Vertical transmission is the infection of the next generation of fish via the movement of pathogens into fertilized eggs from milt or ovarian fluids. Pathogens that enter eggs via vertical transmission are able to avoid standard iodophor inactivation procedures. The potential for a pathogen to be vertically transmitted increases its risk of spreading via egg shipments. Laboratory experiments have demonstrated that vertical transmission can occur with certain viral, bacterial, and eukaryotic pathogens. In this assessment, vertical transmission is designated as “Yes” for pathogens with published evidence to support vertical transmission, “No” for pathogens with published evidence that they cannot be vertically transmitted, “Probable” for pathogens with suggestive evidence for vertical transmission (for example, unofficial reports or studies from closely related taxa), and “Unknown” when a determination cannot be made owing to a lack of information, such as for newly described pathogens (table 1). For the purposes of this assessment, Yes, Probable, and Unknown are treated equally for risk ranking (fig. 1).

Virulence of a pathogen is highly dependent on environmental conditions, species, and other factors, and thus can be challenging to categorize. To broadly evaluate the virulence of pathogens of potential concern (table 1), we considered (1) whether it is a primary pathogen that causes disease in fish with or without the presence of significant stressors, (2) whether it causes significant mortality in aquaculture settings, and (3) whether there is evidence that it causes mortality or morbidity in wild fish. Pathogens were given a “low virulence” designation if they are unlikely to cause mortality in the absence of significant stress (for example, thermal or crowding stress), a “moderate virulence” designation if they cause notable mortality in aquaculture settings but not in wild fish populations, and a “high virulence” designation if they cause high mortality in aquaculture settings or are known to cause disease, morbidity, or mortality in wild populations.

New Zealand’s aquaculture industry, in addition to environmental changes such as climate change, invasive species, and pollution that may alter disease dynamics, highlights the growing need to understand aquatic disease ecology in this region (Lane and others, 2020). Limited availability of pathogen testing data from Chinook salmon in New Zealand, combined with the historical introduction of various salmonid species from a wide geographic range (Closs, 2024), supports the need for pathogen monitoring to better understand fish health risks. New Zealand is the world’s largest producer of farmed Chinook salmon, yet publicly available reports of pathogen testing for this species are limited for both aquaculture and naturally produced populations. As a result, it is unclear whether certain pathogens are truly absent from New Zealand or have simply not been reported. For example, there have not been any published reports of Chinook salmon in New Zealand being infected with Renibacterium salmoninarum, the causative agent of bacterial kidney disease, even though this pathogen has an otherwise worldwide distribution (Brynildsrud and others, 2014). This risk assessment includes whether a pathogen has previously been detected in New Zealand or Australia based on the available public information (table 1), but because of the limited availability of pathogen testing information in New Zealand, reported presence did not influence a pathogen’s risk designation (table 1; fig. 1).

In 2015, a rickettsia-like organism was identified in moribund farmed Chinook salmon from the South Island of New Zealand. The bacterium, designated “New Zealand rickettsia-like organism 1” (NZ-RLO1), was found to be similar to an rickettsia-like organism isolated from diseased Salmo salar (Linnaeus, 1758; Atlantic salmon) in Tasmania, Australia (Corbeil and others, 2005) based on comparison between partial 16S RNA and internal transcribed spacer (ITS) genes (Brosnahan and others, 2017; Gias and others, 2018). A second strain, designated “New Zealand rickettsia-like organism 2” (NZ-RLO2), was subsequently isolated from the same farm (Gias and others, 2018). Both strains were identified during a high mortality event in a period of stressful environmental conditions (seawater temperatures greater than 17.5 degrees Celsius [°C]). A recent vaccine development study found that juvenile Chinook salmon intraperitoneally (IP) injected with NZ-RL01 developed more severe disease and higher morbidity relative to juveniles IP injected with NZ-RLO2 (Jaramillo and others, 2024). For NZ-RLO1, morbidity differed by route of infection, with unvaccinated fish experiencing mortality rates of 79 percent (IP injection) and 12 percent (exposure via bath immersion). IP-infected fish displayed peritonitis, particularly around the adipose tissue, immersion-infected fish displayed dermal lesions, and both exhibited hepatocellular necrosis and degeneration. In a separate study, the pathogenicity of NZ-RLO2 was evaluated by IP injecting Chinook salmon smolts with high, medium, and low doses of bacteria (Brosnahan and others, 2019). Mortality was dose dependent, with 100-percent mortality observed in high and medium dose treatments and 63-percent mortality in the low dose treatment after 30 days. Mortality also increased following a temperature spike, suggesting temperature dependence. Diseased fish consistently displayed inflammation of the pancreas and surrounding adipose tissue, as well as dose-dependent pathology of the kidney, liver, spleen, and to a lesser degree, brain and heart (Brosnahan and others, 2019).

