Aquaculture 432 (2014) 16

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Short communication

Multilocus sequence typing revealed a clonal lineage of Aeromonas

hydrophila caused motile Aeromonas septicemia outbreaks in
pond-cultured cyprinid sh in an epidemic area in central China
Xujie Zhang a,b, Wuming Yang a,c, Han Wu a, Xiaoning Gong a, Aihua Li a,
a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, University of Chinese Academy of Sciences, Wuhan 430072, PR China
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
c
Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Motile Aeromonas septicemia (MAS) has been increasingly prevalent in cultured sh throughout China since
Received 9 March 2014 1989. Over the past two decades, our laboratory conducted two pathogen detection studies in septicemia out-
Received in revised form 7 April 2014 break shponds in an epidemic area in central China. One was conducted from May 1990 to October 1991,
Accepted 8 April 2014
when MAS was beginning to spread in China, and the other was recently conducted from August 2006 to July
Available online 24 April 2014
2009. We found that Aeromonas hydrophila was responsible for these MAS outbreaks. A. hydrophila isolates
Keywords:
were previously known to be phenotypically, serologically, and genetically diverse and no dominant clones
Aeromonas hydrophila were found. In this study, multilocus sequence typing analysis was used to observe a clonal lineage of
Motile Aeromonas septicemia A. hydrophila, which was responsible for MAS outbreaks in pond-cultured cyprinid sh in an epidemic area in
MLST central China for two decades.
Clonal lineage 2014 Elsevier B.V. All rights reserved.
Cyprinid sh

1. Introduction
Aeromonas hydrophila and other motile aeromonads are among the
most common bacteria in freshwater habitats, and these bacteria fre-
quently cause diseases among cultured and feral shes worldwide
(Akinbowale et al., 2007; Cipriano, 2001; da Silva et al., 2012;
Gopalakannan and Arul, 2006; Nielsen et al., 2001). During the past
30 years, the aquaculture industry in China has evolved dramatically,
and the capital-intensive production has become the main culture
mode (Hishamunda and Subasinghe, 2003; Nielsen et al., 2001; Zhang
and Jostein, 2004). The total area devoted to aquaculture increased
from 2.86 million hectares in 1979 to 5.63 million hectares in 2008 in
China. Meanwhile, total production rose from 1.23 to 34.13 million tons,
garnering a 69.7% share of the total aquatic production, making China the
only country in the world where aquaculture production exceeds the
wild catch (Li et al., 2011; National Bureau of Statistics of China, 2009).
With the rapid development of the aquaculture industry, motile
aeromonad infections caused by A. hydrophila have become an increas-
ingly prominent problem (Xiao et al., 2011; Nielsen et al., 2001; Xu
et al., 1993). Based on different clinical symptoms, A. hydrophila can
cause several diseases in sh, which include motile Aeromonas sep-
ticemia (MAS) or hemorrhagic septicemia (da Silva et al., 2012;
Nielsen et al., 2001; Xu et al., 1993), hemolytic ascitesosis (Sun
Corresponding author. Tel./fax: +86 27 68780053.
E-mail address: liaihua@ihb.ac.cn (A. Li).
et al., 1991), intussusception (Liu et al., 2008), tail or n rot (Liu et al.,
1993; Rahman et al., 2001), stigmatosis (Xu et al., 1980), and epizootic
ulcerative syndrome (Austin and Adams, 1996; Roberts, 1997). MAS is
the most serious disease and has frequently caused huge economic losses
in the cyprinid sh industry throughout China since 1989 (Lu, 1992;
Nielsen et al., 2001; Qian et al., 1997).
Many studies have investigated the genetic diversity of human diar-
rhea and environmental isolates of A. hydrophila (Aguilera-Arreola et al.,
2007; Sechi et al., 2002; Szczuka and Kaznowski, 2004). All these studies
revealed a high intra-specic genetic diversity within A. hydrophila, and
no dominant clones were found. As an etiological agent in sh and shell-
sh diseases, A. hydrophila is widely identied and characterized at the
species level (da Silva et al., 2012; Nielsen et al., 2001; Xu et al., 1993).
However, studies concerning the genetic diversity of this widespread
sh pathogen are scarce. Thus, systematic investigations and precise
delineation on the population structure of A. hydrophila from pond out-
breaks in epidemic areas are lacking, making prevention difcult. Clari-
fying the population structure of MAS outbreak isolates of A. hydrophila
and determining an efcient measure for the prevention of this disease
are of utmost importance.
Over the past two decades, our laboratory conducted two pathogen
detection studies in septicemia outbreak shponds in an epidemic area
in central China. One was conducted from May 1990 to October 1991
(Xu et al., 1993), when MAS became widespread in China, whereas
the other was conducted from August 2006 to July 2009 (Zhang et al.,
2013a). We found that A. hydrophila was responsible for these MAS

