Sep 30, 2023

Advancing Aquatic Animal Health Diagnostic Testing in Hawaii

Authors: RuthEllen Klinger-Bowen, Research Corporation University of Hawaii at Manoa, Karin Kurkjian, College of Tropical Agriculture and Human Resources University of Hawaii at Manoa, Taylor Peterson, College of Tropical Agriculture and Human Resources University of Hawaii, Krista Ann Lee, Hawaii Department of Agriculture, and Jenee Odani, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa

The following article reports on the progress for disease detection in the Hawaiian fish aquaculture industry. Under the auspices of the CTSA project “Advancing Aquatic Animal Health Diagnostic Testing in Hawaii, ”the research group tested new diagnostic tools, some of which were successful and some of which many need further development or may be discarded. Surprisingly, the rapidly developing technology of environmental DNA (eDNA) did not yield robust detections, and new DNA amplification technologies were successful only at detecting major outbreaks.

The Food and Agriculture Organization of the United Nations (FAO) reports that total fish consumption in 2020 was 20.2 kg per capita, more than double of 9.9 kg per capita in the 1960s. Global consumption of aquatic foods (excluding algae) is projected to increase to an average of 21.4 kg per capita, a fifteen percent increase by 2030. Due largely to the advantage of sustainable aquaculture, production of aquatic animals is expected to reach 100 million tons in 2027, rising to 106 million tons by 2030 (FAO, 2022). With aquaculture playing such a vital role in food production, significant constraints caused by disease must be addressed. Globally, the industry loses billions of dollars per year to disease. Moreover, the movement of live aquatic animals and feed products, while necessary for industry growth, plays a pivotal role in spreading pathogens to new populations and geographic regions. For example, koi herpes virus (KHV) is infectious to all varieties and subspecies of common carp, Cyprinus carpioand hybrids such as Cyprinus carpiox Carassius auratus(OIE, 2022). All life stages are susceptible, and most mortalities occur when temperatures are 23 to 25° C. The KHV was first reported in the United Kingdom in 1996 but spread to Israel within two years (Haenen et al., 2004). Since then, KHV has been reported from almost all countries that culture koi and/or common carp (OIE, 2022). Fortunately, KHV has never been detected in fish imports to Hawaii.

Francisella orientalis(Fo), previously Francisella noatunensissubsp. orientalisor Fno, a facultative bacterium, has been well studied both in Hawaii and worldwide (Soto et al., 2014; Nguyen et al., 2015; Klinger et al., 2016; Yamasaki et al., 2020). On Oahu, there was a concerted effort to encourage tilapia producers to test for Fo, adapting a protocol from the shrimp broodstock specific pathogen free (SPF) program. However, diagnostic testing is cost prohibitive for most of Hawaii producers. Here we add Fo as part of the PCR testing to determine if it can be detected in system water or in tilapia live feeds, which could potentially reduce the cost of testing and eliminate lethal diagnostic methods.

Vibriosis, caused by Vibriospp., causes severe mortalities in multiple marine fish and shellfish species worldwide. Vibriospp. are rather ubiquitous, and potentially devastating in aquaculture systems (Frans et al., 2011). Zooplankton culture (e. g. copepods) used for fish fry and larval feeds have been shown to harbor bacterial pathogens such as Vibriospp. (Hansen and Bech, 1996). The real-time PCR is a fast methodology that can detect V. parahaemolyticusand V. vulnificusin live feeds. The assay is highly specific and sensitive even if Vibriois present in very small numbers. It reduces the amplification of DNA from nonviable bacteria and therefore results in fewer false-positive results (Pillot, 2010).