Rickettsial bacterial infections are commonly detected from axenic tissue samples with the use of antibiotic-free cell cultures derived from the host species (for example, Chinook salmon embryo [CHSE]-214). Cytopathic effects (CPE) and intracellular bacterial staining methods are used to reveal the presence of rickettsial bacteria to be later identified by genetic sequencing. NZ-RLO1 has also been cultured in CHSE cells at 15 °C (Jaramillo and others, 2024), and NZ-RLO2 has been cultured in CHSE-214, Epithelioma papulosum cyprinid (EPC), and several non-fish cell lines, with optimal growth in fish cell lines observed at 15 °C (Gias and others, 2018). A quantitative polymerase chain reaction (qPCR) test for Piscirickettsia salmonis (Corbeil and others, 2003) has been used for NZ-RLO1 and NZ-RLO2 (Jaramillo and others, 2024). Additionally, two qPCR assays (designed to be used in conjunction) are available that are specific to NZ-RLO2 (Gias and others, 2018).

Pilchard orthomyxovirus (POMV) is a virus that was first isolated from apparently healthy Sardina pilchardus (Walbaum, 1792; pilchards) off the South Australian coast in 1998. The virus was subsequently found in marine farmed Atlantic salmon in Tasmania during routine surveillance in 2006, where it has been linked with disease outbreaks since 2012 (Godwin and others, 2020; Mohr and others, 2020). POMV is related to infectious salmon anemia virus but has relatively low homology with this virus and other orthomyxoviruses, suggesting POMV may represent a new genus (Mohr and others, 2020). POMV can affect all life stages of Atlantic salmon and causes pathological effects to major organs, including the liver, kidney, spleen, heart, and eyes (Godwin and others, 2020). Experimental infections of juvenile Chinook salmon have resulted in relatively high morbidity rates (IP injection: 12–74 percent; immersion: 63 percent; cohabitation: 12.5–46 percent). Laboratory research has shown that transmission can occur indirectly via contaminated water sources, and direct contact between fish is not needed (Samsing and others, 2021). It is not known whether Chinook salmon from the South Island region of New Zealand migrate past affected areas in Tasmania or the South Australian Coast. However, pilchards are highly migratory, and previous epizootics of other pathogens in this species have spanned both Australia and New Zealand (Whittington and others, 1997); thus, it is possible that Chinook salmon in New Zealand could be exposed via the natural migration of pilchards in this region.

POMV causes CPE in the CHSE-214 cell line and can also replicate in Atlantic salmon kidney cells. Virus replication is temperature dependent, with progressively higher in vitro replication between 10 and 20 °C (Godwin and others, 2020). A highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR) test that targets two viral genes has recently been validated and is available for suspected CPE and (or) testing directly from tissues (Samsing and others, 2022). This test can detect concentrations as low as one plasmid copy per microliter and is significantly more sensitive than previously developed tests for detecting infection in field-collected samples.

IPNV is an aquatic birnavirus that has a wide geographical distribution. The disease was first reported in Salvelinus fontinalis (Mitchill, 1814; brook trout) from Canada (M’Gonigle, 1941) and has since been detected on every continent except Antarctica (Dopazo, 2020). IPNV causes necrosis of the pancreatic tissue and may be isolated from the spleen and kidney of affected fish. IPNV tolerates a wide range of environmental conditions and infects multiple host species and life stages, but juvenile salmonids tend to be the most susceptible (Dopazo, 2020). Infected fish can become carriers, thus increasing the risk of vertical transmission (Bootland and others, 1991). The virulence of IPNV also varies significantly among strains and is difficult to predict. Moreover, salmonid hosts may be resistant to one strain of IPNV but susceptible to another (Hillestad and others, 2021). A type of IPNV was isolated from Chinook salmon returning to the Rakaia River in the South Island of New Zealand in the 1980s and has never been detected again (Tisdall and Phipps, 1987), but an IPNV-like virus has since been detected in multiple species in Australia (Crane and others, 2000). It is likely that the birnaviruses detected in New Zealand and Australia are unique from IPNV strains found in the United States and thus could be treated as exotic pathogens to California.

