良好的扩增效果,能够得到明显单一的亮带。对该两种引物的PCR产物克隆后测序比对发现,16s的PCR产物均为千岛湖常见鱼类物种的基因片段,COI的PCR产物则为细菌COI基因的部分片段。综上,我们认为引物16s在通用性和适用性上都更为适合作为鱼类群落结构eDNA研究的通用引物。     

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real‐time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real‐time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real‐time PCR assays determined using genomic DNA templates from three target viruses, 12 non‐target viruses and 25 aquatic bacterial species were 100%. The intra‐run and inter‐run coefficients of variation of both assays were <5%. The real‐time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.