http://dx.doi.org/10.1016/j.aquaculture.2014.04.017
0044-8486/ 2014 Elsevier B.V. All rights reserved.
2 X. Zhang et al. / Aquaculture 432 (2014) 16

outbreaks. A. hydrophila isolates were previously studied to be pheno-
typically, serologically, and genetically diverse (Abbott et al., 2003;
Aguilera-Arreola et al., 2005; Szczuka and Kaznowski, 2004; Thomas
et al., 1990). Thus, we used a recently developed precise method of
multilocus sequence typing (MLST) to evaluate the population structure
of MAS outbreak isolates of A. hydrophila. The results of this study will
help clarify the epidemiological information of outbreak isolates of
A. hydrophila in pond-cultured sh, and further determine an efcient
measure to prevent this pathogen.
ranged from 20% to 40% (data from the owner). Ponds D and E were
200 m apart; Ponds G and H were adjacent to each other but not
connected; and the other ponds were geographically distant from
each other. Ponds E and F reared various cyprinid sh species, including
crucian carp (Carassius carassius), grass carp (Ctenopharyngodon idellus),
silver loweye carp (Hypophthalmichthys molitrix), wuchang bream
(Megalobrama amblycephala), and bighead carp (Aristichthys nobilis).
Ponds C and D reared wuchang bream and silver loweye carp, respec-
tively. Ponds G and H reared crucian carp.

2. Materials and methods

2.2. MLST and phylogenetic analysis
2.1. Bacterial strains
MLST was proposed as a universal method to characterize bacteria
based on sequence polymorphisms within internal fragments of house-
Detailed information about all A. hydrophila strains evaluated in this
keeping genes. Each gene fragment is translated into a distinct allele,
study is listed in Table 1. All strains were isolated from diseased cyprinid
and each isolate is classied as a sequence type (ST) by the combination
sh. Among these strains, one strain (ST78-3-3) was isolated from sh
of alleles of the housekeeping loci (Urwin and Maiden, 2003). MLST can
with stigmatosis in 1978 (Xu et al., 1980). All the other 22 A. hydrophila
achieve precise strain genotyping, and is a powerful tool for outbreak
strains were isolated from pond-cultured cyprinid sh with MAS out-
traceability (Martino et al., 2011). Six housekeeping genes of all strains,
breaks. Among the 22 strains, two strains (NSC90-4-1 and XS91-4-1)
namely, gyrB, groL, gltA, metG, ppsA, and recA, were sequenced with the
were isolated in 1990 and 1991, respectively, when MAS became wide-
previously described primers and procedures (Martino et al., 2011). The
spread in China (Xu et al., 1993). The other 20 A. hydrophila strains
sequences of distinct alleles were deposited in the Aeromonas MLST da-
were isolated from August 2006 to July 2009, when MAS was a high
tabase (http://pubmlst.org/aeromonas) (Jolley and Maiden, 2010) and
risk (Zhang et al., 2013a). The isolation and identication of these strains
GenBank under accession nos. KC767928KC767939. A phylogenetic
were described in the previous studies (Xu et al., 1993; Zhang et al.,
tree was constructed based on the concatenated sequences of the six
2013a,b). Briey, the tissues of diseased shes, which include blood,
housekeeping genes by the neighbor-joining method using MEGA pro-
spleen, liver, kidney or ascites uid, were aseptically inoculated on tryptic
gram (version 4.1) (Tamura et al., 2007). Genetic distances were obtain-
soy agar (TSA; Becton, Dickinson and Company, USA) plates. All plates
ed by Kimura's two-parameter model (Fig. 1). Given that the sequences
were incubated at 28 C for 24 h. Then, two to three single colonies
of subspecies of A. hydrophila in the Aeromonas MLST database are lack-
were randomly selected and re-puried from each TSA plate. Aeromonas
ing, a phylogenetic tree was constructed based on the gyrB gene se-
genus-specic polymerase chain reaction (PCR) detection was carried
quences of these strains and reference Aeromonas strains to further
out as previously described (Lee et al., 2002). All isolates were positively
analyze the subspecies of these strains (Fig. 2).
amplied and were further conrmed as members of the genus
Aeromonas in terms of motility, positive reactions for both oxidase and
catalase, fermentation of glucose and resistance to O/129 on a 150 g 3. Results and discussion
disk (Abbott et al., 2003). To further identify the species of these isolates,
the gyrB gene was amplied and sequenced with previously described Through the MLST and phylogenetic analysis, we conrmed that all
primers (Yez et al., 2003). The results showed that all the isolates the 22 MAS outbreak isolates were A. hydrophila (Table 2, Fig. 1). All
were A. hydrophila, indicating a single species outbreak in these shponds these strains demonstrated identical ST and alleles. Thus, the 3084-bp
(Xu et al., 1993; Zhang et al., 2013a). concatenated sequences of the six alleles in the MLST loci were identical,
The sampled shponds in this study are located in ve cities in cen- whereas the control strain isolated from sh with stigmatosis showed
tral China (Table 1). The mortality rates in ponds C, D, E, F, G, and H different STs and alleles with MAS outbreak isolates (Table 2).