To protect Hawaii’s growing aquaculture industry, we need improved laboratory facilities, diagnostic expertise, control protocols, testing capabilities and permanent data bases. The PCR-based methods of pathogen detection are sensitive and highly specific but they require expensive equipment and reagents, need to be conducted in a highly-controlled laboratory setting, and are time-consuming. Isothermal diagnostic assays such as recombinase polymerase amplification (RPA) or loop mediated amplification (LAMP) are novel approaches of amplifying DNA without the cost and complexities required for PCR (Howson et al. 2017). The loop-mediated isothermal amplification (LAMP) assay is a novel approach of amplifying DNA with high specificity and rapidity under isothermal condition. Additionally, quantification can be achieved by measuring the turbidity of the sample using a photometer (Mekata et al.; 2012). The LAMP method is very rapid, which makes the assay attractive for routine detection, and it is already used to screen shrimp for infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Xia et al. 2015). These properties for the LAMP assay indicate potential for on-site monitoring of infection and preventing spread of disease in aquaculture production. The application of the LAMP assay has the added benefits of reduced analysis time and easy interpretation of results based on a simple colorimetric change, thus offering a faster diagnostic tool compared to PCR. More importantly, LAMP assays are high sensitive and reliable, which has been particularly useful during tilapia lake virus (TiLV) infections (Phusantisampan et al., 2019). The RPA alternative uses recombinase-driven primer targeting strand-displacement synthesis and can be used in point-of-care assays. RPA assays are used with rapid surveillance in livestock outbreaks (Abd El Wahed, 2013) and they are beginning to be used in aquaculture (Liu, 2017). Lobato and O’Sullivan (2017) mentioned that the RPA is an isothermal amplification technique that has already garnered a huge amount of attention and has been exploited for both laboratory-based and portable analysis. It has been used in diverse applications including aquaculture diagnostics. The same authors mentioned applications for hypodermal and hematopoietic necrosis virus (HHNV), Vibrio owensiiand White Spot syndrome virus (WSSV). Xia et al. (2015) developed a highly sensitive real-time RPA assay for rapid pondside IHHNV detection. Shahin et al. (2018) showed that RPA was more robust than qPCR in the detection of Fo in clinical and field samples. Apparently, RPA is more tolerant to common contaminants that inhibit PCR reaction. The RPA technique for the detection of pathogens in aquaculture can be a good option for faster results with good diagnostic sensitivity.

To date, Hawaii does not have a consistent and continually updated database of emerging aquatic diseases that occur regionally, nationally, or internationally. Acquiring the latest information of aquatic animal diseases can be tedious and with the advent of social media, there are many opportunities for misinformation. There are websites offered by national laboratories, such as the Wild Fish Health Survey, by US Fish and Wildlife Service, which keeps track of freshwater fish diseases in the U.S. International databases include sites such as EBSCO and World Fish Center, and the Office International des Epizooties or OIE (recently renamed World Organisation for Animal Health (WOAH)) (2023), the predominant world organization for emerging pathogens for all animals. OIE is regarded as the international standard for allowing animals to move across borders based on disease events and history. Utilizing such a global database is essential for tracking the latest emerging pathogen (Tedesco et al 2017; Chen et al., 2018). With the assistance of the National Marine Fisheries Service Honolulu office, which tracks regional fish disease outbreaks, along with HDOA Animal Diagnostic Laboratory, HDOA PQ, USDA, and OIE, it is possible to compile all the current information relevant to Hawaii producers in a timely confident manner. This information can be posted to a listserv for anyone interested in receiving pertinent disease information and guidance. Similarly, historical and contemporary data available in such a database, which would also include case information such as environmental and management variables, would be valuable for analysis of trends that could inform future management practices. Preliminary results for detection of most of these pathogens via non-lethal methods has so far proved unsuccessful with real-time or conventional PCR; however, using environmental DNA (eDNA) has potential to detect pathogens with just a water sample (van der Heijden et al. 2008; Zhang et al. 2020).

This project expands upon previous works by using eDNA to directly assess imported koi shipment water, system water, and live feeds used in fish culture as potential disease vectors. The ultimate goal is to develop a state/region wide database/listserv to communicate up to date information on aquaculture diseases around the world. A quantitative PCR for V. parahaemolyticusand V. vulnificuswas developed and added to the panel of assays available at the UHADL. The UHADL/HDOA became a diagnostic program for aquaculture producers to include histopathologic examination and bacteriology, as needed. Also the expanse of other technology such as RPA or LAMP, which provides quick turnaround results, was compared to traditional diagnostic methods.

This project utilized eDNA assays to detect pathogens in shipping water in the hope of allowing broader non-lethal sampling of the consignment, as opposed to a random sampling of individual fishes to represent the whole population. This eDNA/PCR methodology was also used to detect specific pathogens in live feeds prior to entering the fish systems. The impact of the project was apparent by the expansion of diagnostic testing for diseases affecting aquacultured animals, the determination of potential risks associated with importing animals and feed, and dissemination of knowledge related to aquaculture pathogens to guide biosecurity practices or other methods of mitigating risk.

During the project, two separate importations of koi arrived in Hawaii from multiple farms in Japan. The first consignment of ten fingerlings were collected and gill, spleen, and posterior kidney were sampled for PCR analysis of KHV. Water (3 – 1L bottles) was also collected for eDNA to determine if KHV could be detected in the imported shipment water. The second importation of koi from Japan were too valuable to sample tissues, so only water from the shipping bags were sampled for eDNA extraction (Figure 1). Neither sampling effort detected KHV in any of the tissues or shipment water.

To determine the presence of Fo, three tilapia farms participated in the summer collections, while five farms participated in the late winter/early spring collections. Table 1 describes Fo presence in tilapia from each farm. Note that eDNA from system water was negative for Fo at all farms during both collecting seasons.