IPNV causes CPE in CHSE-214 and other fish cell lines (Kelly and others, 1978). Multiple molecular diagnostics are available, including RT-qPCR and nested polymerase chain reaction (Eissler and others, 2025). Virus serotypes can also be identified via a neutralization test following isolation from cell culture (Dopazo and Barja, 2002).

Yersinia ruckeri is a widely distributed bacterial pathogen that was originally isolated from rainbow trout in the United States (Ross and others, 1966) and has been detected in trout above Shasta Dam (U.S. Fish and Wildlife Service, 2001). Virulence mechanisms differ among strains of Y. ruckeri, with differences often corresponding to geographic origin (Kumar and others, 2015). The Y. ruckeri lineages isolated from Australia and New Zealand are distinct from North American strains, and it is unclear how these strains would affect salmonid species in the Sacramento River Basin if they were to be introduced (Barnes and others, 2016). Y. ruckeri has consistently caused mortality in Atlantic salmon aquaculture in Tasmania since 1987, but an effective vaccine is now available for aquacultural use (Barnes and others, 2016). In New Zealand, Y. ruckeri has occurred sporadically in Chinook salmon since 1989 (Anderson and others, 1994), but this strain appears to have low virulence and has not been a major cause of mortality; therefore, vaccination has not been considered necessary in New Zealand (Barnes and others, 2016).

In general, diseased fish may become anorexic and lethargic, with hemorrhage around the mouth (leading to the common name, enteric redmouth disease) and develop inflammation and septicemia in most organs (Kumar and others, 2015). Transmission rates for Y. ruckeri are highly dependent on environmental conditions, as the bacteria are typically released from the lower intestine when carrier fish become stressed (for example, by elevated temperatures or crowding; Kumar and others, 2015). The bacteria can survive in fresh or brackish water for more than 4 months (Thorsen and others, 1992), and some strains are able to form biofilms that may cause recurrent outbreaks. Although early vaccination treatments in aquaculture systems are generally very protective, such preventative measures are impractical for the protection of wild fish populations. In addition to horizontal and environmentally mediated transmission, there is a report that Y. ruckeri has the potential to be vertically transmitted (Glenn and others, 2015). Glenn and others (2015) detected Y. ruckeri DNA in unfertilized eggs and ovarian fluid; however, they did not detect viable bacteria from eggs, and therefore, vertical transmission remains unconfirmed.

Multiple methods and diagnostic tests are available for Y. ruckeri. The bacterium can be isolated using several different growth media and at a wide range of temperatures, with optimal growth occurring between 20 and 28 °C (Kumar and others, 2015). Multiple serology and molecular detection methods exist, including PCR-based tests that can detect low levels of infection that may occur in asymptomatic carriers (Gibello and others, 1999; Bastardo and others, 2012).

Designing an efficient sampling scheme to provide confidence in the disease-free status of transported eggs is critical because of the limited number of New Zealand Chinook salmon potentially available for disease testing. One approach that may reduce the need for large-scale population-level surveillance when eggs are the transported life stage is testing all parents for vertically transmitted pathogens, combined with egg disinfection to reduce the possibility of external contamination from water sources. In this manner, a high degree of confidence (constrained only by testing sensitivity) in the disease-free status of the transported gametes can be achieved. To overcome issues with unknown or inadequate testing sensitivity, multiple tissue samples from each individual can be collected and tested to reduce the possibility of generating a false negative result. In some scenarios, 100-percent testing of broodstock combined with appropriate disinfection protocols may provide sufficient confidence in the disease freedom of transported fish or gametes. An important consideration for this approach is the need to contain the eggs and hatched fish in quarantine until the testing has been completed. This provides an additional opportunity for sampling and testing this population directly (more information on this in section 5.3).

