Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT‐PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman‐based real‐time RT‐PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real‐time RT‐PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell‐based methods. In conclusion, the presented real‐time RT‐PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.     

Improvement of a diagnostic procedure in surveillance of the listed fish diseases IHN and VHS

Infectious haematopoietic necrosis (IHN) and viral haemorrhagic septicaemia (VHS) are OIE‐listed and notifiable viral fish diseases which are controlled by eradication and surveillance programmes globally. The present study provides improved RT‐qPCR procedures based on recently described OIE protocols. Improvements comprise the design of a new TaqMan® probe, replacing a TaqMan® MGB probe that turned out to show impaired binding. Reason for this is SNPs detected in the nucleoprotein N gene sequences of IHNV strains targeted by the RT‐qPCR. Furthermore, the IHNV and VHSV RT‐qPCR assays were realized as one‐step and one‐run procedures supplemented by an endogenous control system. The IHNV and VHSV RT‐qPCR assays are characterized by a technical sensitivity of 19 and 190 gene equivalents (cRNA) and an analytical sensitivity of 2–7 and 13 TCID₅₀/ml, respectively. For verification purposes, 105 IHNV and 165 VHSV isolates and several non‐targeted viral and bacterial pathogens were included and returned adequate results. However, in field samples divergent results left 14 samples of 154 undetected for IHNV and one sample of 127 for VHSV using cell culture. The study shows that RT‐qPCR assays ensure facilitated and reliable testing on IHNV and VHSV in eradication and surveillance programmes.     

Investigation of round goby viral haemorrhagic septicaemia outbreak in New York

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT‐qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream,Pagrus auriga Valenciennes,and common seabream,Pagrus pagrus (L.), using a non‐destructive procedure

A non‐destructive procedure based on nested RT‐PCR and dot‐blot hybridization has been developed for the detection of asymptomatic IPNV‐carrier fish. The pair of primers designed for RT‐PCR amplified a 599‐bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT‐PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191‐bp probe was designed for hybridization studies used in combination with the nested RT‐PCR. The application of the nested RT‐PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV‐carriers, respectively. The combination of nested RT‐PCR and dot‐blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Trade practices are main factors involved in the transmission of viral haemorrhagic septicaemia

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus viral haemorrhagic septicaemia virus (VHSV), causes significant economic problems to European rainbow trout, Oncorhynchus mykiss (Walbaum), production. The virus isolates can be divided into four distinct genotypes with additional subgroups. The main source of outbreaks in European rainbow trout farming is sublineage Ia isolates. Recently, this group of isolates has been further subdivided in to two subclades of which the Ia‐2 consists of isolates occurring mainly in Continental Europe outside of Denmark. In this study, we sequenced the full‐length G‐gene sequences of 24 VHSV isolates that caused VHS outbreaks in Polish trout farms between 2005 and 2009. All these isolates were identified as genotype Ia‐2; they divided however into two genetically distinct subgroups, that we name Pol I and Pol II. The Pol I isolates mainly caused outbreaks in the southern part of Poland, while Pol II isolates predominantly were sampled in the north of Poland, although it seems that they have been transmitted to other parts of the country. Molecular epidemiology was used for characterization of transmission pathways. This study shows that a main cause of virus transmission appears to be movement of fish. At least in Polish circumstances trading practices appear to have significant impact on spreading of VHSV infection.     

Application of a sensitive,specific and controlled real‐time PCR assay to surveillance indicates a low prevalence of viral haemorrhagic septicaemia virus (VHSV) in wild herring,Clupea harengus L., in Scottish waters

Surveillance data on the distribution of viral haemorrhagic septicaemia virus (VHSV) in the North Sea (UK), targeting Atlantic herring in areas with previous virus detection, were obtained from research cruises conducted during 2005. The sensitive molecular approach of real‐time RT‐PCR (qRT‐PCR) was applied alongside a newly developed endogenous positive control assay specific for herring (elongation factor 1α) to ensure integrity of template. Three hundred and five pools from 1937 individual herring were tested, and no evidence of VHSV in association with wild Atlantic herring was detected. Samples were obtained from Scottish waters where marine aquaculture is conducted. The results confirm that previous tissue culture studies have most likely not significantly underestimated the prevalence of carrier herring in this area. The significance of migratory species such as herring as a reservoir species for VHSV, with the potential to translocate virus genotypes between geographical areas, is discussed.     

PCR‐based prevalence of a fatal reovirus of the blue crab,Callinectes sapidus (Rathbun) along the northern Atlantic coast of the USA

There is a need for more information on the relationship between diseases and fluctuations of wild populations of marine animals. In the case of Callinectes sapidus reovirus 1 (CsRV1, also known as RLV), there is a lack of baseline information on range, prevalence and outbreaks, from which to develop an understanding of population‐level impacts. An RT‐qPCR assay was developed that is capable of detecting 10 copies of the CsRV1 genome. In collaboration with state, federal and academic partners, blue crabs were collected from sites throughout the north‐eastern United States to assess the northern range of this pathogen. In addition, archived crab samples from the Chesapeake Bay were assessed for CsRV1 by RT‐qPCR and histology. PCR‐based assessments indicate that CsRV1 was present at all but one site. Prevalence of CsRV1 as assessed by RT‐qPCR was highly variable between locations, and CsRV1 prevalence varied between years at a given location. Mean CsRV1 prevalence as assessed by RT‐qPCR was >15% each year, and peak prevalence was 79%. The wide geographic range and highly variable prevalence of CsRV1 indicate that more study is needed to understand CsRV1 dynamics and the role the virus plays in blue crab natural mortality.     

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.     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

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.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.