While the detection of Fo with qPCR was the primary objective, tilapia tissues were also taken for histology to create a pictural publication of normal tilapia tissues. Granulomas in spleen tissues were categorized from ND (not detected), 1 (light infestation), 2 (moderate infestation), or 3 (heavy infestation). Tilapia spleens were also sectioned for gross examination under light microscopy and microbiology. Although granulomas could be observed under the microscope, no Fo growth was evident with bacteriological (culture) methods. However, it is well known that Fo is difficult to grow in culture when no outbreaks are occurring.

The observation of spleen granulomas with histology and/or by light microscopy in cases that were not positive for Fo with PCR merely shows granulomas can be caused by a wide variety of pathogens or environmental insults. Therefore, scoring granulomas by histological or light microscopy should not be used solely to assess the Fo status of tilapia.

A new loop-isothermal amplification (LAMP) assay, designed by graduate student Taylor Peterson, provides results in as little as 10 minutes. The LAMP assay targets the iglC gene of Francisella orientalis, which is the same target as the qPCR created by Soto et al (2010). A limit of detection test was tested in triplicate using a 1:10 serial dilution for a total of ten dilutions of genomic DNA of the Fo LADL-0735A. The LAMP was able to detect 100 pg (picograms) of DNA, therefore during a large disease outbreak, it can detect Fo if disease is rampant and at a high level. We also verified the specificity of the assay by running the LAMP against a panel of closely related species to Fo, as well as bacterial species that are common in fish. The LAMP was highly specific, only detecting strains of Fo and had zero cross-reactivity to the other pathogens.

We also used the samples acquired in the farm survey to test the LAMP as a surveillance tool in the future. The LAMP was not able to detect low level infection that qPCR was able to detect, which is 1 pg. Therefore, for future surveillance work, UHADL should use the qPCR by Soto et al. (2010) instead of Peterson’s LAMP. However, we emphasize that the LAMP would be good for fast and reliable detection during a disease outbreak.

In the course of this research, we developed qPCR assays for Vibrio parahaemolyticus and Vibrio vulnificus. Live feeds from two facilities in summer 2022 and three facilities in March 2023 were sampled. System water was also collected in triplicate for eDNA analysis. The DNA of live feeds including Artemia, rotifers, copepods, diatoms, and marine algae were extracted (Figure 2) and tested for V. parahaemolyticus and V. vulnificususing real time PCR methodology. eDNA from system water was also tested for both Vibrios. All samples collected at both time intervals were negative for V. parahaemolyticusand V. vulnificus.

Moina and Gammurus that are used to feed tilapia were collected and tested for Fo. These live feeds and system water (3-1L bottles plus one L DI water used as a control) were negative for Fo with qPCR methods.

The maintenance of a University-housed database/listserv of morbidities/ mortalities occurring regionally, nationally and internationally will alert our producers to what pathogens are occurring and where (location will only be given at the national and international) in a timely and efficient manner. Similar to the listservs UH provides to swine and poultry producers, maintaining a database and listserv to communicate with aquaculture producers will help develop an efficient and integrated industry state-wide and region-wide.

Trends for morbidities/mortalities outbreaks were determined through coordination with the Hawaii aquaculture producers and the USGS National Wildlife Health Center Honolulu Office. Data was coded by assigning a unique accession number for each event and results were secured in keeping with HDOA and UH-ADL policies on client confidentiality. De-identified information will continue to include the pathogens detected, environmental factors (water temperature, D.O., pH, and biomass), farm records (operation type, species, origin of animals, etc.), history of diseases (year, diagnostic methods utilized, and results), and treatment applied (drugs, probiotics, etc.) to determine factors associated with outbreaks. Confidential data collected included GPS coordinates of farms to facilitate tracking of pathogen spread. Information from national/international disease databases was also incorporated to serve as an early warning system for our aquaculture producers via a listserv for rapid communication.

Sampling water for eDNA analysis during a non-disease event yielded negative results for Fo and KHV. Therefore, using eDNA to detect these pathogens during non-outbreaks is not a useful tool, and sampling fish tissues is still the best method for PCR detection. Also, this project assured the participating producers thatthe biosecurity measures put in place are working to prevent specific pathogens entering their facility. They also have the wherewithal to mitigate pathogen spread if fish morbidities/mortalities occur. When these individual farms received their complete test results, they were satisfied that their farm samples were coded by a unique accession number and results are secured per HDOA and UHADL policies on client confidentiality. In the future, veterinarians, other health professionals, or producers will benefit from a manual on fish sampling and histology to ensure the best diagnostic technologies are applied.

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