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.     

A comparison between non-destructive and destructive testing of Atlantic salmon, Salmo salar L., broodfish for IPNV--destructive testing is still the best at time of maturation.

Two populations of Atlantic salmon broodstock, previously identified as infectious pancreatic necrosis virus (IPNV) carriers, were screened for IPNV at the time of stripping. Four hundred and ten broodfish were individually sampled of which 91 were detected as IPNV positive by virus culture of sonicated kidney homogenates combined with gonadal fluid, but none tested positive by the blood leucocyte assay. Thirty fish identified as IPNV carriers prior to maturation by the blood leucocyte assay were used in a separate study to compare non-destructive vs. destructive testing methods at stripping. IPNV was not detected using the blood leucocyte method at the time of stripping. RT-PCR and real-time PCR assays failed to detect IPNV from 13 blood samples, the virus was not isolated from milt (0/14) or sonicated ovarian fluid cell pellets (0/16) and only three fish tested positive by the standard culture of kidney homogenates. A third study of Atlantic salmon broodfish compared the IPNV isolation rates prior to maturation with the isolation rates at spawning during 1999-2001. In each year the percentage of IPNV-positive broodfish was significantly lower than in the pre-broodstock sample. While in pre-broodfish samples IPNV was detected by the blood leucocyte assay, no culture isolations or PCR positives were detected from non-destructive samples of the same individual broodfish at stripping. A consistent finding was that even for the kidney assay, the percentage of IPNV-positive fish in carrier populations was higher in pre-broodstock than in broodfish at stripping. These results indicate that destructive kidney sampling is still the most sensitive method for detecting IPNV carrier Atlantic salmon broodfish and that a change in IPNV carrier-status occurs during the maturation period.     

Detection of Flexibacter maritimus in fish tissue using nested PCR amplification

A nested polymerase chain reaction (PCR) system was developed for the detection of Flexibacter maritimus from fish tissue. The total procedure for the diagnosis of marine flexibacteriosis, from the point of DNA extraction to the electrophoretic analysis, can be performed in < 4 h. This was achieved by the combination of a short thermal cycling programme with a rapid DNA extraction procedure. The assay was extremely sensitive, capable of detecting as few as 75 cfu mg(-1) fish tissue. The accuracy of the nested PCR was confirmed under field conditions using tissue samples recovered during 1993-2002 from fish suffering marine flexibacteriosis. The nested PCR method proved to be efficient for the rapid and sensitive detection of F. maritimus from fish tissues and can be used for routine diagnosis of the disease caused by this pathogen.     

同步检测7种鱼类病毒的扩增子拯救多重PCR(Arm-PCR)方法的建立和应用

淋巴囊肿病毒(LCDV)、肿大细胞病毒属虹彩病毒(Mega)、赤点石斑鱼神经坏死病毒(RGNNV)、传染性造血器官坏死病毒(IHNV)、传染性胰脏坏死病毒(IPNV)、病毒性出血败血症病毒(VHSV)和传染性鲑鱼贫血症病毒(ISAV)是养殖鱼类主要的病毒性病原,危害巨大。为实现这7种病原的高通量、同步检测,本研究在分析这7种病毒基因序列的基础上,设计了9组扩增子拯救多重PCR(Arm-PCR)引物,并对扩增体系中的Taq酶、Mg2+、dNTP、Primer Mix浓度及退火温度等参数进行调整和优化,结合基因芯片检测技术,建立了同步检测7种鱼类病毒的Arm-PCR方法。优化后的Arm-PCR方法第一步PCR体系为：Taq酶(2.5 U/μl)1.0μl,10×PCR Buffer(含20 mmol/L的Mg2+)5μl,dNTP(各2.5 mmol/L)5μl,10×Primer Mix(各2μmol/L)9μl,模板1μl,ddH2O补足至50μl,退火温度为56℃。研究结果显示,该方法可以在1支反应管内对上述7种病毒的9个致病基因同步进行扩增和检测,检测灵敏度分别为101 copies/μl (RGNNV、VHSV、ISAV-NS、ISAV-MA)、102 copies/μl (LCDV、Mega、IHNV、IPNV)和103 copies/μl (大菱鲆红体病虹彩病毒,TRBIV)。该方法特异性强,与半滑舌鳎、石斑鱼、大菱鲆和牙鲆基因组DNA不产生交叉反应。本研究建立的可同步检测7种鱼类病毒的Arm-PCR方法具有高通量、高灵敏度、高准确性的优势,能有效提高工作效率,在鱼类病毒的筛查和流行病学调查领域有广泛的应用前景。     

A sandwich ELISA to detect VHSV and IPNV in turbot

The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.     

