1. INTRODUCTION
The abalone is an important aquaculture species worldwide, both due to
its high levels of nutrients and to its high economic value (Nam et al.,
2017). China produces more abalone than any other country in the world
(Cook, 2019), and the most common abalone farmed in China is the Pacific
abalone, Haliotis discus
hannai, as this species has excellent growth performance and survival
rates
(Chen
et al., 2016; Liang, Luo, You, & Ke, 2018; You et al., 2015).
However,
during
a record-breaking heatwave (mid-May to early-June, 2018) a serious
epidemic, which did not cause
obvious tissue injury,
led
to the massive die-off of juvenile abalone (6 to 7‐month‐old) in Fujian
Province,
south China (K. Deng, Yang, Lin, Li, & Hu, 2019; K. Q. Deng, Yang, Gu,
Lin, & Li, 2020).
Previous studies have identified the pathogens causing several abalone
diseases and die-offs, such as Francisella halioticida , which
caused a mass mortality of H. gigantea in 2005 (Brevik, Ottem,
Kamaishi, Watanabe, & Nylund, 2011; Kamaishi, Miwa, Goto, Matsuyama, &
Oseko, 2010); the bacterium Vibrio harveyi , which caused numerous
deaths of adult H.tuberculata in France in
2002 (Nicolas, Basuyaux, Mazurie, & Thebault, 2002), of H. discus
hannai in 2007 (Fukui, Saitoh, & Sawabe, 2010; Sawabe et al., 2007),
and of H. discus hannai in 2018 (as foot pustule disese; R. X.
Wang, Yao, Liu, & Wang, 2018); the
intracellular bacterium CandidatusXenohaliotis californiensis,which caused withering syndrome in H. spp . in 2000 (Friedman et
al., 2000); and a herpes-like virus, which caused high mortality rates
in
mariculturalH. diversicolorsupertexta in 2005 (Chang et al., 2005). Infections of otherVibrio species, including V.carchariae (Nicolas et al.,
2002; Nishimori, Hasegawa, Numata, & Wakabayashi, 1998),V.parahaemolyticus (Bathige et
al., 2016; Hu et al., 2018), V.anguillarum(Maldonado-Aguayo, Teneb, & Gallardo-Escarate, 2014), and V.alginolyticus (C. J. Wu,
Wang, Chan, & Li, 2011; Y. S. Wu, Tseng, & Nan, 2016), have also
caused abalone deaths. As is known to all, numerous bacterial strains
can be isolated from unhealthy individuals using plate culture methods,
not all the culturable bacteria are pathogenic. And it is challenging to
link a certain pathogen to a specific disease because opportunistic
pathogens exist naturally, and pathogenic behaviors can be induced by
external factors, such as anthropogenic stressors and climate change
(Zozaya-Valdes, Egan, & Thomas, 2015).
However,
most authors assumed possible
pathogen
based on previous experience (Tavares et al., 2018), or only briefly
described the process by which the causative pathogen was identified
from among dozens of
candidates
(Ren et al., 2019).
It is well known that the mucosal
epithelial
tissues serve as the most important interface in the host defense
against
dynamic physical forces (Ángeles Esteban, 2012; Buchmann, 2014; Lang,
Hansson, & Samuelsson, 2007). The diverse cell types
lining
the mucosal epithelial tissues synthesize and
release
bioactive
molecules, including mucins, lectins, lysozymes, immunoglobulins,
C-reactive
proteins,
proteolytic enzymes, antimicrobial peptides, and lipids (Allam &
Espinosa, 2015; Bakshani et al.,
2018; Buchmann, 2014). Mucus, secreted by mucous membranes, which plays
many roles in innate immunity,
also
inhibits microorganismal growth and reproduction (Bakshani et al.,
2018). For example, the skin-secreted mucus of sea bream and sea bass
was
shown to block or limit bacterial growth (i.e., lytic activity) based on
24 h growth curves (Sanahuja et al., 2019); the skin-secreted mucus ofSalmosalar had high levels of
NK-lysin, and strong bacteriostatic properties (Valero, Cortes, &
Mercado, 2019); and the skin-secreted mucus of Amphiprionclarkii showed robust time-
and dose-dependent bactericidal and antiparasitic activity (H. Wang,
Tang, Zhang, & Ding, 2019). In addition,
skin mucus showed higher
antimicrobial activity against tested pathogens in experimental rainbow
trout with beneficial dietary additive (Adel, Pourgholam, Zorriehzahra,
& Ghiasi, 2016; Mansouri Taee, Hajimoradloo, Hoseinifar, & Ahmadvand,
2017). It was also shown that
stressed corals produced mucus with
higher bioactive content and
increased antibacterial activity (Wright, Strader, Genuise, & Matz,
2019); that the mucus ofEiseniafoetida exhibited strong
antimicrobial activity against human bacterial and fungal pathogens
(Andleeb et al., 2016); and thatHelix aspersa mucus had strong antibacterial effects
on Pseudomonasaeruginosa and weak effects
on Staphylococcusaureus (Fuochi et al.,
2017). Finally, the mucus and liquified tissues of the cuttlefishSepiapharaonis strongly inhibitedV. harveyi strain Wz211 and weakly inhibited V.
alginolyticus strain Wz11 after separate co-incubation (Lv et al.,
2019).
Previous
studies have shown that the mucus secretions of marine animals
can
favor the attachment and growth of specific
symbiotic microbes, helping the host
to maintain a
well-balanced
microbial
population (Allam & Espinosa, 2015). Thus, the viability of bacteria in
host mucus might be a useful metric by which to identify pathogenic
bacteria. Symbiotic relationships between beneficial microbes and mucus
have been observed in abalone (Choresca, Choi, Gomez, Kim, & Park,
2010; Guo, Huang, Huang, Zhao, & Ke, 2009), corals (Shnit-Orland &
Kushmaro, 2009), and fish (Carda-Dieguez, Ghai, Rodriguez-Valera, &
Amaro, 2017; Minniti et al., 2017).
However,
changes in environmental
conditions, especially temperature increases, may cause common microbes
to become more virulent and to exhibit conditional pathogenic behaviors
(Cohen et al., 2018; Gardiner, Bournazos, Maturana-Martinez, Zhong, &
Egan,
2017).
As the distinction between pathogenic and non-pathogenic bacteria may
thus be
non-obvious
for many widespread strains (Carda-Dieguez et al., 2017; Choresca et
al., 2010; Minniti et al., 2017; Shnit-Orland & Kushmaro, 2009), it is
urgent
to
develop
a more rapid, efficient approach for the detection and identification of
pathogens,
especially opportunistic pathogens.
Herein,
we developed a non-invasive,
simple, and rapid method to address this knowledge gap. We first
isolated
bacteria
from
body mucus, and selected the
isolates
enriched on diseased individuals, as compared to healthy individuals.
Then, we tested the mucus resistance of each of the selected isolates.
Candidate isolates were then selected for tests of Koch’s postulates.