Table 1
Information of tested A. hydrophila strains in this study.

Strain Host sh species Source Disease Pond Location Isolation time

NSC90-4-1 Silver loweye carp Spleen or liver or kidney MAS A Changsha 1990
XS91-4-1 Silver loweye carp Spleen or liver or kidney MAS B Xiaogan 1991
IB101 Wuchang bream Blood MAS C Hanchuan 08/2006
IB336 Wuchang bream Blood MAS C Hanchuan 08/2006
4LNG101 Silver loweye carp Liver MAS D Jingmen 06/2008
4LNG201 Silver loweye carp Liver MAS D Jingmen 06/2008
4LNS301 Silver loweye carp Kidney MAS D Jingmen 06/2008
JG101 Crucian carp Liver MAS E Jingmen 06/2008
CG101 Grass carp Liver MAS E Jingmen 06/2008
LNB101 Silver loweye carp Blood MAS E Jingmen 06/2008
DWCG101 Wuchang bream Liver MAS F Wuhan 06/2009
DLNG101 Silver loweye carp Liver MAS F Wuhan 06/2009
DLNG201 Silver loweye carp Liver MAS F Wuhan 06/2009
DBHS101 Bighead carp Kidney MAS F Wuhan 06/2009
JBN1001 Crucian carp Blood MAS G Wuhan 06/2009
JBN1101 Crucian carp Blood MAS G Wuhan 06/2009
JBN1201 Crucian carp Blood MAS G Wuhan 06/2009
JBN1301 Crucian carp Blood MAS G Wuhan 06/2009
2JBN101 Crucian carp Blood MAS H Wuhan 07/2009
2JBN301 Crucian carp Blood MAS H Wuhan 07/2009
2JFN201 Crucian carp Ascites uid MAS H Wuhan 07/2009
2WCL101 Wuchang bream Liver MAS H Wuhan 07/2009
ST78-3-3 Silver loweye carp Lesions or blood Stigmatosis I 1978
 X. Zhang et al. / Aquaculture 432 (2014) 16 3

Fig. 1. Unrooted neighbor-joining phylogenetic tree of A. hydrophila strains constructed from the concatenated sequences of the six housekeeping genes analyzed in this study.

A. hydrophila is prevalent in aquatic habitats with cosmopolitan dis-
tribution, and has resulted in heavy mortalities in farmed and feral sh-
es worldwide (da Silva et al., 2012; Harikrishnan and Balasundaram,
2005; Janda and Abbott, 1998). Vaccines are being developed against
A. hydrophila, unfortunately, these are not yet commercially available
(Yin et al., 2009). A. hydrophila is such a heterogeneous species, having
diverse antigens, that vaccine development is extremely complex (Ard
et al., 2008; Karunasagar et al., 1991; Stevenson, 1988; Yin et al., 2009).
Thus, the epidemic information underlying the outbreaks should be
claried before vaccines against this genetically diverse sh pathogen
are developed. For this purpose, we pioneered the use of the MLST
method to conduct a clone-level examination of the outbreak isolates
of A. hydrophila. In this study, all strains, except the control strain,
were isolated from pond cultured cyprinid sh with MAS outbreaks
from an epidemic area in central China. Previously, only several studies
have used ERIC or RAPD-PCR to investigate the genetic diversity of sh
pathogenic A. hydrophila strains, and no dominant clones were
observed (Lee et al., 2000; Thomas et al., 2009; Xiao et al., 2011). How-
ever, our study showed different results. We found that the previous re-
search had the same limitations, namely, the diseases of the sampled
sh were unclear and not all strains were outbreak isolates. Certain
strains may even be the secondary invaders. Given that A. hydrophila
can cause several diseases in a wide range of sh species, our results sug-
gest that we should rst classify the pathogenic strains according to the
host sh species and sh diseases in future epidemiologic study on
A. hydrophila. We can then clarify the epidemiology of A. hydrophila un-
derlying various infections. Epidemiological studies based on randomly
isolated strains are not advantageous, because the results cannot reect
the hidden epidemiologic information of A. hydrophila and will result in
confusion for the selection of vaccine strains.
The taxonomy of the genus Aeromonas is complex and accompa-
nied with confusion and controversy (Janda and Abbott, 2010;
Martinez-Murcia et al., 2011; Martino et al., 2011). At present, 24
validly published species in the genus Aeromonas have been reported
4 X. Zhang et al. / Aquaculture 432 (2014) 16

