Leucocytes from Atlantic salmon, <i>Salmo salar</i> L., experimentally infected with infectious salmon anaemia (ISA) exhibit an impaired response to mitogens

Infection models for both fresh and seawater salmon were established using a Scottish isolate of infectious salmon anaemia virus (ISAV). Modes of infection were intra-peritoneal injection, cohabitation and immersion exposure, and a range of doses was tested. Development of these models using a Scottish isolate of ISAV provided an approximation of the minimum infective dose leading to mortality under different infection regimens. The models also allow prediction of the time to first mortality and an estimation of expected total mortality following the various routes of infection. Such knowledge is important to the development of anti-ISAV vaccines and to future studies aimed at understanding the biology of ISAV in general. KEY WORDS: Infectious salmon anaemia virus · Atlantic salmon · Salmo salar · Experimental infection Resale or republication not permitted without written consent of the publisherDis Aquat Org 47: [169][170][171][172][173][174] 2001 MATERIALS AND METHODS Cell culture and virus propagation. The isolate of ISAV used in this study was 0390/98, which originated from a clinical outbreak of ISAV on a salmon farm in west Scotland. The salmon head kidney-1 (SHK-1) cell line was used to propagate virus for transmission experiments according to previously described methods (Dannevig et al. 1995). Harvested virus was stored at -80°C following 2 passages in SHK-1 cells. A single aliquot of virus was titrated using the tissue culture infectious dose method (TCID 50 ) (Reed & Muench 1938, Burleson et al. 1992) following a single freezethaw cycle.Pathogen-free fish. Atlantic salmon parr (mean weight 27.5 g) and Atlantic salmon post-smolts (mean weight 273 g) were reared at the FRS Marine Research Unit, Aultbea, Ross-shire, Scotland. Before the infection experiments fish were screened for the presence of ISAV, viral haemorrhagic septicaemia virus, infectious haematopoietic necrosis virus and infectious pancreatic necrosis virus by standard virology tissue culture methods as previously described (Dannevig et al. 1995, Snow & Smail 1999 and, for ISAV, by reverse transcription polymerase chain reaction (RT-PCR) (Mjaaland et al. 1997).Experimental infection with ISAV. Fish were allowed to acclimate for 7 d and starved for 24 h before all experimental infections. Water temperatures were maintained at 10°C for the duration of each experimental infection. Freshwater was supplied from Aberdeen city drinking water, which originates from the River Dee and passes through an activated carbon filter and UV treatment before entering fish tanks. Seawater was extracted from the North Sea at a salinity of 32 ‰ and passed through a sand filter and UV disinfection treatment before entering fish tanks.Experimental infection of freshwater Atlantic salmon parr with ISAV. Atlantic salmon parr were stocked in 30 l aquaria supplied with flow rates of 40 l h -1 freshwater, at a density of 15 fish per tank. Experimental infections were obtained via immersion, i.p. and cohabitation routes. Fish were anaethetised before i.p. i...

Section: Discussionsupporting

confidence: 84%

Experimental infection models and susceptibility of Atlantic salmon Salmo salar to a Scottish isolate of infectious salmon anaemia virus

Raynard

Snow²,

Bruno³

2001

Section: Resultssupporting

confidence: 92%

“…While the physical relationship between ISAV particles in blood and the circulating erythrocytes is not known, it is clear from our study that ISAV does not replicate in circulating red blood cells. This is consistent with data cited by Dannevig et al (1993) which showed that in vitro inoculation of salmon erythrocytes or reticulocytes with infective ISA material did not result in ISAinfective cells as judged by transmission trials in fish.In conclusion, this study demonstrates that viremia in ISAV infection is associated with virus replication in leucocytes, and that erythrocytes are not target cells for virus replication. Studies are in progress to establish the physical relationship (if any) between ISAV particles in blood and the circulating erythrocytes in ISAV-infected fish.…”

supporting

confidence: 92%

Viremia during infectious salmon anemia virus infection of Atlantic salmon is associated with replicating virus in leucocytes

