Heritability of Disease Resistance and Immune
Function in Chinook Salmon, with Special
Emphasis on Broodstock Culling to Control
Bacterial Kidney Disease

The Problem

Many diseases of trout and salmon persist in our cultured fish stocks today, despite improvements in fish culture practices and years of research on vaccines and chemotherapeutants. An excellent example is bacterial kidney disease (BKD), caused by the bacterium Renibacterium salmoninarum. Infections by R. salmoninarum are considered by many to be one of the most detrimental problems of Pacific salmon. The kidney disease bacterium manifests itself early in the growing fry, and infections may

Individual egg lots are held separately until BKD testing is complete.

progress to kill the fish or enter a chronic, systemic phase that can persist as a life-long disease. We know healthy fish can become infected with R. salmoninarum following their contact with water or hatchery equipment contaminated with bacteria shed from infected fish. Fish health managers have consequently developed new hatchery practices that minimize or eliminate fish-to-fish spread of BKD. Unfortunately, mortality due to BKD continues today in many trout and salmon programs, and there are no effective vaccines or antibiotic therapies to completely stop the losses. We now believe that other factors, such as vertical transmission of the bacterium, may be especially important for the persistence of R. salmoninarum in salmonid stocks. Vertical transmission only occurs with a very small fraction of the fish pathogens, and is unique in that it results passage of the microorganism from generation-to-generation in the maturing egg - R. salmoninarum is a vertically transmitted fish pathogen, and in this case, the successful egg-associated transmission can be correlated with high levels of infection in at least the female parent. Although the male parent is assumed to play a minor role in the vertical transmission of R. salmoninarum, there is very little scientific information available to support this belief. In an attempt to control vertical tranmission, most hatcheries in the western U.S. and elsewhere have aggressive R. salmoninarum monitoring programs that target adult fish as they reach sexual maturity. The goal is to screen female parents at the time of spawning to determine if they are infected with the kidney disease bacterium, and if so, whether the level of infection is high enough to warrant concern that they may transmit BKD to the next generation with their eggs. If the answers are "yes" in both cases for a given female, her eggs are culled from the spawning population to later be segregated for separate rearing, or destroyed. This culling procedure has proven to be one of the most significant advances in the control of BKD in hatchery salmonids.

Genetic effects of hatchery management practices have become a major concern in recent years. In particular, questions have been raised regarding the effects of broodstock culling to prevent outbreaks of BKD. This practice may reduce the future fitness of the affected fish populations if general disease resistance is heritable and somehow correlated genetically with high R. salmoninarum infection levels in one, or both, parents. This places fish health managers in a difficult situation, as they may successfully control BKD through broodstock culling, but at the same time select for fish that may be poorly equipped to resist infection by other microorganisms. Unfortunately, we neither understand the exact efficiency of vertical transmission by R. salmoninarum relative to the level of infection in the parents, nor the possible genetic impacts of broodstock culling on the long-term fitness of progeny fish. These data are critical for optimizing broodstock maintenance procedures, particularly where artificial propagation may be necessary to help recover populations listed under the Endangered Species Act.

Objectives

The goal of this study is to use tools in quantitative genetics, molecular biology, and fish microbiology to evaluate the relative value and genetic risks associated with broodstock culling to control BKD. To do so, the study has been divided into two parts: (1) First, to more completely understand the vertical transmission of R. salmoninarum in spring chinook salmon, individual eggs from females with different R. salmoninarum infection levels are being tested to determine if they contain quantifiable levels of the bacterium. This information will help fish health biologists more accurately identify which egg lots should be segregated to reduce future BKD-related losses. (2) In the second part of this study, laboratory disease challenges and genetic analyses are being used to determine if there is a correlation between disease resistance in progeny fish and the R. salmoninarum infection levels in the male parent. The relative resistance of progeny from each family group to infection by either R. salmoninarum, or another fish pathogen typically encountered by salmon entering seawater (Vibrio anguillarum), will be measured in separate laboratory challenges.

