Carp edema virus surveillance in the koi trade: early detection through shipping environment sampling and longitudinal monitoring of CEV outbreaks in a wholesaler facility

Abstract

Carp edema virus (CEV), a member of the Poxviridae family, has been a significant pathogen in koi and common carp since its initial identification in Japan during the 1970s. CEV, the causative agent of Koi Sleepy Disease (KSD), can cause high mortality rates and has been reported in many countries and is often linked to the fish trade. The virus is typically detected through DNA analysis of gill tissues, where the highest viral loads are found. However, traditional sampling methods, such as gill sampling, are lethal, complicating routine surveillance, particularly in asymptomatic or high-value koi. This study aimed to evaluate nonlethal sampling methods for CEV surveillance in the koi trade. We analysed various shipping environment samples, such as shipping water and fish bag swabs, alongside gill swabs from anaesthetised fish and gills from naturally deceased fish. Using qPCR, we found that the sensitivity of environmental samples, particularly shipping water, was greater than that of direct fish samples. Latent class modelling estimated that the sensitivity associated with 1.5 mL shipping water samples was greater than 89%, making them a reliable alternative for early detection. All detected variants belonged to genogroup II. Some post-import outbreaks shared variants with earlier outbreaks or shipping environment samples, suggesting that the detected DNA generally reflected infectious particles rather than just free environmental DNA and indicating that CEV can go unnoticed for several months after importation. These findings highlight the utility of environmental samples for effective, non-invasive surveillance and improved biosecurity management in the koi trade.

Keywords: Cyprinus carpio; Poxviridae; Carp; genotyping; transboundary animal disease.

Figure 1

CEV DNA load in shipping water and fish bag swabs. A Comparison between shipping water and fish bag swabs collected in 2019, 2020 and 2022. Negative samples were displayed at 1 copy/2 µL for visual purposes on a logarithmic scale. **p < 0.01. B Distribution of the CEV load by total DNA concentration for shipping water across 2019, 2020 and 2022 shipments and for fish bag swabs in 2019 and 2022 shipments.

Figure 2

CEV DNA detection in shipping bags and koi fish. Sampling was conducted during three koi fish imports from Japan in 2019, 2020, and 2022. Dead fish were examined: gills of fish that were found dead in shipping bags on day 0 were analysed by a batch pool, whereas those that died in their aquaria between 1 and 4 days after arrival were individually analysed. Gill swabs were collected from five anaesthetised fish per batch and analysed in pools (except for batches 19-F1, which were analysed individually) four days after arrival in 2019 and within 12 h in 2022. The results of the fish bag swabs are not shown except when they were positive despite being negative for shipping water. Real-time PCR targeting carp DNA was used to confirm the amplification of DNA extracted from the fish samples. All tested samples, both CEV-negative and the majority of CEV-positive, were positive for carp DNA. Logistic regression analysis revealed no statistically significant association between CEV qPCR positivity and the Ct values of carp qPCR (p > 0.05).

Figure 3

Distribution of the CEV load in shipping bags and imported dead fish (pools by batch) according to the shipping mortality rate (restricted to the 2020 shipment). The total DNA quantity in 100 µL of shipping water ranged between 7 and 2.10³ ng, with a mean of 3.10² ng.

Figure 4

Comparison of the estimated sensitivity of five sampling techniques used to detect CEV DNA from 64 Koi fish batches (A) and estimation of the prevalence among different imports (B) using latent class modelling. The mass probability is represented by areas, with the shaded area representing 50% mass probability and the dark blue line representing the median. The various methods for sampling DNA include extracting it from 1.5 mL of shipping water and from 200 µL of shipping water (or negative from a 20 mL pellet), from a fish bag swab taken upon arrival, from the gills of naturally dead fish from 0 to 4 days after arrival, and from a pool of 5 gill swabs taken under anaesthesia from apparently healthy koi fish within 4 days of their arrival. The prevalence of DNA was estimated for each shipment studied, identified by their associated year.

Figure 5

Pairwise comparison (number of substitutions) of sequences (A) from the shipping environment and very early deceased fish and (B) from major post-import outbreaks. SW: shipping water; BS: fishbag swab; gills: gills of naturally dead fish. The greener the color of the cell is, the fewer substitutions are present; the redder the color is, the more substitutions are observed.