Moneke¹,

Ikede²,

Kibenge³

2005

ABSTRACT:In situ hybridization (ISH) was used to investigate the presence of viral mRNA in different fractions of blood cells of Atlantic salmon between 6 and 12 d following experimental infection with infectious salmon anemia virus (ISAV). Using a riboprobe targeting ISAV segment 7 mRNA, hybridization signals were observed only in leucocytes in buffy coat smears from samples collected from 8 to 12 d post-infection (dpi). None of the red blood cell smears from any sample showed ISH signal. These observations allow us to conclude that viremia in ISAV infection is associated with virus replication in leucocytes, and that erythrocytes are not target cells for virus replication. KEY WORDS: ISAV · Cell-associated viremia · In situ hybridization · Atlantic salmon Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [153][154][155][156][157] 2005 cides with the peak viremia period (Moneke et al. 2005). Previous in situ hybridization (ISH) studies on ISAV-infected fish have not reported on viral mRNA in blood cells (Gregory 2002, Moneke et al. 2003 although we have occasionally observed ISH signals in some circulating blood cells suspected as erythrocytes and in some individual cells in the head kidney suspected to be hematopoietic cells (Moneke et al. 2004). It was therefore decided to use ISH to more specifically identify the circulating blood cell(s) carrying virus during viremia. MATERIALS AND METHODS Cells and viruses.The ISAV isolate Norway 810/9/99 used for this study was propagated and titrated in the TO cell line as described previously (Kibenge et al. 2001).Riboprobe preparation. Preparation of the ISAV riboprobe was carried out as previously described (Moneke et al. 2003). Briefly, total RNA extracted from ISAV-infected cell culture lysate with Trizol Reagent (Invitrogen Life Technologies) was used in reverse transcription-polymerase chain reaction (RT-PCR) to obtain ISAV cDNA. The PCR primers consisted of ISAV RNA segment 7 (Ritchie et al. 2002, GenBank Acc. No. AF328627) forward primer 5'-ATG TCT GGA TTT AAC CTC GAG G-3' (nucleotides 133-154) and segment 7 reverse primer 5'-CAT AAC AAG TTT TCA ACC AAT C-3' (nucleotides 902-923) that yield a product 792 bp long. One-step RT-PCR was carried out as previously described (Kibenge et al. 2000), using Titan-one tube RT-PCR kit (Roche Molecular Biochemicals). The RT-PCR product was first cloned in the pCR ® II-TOPO ® Vector (Invitrogen Life Technologies) following the manufacturer's protocol. The insert DNA was then subcloned into pGEM-3Z vector (Promega) using the Rapid DNA Ligation Kit (Roche Molecular Biochemicals). The orientation of the DNA insert in the plasmid was determined by restriction enzyme analysis in order to allow generation off the T7 promoter, an antisense riboprobe of approximately 405 bases long. Thus the plasmid DNA was digested with Xba I prior to use. The in vitro transcription reaction was carried out in presence of digoxigenin-11-deoxyuridine triphosphate (Digoxigenin-11-UTP, Roche Molecular Bi...

“…Atlantic salmon presmolts with an average weight of about 100 g were obtained from a salmon hatchery where ISA had never been diagnosed. Each of 200 fish was injected intraperitoneally with 0.5 m1 ISAV-infective tissue homogenate prepared from experimentally infected salmon, as described elsewhere (Dannevig et al 1993). The fish were kept at 12°C in fresh water in a 1 m* tank.…”

Section: Methodsmentioning

confidence: 99%

Time course tissue distribution of infectious salmon anaemia virus in experimentally infected Atlantic salmon Salmo salar

Rimstad

Falk²,

Mikalsen³

et al. 1999

Atlantic salmon Salmo salar L. were injected intrapentoneally with infectious salmon anaemia virus (1SAV)-infective tissue homogenate to clarify the tissue distribution of ISAV in a time course study. Fish were sampled at 11 different intervals between 1 and 40 d post-infection (p.i.) and mid-kidney, head kidney, liver, spleen, intestine, gills, muscle and heart were tested for the presence of ISAV by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that during a disease outbreak, ISAV is present in most organs. It was possible to detect ISAV at all sampling times in at least 1 of the fish examined. However, for the first 8 d p.i. positive RT-PCR results were predominantly found in samples from the head hdney and mid-kidney. Fish giving positive samples after Day 13 p.i. were RT-PCR positive in most organs. These results indicated that between Days 8 to 13 p.i. considerable replication of the virus occurred, combined with wide tissue dissemination.