If managers require information on disease prevalence in the source population in addition to the transported individuals or gametes, surveillance strategies should be carefully considered. Sample size needs can be calculated given the size of the population of interest, expected disease prevalence (percentage of infected individuals), test sensitivity (percentage of infected individuals that will test positive), and desired level of confidence (Cannon, 2001). In scenarios where there is insufficient baseline data to define expected disease prevalence, the maximum acceptable level of prevalence (and associated level of confidence) may be determined based on the risk tolerance of the decisionmakers. The percentage of the population that needs to be sampled increases as population size, test sensitivity, or expected prevalence decrease or as the desired confidence level increases. The American Fisheries Society–Fish Health Section Blue Book recommends sampling sufficient numbers of fish to provide 95-percent confidence of disease freedom. For large population sizes (like those greater than 100,000) and an assumed pathogen prevalence of 5 percent, this generally translates to 60 fish (American Fisheries Society–Fish Health Section, 2014). If pathogen prevalence is assumed to be 2 percent, then a sample size of 150 is required to achieve the same confidence level. For sampling free-ranging populations, additional factors such as geography, life history, and age structure of the population may impact sample size considerations.

For population-level pathogen surveillance, sample size needs may be reduced through several considerations. The first of these is risk-based sampling, where individuals from a high-risk group (for example, post-spawned adults) are sampled preferentially (Stärk and others, 2006). This can result in higher detection rates, and thus a smaller number of fish need to be sampled to determine whether a disease is present in the population. For example, Gustafson and others (2005) found that targeting moribund fish for infectious salmon anemia virus surveillance resulted in significantly higher detection probability than sampling randomly from the population. In Chinook salmon, adult fish become immunosuppressed and highly susceptible to infections prior to spawning (Dolan and others, 2016); therefore, risk-based sampling targeted at sexually mature or post-spawned moribund adults could reduce the number of samples needed to detect disease in a population. The strategy of sampling moribund fish to reduce overall sample size needs has been recommended for research facilities (Kent and others, 2011) and aquaculture facilities (Gustafson and others, 2015). Because sample size calculations depend on expected prevalence, sampling moribund fish or an age class that is more likely to be infected can significantly decrease sample size needs. Sample size can be adjusted if information is available on the expected prevalence of each pathogen within the population as a whole versus the subpopulation targeted for sampling. Gustafson and others (2015) provided a hypothetical scenario in which a moribund group was expected to have a 5-percent prevalence of a disease, relative to the expected 2 percent in the population at large. In this scenario, the same confidence level could be achieved by sampling 69 moribund fish versus a random sample of 175 fish from the wider population. On the other hand, if a pathogen is presumed to be equally distributed across host life stages, then sampling could likewise include both juveniles and adults to achieve confidence in disease-free status. The second consideration is sampling over time, or the use of existing surveillance data, as long-term data can enhance confidence in the disease-free status of a population (Gustafson and others, 2024). Although the frequency and sample size required for testing depend on the distribution of the target pathogen, repeated sampling that is carried out regularly over time with negative results tends to decrease the intensity of sampling that must be conducted to maintain confidence in disease freedom.

Disinfection treatment is an important biosecurity measure that is critical for reducing the risk of transporting infectious bacteria, viruses, and parasitic organisms in egg shipments. The efficacy of these treatments depends on whether the pathogen is vertically transmitted. For example, infectious pancreatic necrosis virus (IPNV; Bootland and others, 1991), infectious hematopoietic necrosis virus (Mulcahy and Pascho, 1985), and salmonid alphaviruses (McLoughlin and Graham, 2007; Bratland and Nylund, 2009) all have shown the potential for vertical transmission, indicating that external disinfection is not considered sufficient to prevent transmission of the pathogen via infected eggs. For pathogens that are not vertically transmitted, contamination of eggs can often be eliminated via external disinfection. Chinook salmon eggs can be disinfected by povidone iodine immediately post-fertilization during water hardening prior to transport and again at the eyed egg stage upon receipt at the target facility to significantly reduce potential biological contaminants on the surface of the egg (Huysman and others, 2018). The U.S. Fish and Wildlife Service (2022)’s recommended treatment protocol is to use a dual disinfection of fish eggs in 50-milligram-per-liter (mg/L) iodine solution for 30 to 60 minutes during water hardening, followed by a secondary disinfection in 100 mg/L iodine for 10 to 30 minutes upon arrival. External horizontally transmitted pathogens, such as Aeromonas salmonicida, on the surface of eggs from infected broodstock or water sources can be consistently inactivated if the recommended disinfection protocol is followed without reusing iodine solutions for each batch of eggs (Cipriano and others, 2001). Although vertically transmitted pathogens may be resistant to iodine egg treatments, following established protocols for disinfection is still critical for minimizing the risk of other pathogens. Care must be taken to follow protocols and achieve the proper concentration of iodine solution to ensure effective treatment of the egg surface. Following treatment, any water used with eggs prior to transport should be sterilized to avoid contamination of eggs with the untreated source water.