Methodology

Spawning spring chinook salmon returning to Carson National Fish Hatchery on the lower Columbia River were selected to provide eggs for this study. The goal was to create families from specific mating, such that 24 families would be the progeny of males with high levels of R. salmoninarum infection, and 24 families would be the progeny of males with low or undetectable levels of infection.

A quantitative PCR is being used to determine how many eggs are infected with the kidney disease bacterium.

To complete the first goal of this study, the prevalence of R. salmoninarum among individual eyed eggs from each family will be determined using a quantitative polymerase chain reaction that detects a gene of R. salmoninarum. When complete, this testing should provide a quantitative estimate of the rate and magnitude of vertical transmission relative to the parental level of R. salmoninarum infection. For the second part of this study, the relative abilities of fish from each family to resist infection by R. salmoninarum and Vibrio anguillarum will be measured under controlled laboratory conditions. Quantitative genetic methods will be used to estimate genetic correlations between the level of R. salmoninarum infection in parent fish and the level of infection and disease resistance among their progeny.

Highlights and Key Findings

During the fall of 2001, researchers from the Western Fisheries Research Center set up a mobile fish health laboratory at Carson National Fish Hatchery. The purpose of this laboratory was to screen spring chinook salmon returning to the hatchery for the presence and levels of R. salmoninarum by testing tissues with highly sensitive serological and molecular assays. Kidney tissue samples from over 400 male fish and 84 female fish were processed and tested overnight for the presence of R. salmoninarum - during this testing period sperm and egg lots were held separately for selected mating based on the testing results. Sperm from males with the 13 lowest R. salmoninarum infection levels were used to separately fertilize the eggs from each of three random females (1 male x 3 females). Similarly, sperm from males with the 15 highest R. salmoninarum infection levels were used to fertilize the eggs from each of three additional, random females (1 male x 3 females). These crosses resulted in a total of 84 full-sib families: 39 families representing the progeny of the 13 "lowest" males and 45 families representing the progeny of the 15 "highest" males. Fertilized eggs from each cross were reared in separate, isolation-type incubators on pathogen-free water.

Passive integrated transponder (PIT) tag used to give individual fish a unique identifier for the bacterial challenges.

A system was developed so that progeny fish from all the families could be combined into a single tank for a given bacterial challenge. To accomplish this, progeny fish in each family group were given a unique identifier by injection of a passive integrated transponder (PIT) tag. PIT tags are very small glass tubes that encapsulate both an integrated circuit and a coil (antenna). Each integrated circuit is programmed with a unique code that can be read by passing the fish through a a special apparatus that energizes the circuit. All of the fish were injected with a PIT tag prior to the challenges, permitting us to identify the family of origin of any fish that die from among the approximately 1,600 fish held together in a challenge or control tank.

PIT tag is activated by a special reader.

A quantitative polymerase chain reaction (QPCR) was developed to test the individual eggs for R. salmoninarum. It provides us with an extremely sensitive method to determine how much R. salmoninarum DNA is inside an egg. Extraction of DNA from individual eggs is ongoing, and to date, more than 1,000 eggs have been processed for analysis.

Where Are We Headed In 2003

During 2003 the bacterial challenges will be completed, and the QPCR will be used to look for R. salmoninarum DNA in each of the processed eggs. This testing will allow a more accurate estimate: (1) of the probability of vertical transmission relative to parental R. salmoninarum infection levels, (2) if there is an additional genetic factor involved in transmission, and (3) if the rate of transmission can be correlated with either general disease resistance or only resistance to R. salmoninarum.

Project Contact

John Hansen
U.S. Geological Survey
Western Fisheries Research Center
6505 NE 65th St.
Seattle, WA 98115

Email: jhansen@usgs.gov
Phone: 206-526-6282
Fax: 206-526-6654

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