A survey of viral diseases in farmed and feral salmonids in Switzerland

A field survey was carried out to study the occurrence and distribution of viruses causing diseases of major impact in fish farming, namely viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) in farmed and wild fish in Switzerland. The presence of VHS virus (VHSV), IHN virus (IHNV) and IPN virus (IPNV) in the tissue samples was tested by virus isolation in cell cultures, and subsequent virus identification by immunofluorescence. The sera were screened for anti-VHSV antibodies (VHSV-AB) using a serum plaque neutralization test with complement addition. These data were then compared with results of a similar survey performed in 1984/85, and with data from routine diagnostic work completed at the Centre for Fish and Wildlife Health (FIWI) of the University of Bern from 1978 to 2001. Sampling sites included private and government fish farms as well as natural habitats from all major river catchments in Switzerland. In 2000/01, 522 tissue samples and 1910 sera were collected from 3400 fish. In 1984/85 1239 tissue samples and 694 sera were collected from 1628 fish. During the last 24 years of routine diagnostics at the FIWI, 1776 tissue samples were examined for presence of viruses. The results of the tissue analysis from the surveys in 1984/85 and 2000/01 showed low numbers of sites with virus-positive fish (five VHSV, three IPNV and three VHSV, one IPNV, respectively) in Swiss fish farms and rivers. The sites with virus-positive fish were located throughout the country. The decline in virus-positive cases observed between the two surveys agrees with data from the routine diagnostic work of the FIWI which show a decrease in total virus isolations from approximately 35 cases per year in the late 1970s, to approximately 10 cases per year during the last 10 years. However, in 1984/85 8.3% (58 of 694 serum samples) and in 2000/01 6.3% (121 of 1910 serum samples) proved to be positive for VHSV-AB. The 58 positive samples in 1984/85 originated from 40 of 175 sites (23%) and the 121 positive samples in 2000/01 were from 84 of 217 (29%) sites. These results are indicative of a wider distribution of VHSV than expected from the results of the virus isolations.     

Genogrouping of birnaviruses isolated from marine fish: a comparison of VP2/NS junction regions on genome segment A

Yellowtail ascites virus and related strains isolated from marine fish have been shown to be similar to infectious necrosis virus (IPNV) in terms of biological and serological characteristics. This paper explores the relationship of aquatic birnaviruses at the genetic level. The junction region on the genome segment A coding viral capsid protein VP2 and viral protease NS was amplified by PCR in six marine strains. Analysis of nucleotide and the deduced amino acid sequences revealed that the six marine strains have amino acid variations in the possible amino terminus of NS when compared to IPNV. The six marine strains form a new genogroup which is distinguished from three serotypes ofPNV.     

Detection and identification of Flavobacterium psychrophilum from gill washings and benthic diatoms by PCR-based sequencing analysis

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S-rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S-rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.     

Infections of nervous necrosis virus in wild and cage‐reared marine fish from South China Sea with unexpected wide host ranges

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage‐reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage‐reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage‐reared fish, respectively. However, by nested RT‐PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage‐reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage‐reared fish by seminested PCR assay, respectively. However, by nested RT‐PCR, the positive signal was observed in 65.2% and 100% species of wild and cage‐reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red‐spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty‐five species of the marine fish were the new hosts of NNV.     

A sensitive non-destructive method for detecting IPNV carrier Atlantic salmon, Salmo salar L., by culture of virus from plastic adherent blood leucocytes

In populations of Atlantic salmon in sea water, infectious pancreatic necrosis virus (IPNV) could be detected by standard virological culture methods in sonicated kidney homogenates and in mucus samples (gill, skin and rectum) from 14 and nine of 25 fish, respectively, but all fish were positive by virus culture from lysates of kidney macrophages and adherent blood leucocytes. In fish which tested negative for IPNV by the standard method of detection, the virus could be detected using adherent blood leucocytes isolated on a Percoll gradient from as little as 10 microL of blood. The blood sample could be stored for at least 3 days in a heparinized tube on ice before preparing the plastic adherent leucocytes. Furthermore, the latter could be prepared without prior fractionation on Percoll simply by incubating whole blood (33 microL) in cell culture medium (66 microL) in 96-well plates overnight and washing away the non-adherent cells before lysing the adherent cells and inoculation of the lysate onto CHSE-214 cells. This highly sensitive method for detecting IPNV-carriers is therefore very suitable for non-destructive sampling of fish in the field.     

Evaluation of a rapid coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of Atlantic salmon, Salmo salar L.

The field use of a staphylococcal coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples from Atlantic salmon, Salmo salar L., was evaluated. The COA test was compared with an immunohistochemical (IHC) method for the detection of clinical outbreaks of infectious pancreatic necrosis (IPN). The present paper describes the evaluation of 320 COA test results performed at local fish health laboratories in Norway from 1994 to 1996, and COA test results from two infection trials with IPNV. The agreement between the COA test and the IHC was very good. The agreement beyond chance, measured as kappa values, was 0.74 in individuals and 0.90 in pooled samples. Thus, the COA test was suited for the detection of outbreaks of IPN. Covert infections with IPNV remained undetected by the COA test. The minimum IPNV titre needed to obtain a positive COA test was ≈ 10⁵ TCID₅₀ mL^–1.     

Study of the interaction between a persistent infectious pancreatic necrosis virus (IPNV) infection and experimental infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L.

Abstract. An infectious pancreatic necrosis virus (IPNV) carrier stock of Atlantic salmon parr (100 g) was divided between two tanks and inoculated experimentally with tissue homogenate containing the aetiologic agent of infectious salmon anaemia (ISA) and non-ISA tissue homogenate (control), respectively. Plasma and kidney samples from ISA-infected and control fish were taken twice weekly for 25 days. In the kidney samples, IPNV was quantified by a plaque assay. In plasma, anti-IPNV antibodies were measured using an indirect ELISA. The ISA-infection did not seem to activate the IPNV-infection. Neither the proportion of fish with IPNV or anti-IPNV antibodies, nor the IPNV titre or level of anti-IPNV antibodies showed any specific trend during the study. Independently of ISA, IPNV was detected in 54 out of 132 fish (41%), while 71 out of 195 fish (36%) had plasma antibodies against IPNV. No association was found between detection of IPNV, and presence or level of anti-IPNV antibodies in individual fish.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.