Holding transported eggs and juvenile fish in quarantine for a period after transport can enhance confidence in pathogen freedom and reduce the risk of pathogen introduction via movement of eyed eggs. This may be particularly important when adequate testing of the source fish population and (or) broodstock is not available. Appropriate quarantine periods may vary among pathogens depending on the relative risk and incubation period of each pathogen within the host species of interest (Humphrey, 1994). It is also important to note that holding temperature can affect pathogen incubation time; therefore, longer quarantines may be necessary for fish held at low temperatures. For example, standard holding periods of 30 days are often used for zoo and aquarium fish, but Boerman and others (2006) recommend doubling this holding period when fish are held at 12–15 °C. Pathogens that have a long incubation period, such as R. salmoninarum, may extend the quarantine period several more weeks if there is an indication that the broodstock were infected (Munson and others, 2010). During quarantine, risk-based sampling can be applied by testing any fish that develop signs of disease. If no signs of disease emerge in the transported population, random sampling of fish using the epidemiological principles discussed in section 5.1 may be used to achieve the desired level of confidence in pathogen freedom. Exposing a subpopulation of the quarantined fish to a stressor before sacrificing them for fish health testing could improve the chances of isolating a pathogen, if one is present at levels lower than the detection threshold. Temperature stress or treating fish with dexamethasone, known to lower the innate immune system of fish (Lovy and others, 2008), may increase the replication or pathological effects of low-level pathogens, thus improving their detection capability. The impacts of temperature and dexamethasone may vary based on the biology of each pathogen.

Because of the possible presence of pathogens not specifically tested for and the large geographic distance of the proposed egg movement, managers may wish to include additional methods to detect infectious agents not considered in this pathogen risk assessment. Many pathogen testing protocols are specific to the detection of only that pathogen species, though methods that are less specific have the advantage of detecting a range of infectious agents. For example, the culture of viruses and bacteria in cell culture and (or) growth media can provide a broad assessment of potential pathogens and could be conducted alongside other diagnostics for specific pathogens. Considering that unknown pathogens may be a risk associated with the international movement of eggs, managers may require the characterization of unknown agents that cause CPE in viral cell culture assays and the identification of any bacteria isolated. These organisms could then be characterized to better understand if they are considered pathogens of fish and thus if they pose a risk for egg movements. Care should be taken while interpreting bacterial isolates that come from spawning adult fish, as it is possible to isolate a variety of opportunistic and inconsequential microbes. If a pathogen is detected, characterization may include identification of the strain or genetic type to determine whether it differs from strains found in the receiving watershed.

Methods that are not biased towards detecting certain pathogens and instead focus on recognizing a disease condition in the fish can alert investigators to a disease and potential unknown infectious agent. This may be conducted by careful examination of fish for gross external and internal lesions during necropsy. Disease-causing agents may be associated with certain gross lesions, like enlarged organs, hemorrhage, ulcerations, cysts, granulomas, and other inflammatory processes, which may be identified through careful observation during a necropsy. If gross lesions are observed, histology is useful to further determine the etiology of these lesions. Histological examination of organs may be used to evaluate for microscopic anomalies or pathology, which may be otherwise missed in gross observation. This enhanced evaluation of samples can complement fish health testing to improve confidence that unknown disease agents are not present in the source fish population.