Diane G. Elliott

Infectious haematopoietic necrosis virus genogroup‐specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control.

Brood stock segregation for the control of bacterial kidney disease can affect mortality of progeny chinook salmon (Oncorhynchus tshawytscha) in seawater.

Abstract Segregation of spring chinook salmon ( Oncorhynchus tshawytscha ) brood stock based on the measurement of maternal Renibacterium salmoninarum infection levels by the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) was previously shown to affect the prevalence and levels of bacterial kidney disease (BKD) in progeny fish during hatchery rearing. Smolts from that study were subjected to standardized fish health and condition evaluation procedures 2 weeks before the conclusion of hatchery rearing and release of the fish for migration to the Pacific Ocean. The results suggested that the general health of the smolts in the progeny group from parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group) was better than that of the smolts in the progeny group from female parents with high R. salmoninarum infection levels (high-BKD group). Testing by the ELISA showed that the overall severity of R. salmoninarum infection also was lower in the smolts from the low-BKD group. Subgroups of smolts from the study were acclimated to tanks of seawater for extended holding. After a 22-day acclimation period and 98 days in full-strength (29 ppt salinity) seawater, total mortality was 12% in the low-BKD group and 44% in the high-BKD group. All of the mortality in the low-BKD group and 85% of the mortality in the high-BKD group occurred after the fish were transferred to full-strength seawater. Testing of kidney tissues from all dead fish by the FAT revealed that 85% of the fish that died in the high-BKD group had high R. salmoninarum numbers, indicating that BKD was the cause of death. In contrast, none of the fish that died in the low-BKD group had detectable numbers of R. salmoninarum . We concluded that brood stock segregation by use of the ELISA and the FAT can affect mortality and the R. salmoninarum status of progeny chinook salmon for as long as 21 months after hatching, even after the fish have been transferred to seawater.

Renibaeterium salmoninarum in Spring–Summer Chinook Salmon Smolts at Dams on the Columbia and Snake Rivers

Abstract We evaluated Renibaeterium salmoninarum infection in smolts of hatchery and wild spring-summer Chinook salmon Oncorhynchus tshawytscha sampled during most of the outmigration at Little Goose (1988) and Lower Granite dams (1988–1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988–1990). We sampled 860–2,178 fish per dam each year. Homogenates of kidney–spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1–11% of fish sampled at a given dam during any l year exhibited lesions characteristic of bacterial kidney disease, 86–100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4–17% of the fish tested positive by the FAT. During most years, a majority (68–87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, ...

Evidence that Coded-Wire-Tagging Procedures Can Enhance Transmission of Renibacterium salmoninarum in Chinook Salmon

Abstract Binary coded wire tags (CWTs) are used extensively for identification and management of anadromous salmonid populations. A study of bacterial kidney disease (BKD) in two brood year groups of hatchery-reared spring chinook salmon Oncorhynchus tshawytscha provided strong evidence that horizontal transmission of Renibacterium salmoninarum, the causative agent of BKD, might be enhanced by CWT-marking procedures. About 4 months after CWTs were implanted in the snouts of juvenile fish, 14–16 different tissues were sampled from each of 60 fish per brood year group for histological analysis. Of the fish that were positive for R. salmoninarum by histological examination, 41% (7 of 17) of the 1988 brood year fish and 24% (10 of 42) of the 1989 brood year fish had BKD lesions confined to the head near the site of tag implantation. These lesions often resulted in the destruction of tissues of one or both olfactory organs. No focal snout infections were observed in fish that had not been marked with CWTs. Fur...

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

ABSTRACT Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.

Comparison of Traditional and Molecular Methods for Detection of Renibacterium Salmoninarum

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum occurs in most parts of the world where wild or cultured salmonid fishes are present. Several extensive reviews have been written on the pathogen and the disease (Fryer and Sanders, 1981; Austin and Austin, 1987; Elliott et al., 1989; Evelyn, 1993; Evenden et al., 1993; Fryer and Lannan, 1993). Bacterial kidney disease can cause serious mortality in juvenile salmonids in both fresh water and seawater, and also in prespawning adults. Although the chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral fish populations, BKD is one the most important bacterial diseases affecting cultured salmonids, with reported losses as high as 80% in stocks of Pacific salmon (Oncorhynchus spp.) and 40% in stocks of Atlantic salmon (Salmo salar) (Evenden et al., 1993).

Decreased Mortality of Lake Michigan Chinook Salmon after Bacterial Kidney Disease Challenge: Evidence for Pathogen-Driven Selection?

In the late 1960s, Chinook salmon Oncorhynchus tshawytscha from the Green River, Washington, were successfully introduced into Lake Michigan. During spring from 1988 to 1992, large fish die-offs affecting Chinook salmon occurred in the lake. Multiple ecological factors probably contributed to the severity of the fish kills, but the only disease agent found regularly was Renibacterium salmoninarum, the causative agent of bacterial kidney disease. In this study, survival after challenge by R. salmoninarum was compared between two Chinook salmon stocks: a Lake Michigan stock from Wisconsin (WI) and the progenitor stock from the Green River. We found that the WI stock had significantly greater survival than the Green River stock. Next, the WI and Green River stocks were exposed to the marine pathogen Listonella anguillarum (formerly Vibrio anguillarum), one of the causative agents of vibriosis; survival after this challenge was significantly poorer for the WI stock than for the Green River stock. A close genetic relationship between the Green River and WI stocks was confirmed by analyzing 13 microsatellite loci. These results collectively suggest that disease susceptibility of Lake Michigan Chinook salmon has diverged from that of the source population, possibly in response to pathogen-driven selection.

Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge.

Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-gamma, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (> or = 28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

Management of Bacterial Kidney Disease in Chinook Salmon Hatcheries Based on Broodstock Testing by Enzyme-Linked Immunosorbent Assay: A Multiyear Study

Abstract From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen le...