99

27

28 91

28

88 99

73

0.005

Fig. 2. Unrooted neighbor-joining phylogenetic tree based on the gyrB gene sequences of Aeromonas strains.

(Janda and Abbott, 2010). In the species A. hydrophila, ve common sub-
species exist, namely, subsp. hydrophila, subsp. ranae, subsp. dhakensis,
subsp. anaerogenes and subsp. proteolytica (Huys et al., 2002; Huys
et al., 2003). Thus, false identication results of species can produce un-
real epidemiologic information on A. hydrophila. According to the phy-
logenetic tree based on the gyrB gene sequences of Aeromonas strains,
we observed that the 22 MAS outbreak isolates belonged to
A. hydrophila subsp. hydrophila, whereas the control strain belonged to
A. hydrophila subsp. dhakensis (Fig. 2). These ndings indicate that, in
the future epidemiologic study of A. hydrophila, we should identify the
pathogenic strains to the subspecies level corresponding to various
diseases.
Enhancement of the immune system is the most promising method
for preventing sh diseases. This can be achieved with vaccines, which
are considered to be the most effective immunotherapy agents and
can enhance the specic immune response of the sh. But a single vac-
cine is effective against only one type of pathogenic A. hydrophila, and in
the case of many different strains, no effective vaccine is available due to
the complex antigenic structure (Ard et al., 2008; Karunasagar et al.,
1991; Stevenson, 1988). In this study, the MLST method was used to
investigate the MAS outbreak isolates of A. hydrophila in an epidemic
area at the clone level. Although the origin of MAS outbreak isolates
assayed covered ve sh species in eight ponds from ve cities isolated
at six different time points for two decades, the isolates belonged to a
conserved clonal lineage. This nding not only provides a guide for fur-
ther epidemiological investigations of this heterogeneous species, but
also deepens the understanding of this pathogen. The results suggest
that a common whole cell vaccine may develop to prevent the MAS out-
breaks in the investigated epidemic area in central China. Future system-
atic investigations should be conducted at the clone level corresponding
to host sh species and sh diseases at broad geographical scales for the
improved prevention of this widespread sh pathogen.
Acknowledgments
This work was supported by grants from the National Science and
Technology Pillar Programs (Grant no. 2011BAI15B01-41), the FEBL
project (2011FBZ26) and the National Natural Science Foundation of
China (Nos. 30670112 and 31070112).
 X. Zhang et al. / Aquaculture 432 (2014) 16 5

Table 2
Multilocus sequence typing data of A. hydrophila strains analyzed in this study.

Strain Host sh disease Sequence type (ST) Allele

gyrB groL gltA metG ppsA recA

NSC90-4-1 MAS 251 210 214 122 211 221 217

XS91-4-1 MAS 251 210 214 122 211 221 217
IB101 MAS 251 210 214 122 211 221 217
IB336 MAS 251 210 214 122 211 221 217
4LNG101 MAS 251 210 214 122 211 221 217
4LNG201 MAS 251 210 214 122 211 221 217
4LNS301 MAS 251 210 214 122 211 221 217
JG101 MAS 251 210 214 122 211 221 217
CG101 MAS 251 210 214 122 211 221 217
LNB101 MAS 251 210 214 122 211 221 217
DWCG101 MAS 251 210 214 122 211 221 217
DLNG101 MAS 251 210 214 122 211 221 217
DLNG201 MAS 251 210 214 122 211 221 217
DBHS101 MAS 251 210 214 122 211 221 217
JBN1001 MAS 251 210 214 122 211 221 217
JBN1101 MAS 251 210 214 122 211 221 217
JBN1201 MAS 251 210 214 122 211 221 217
JBN1301 MAS 251 210 214 122 211 221 217
2JBN101 MAS 251 210 214 122 211 221 217
2JBN301 MAS 251 210 214 122 211 221 217
2JFN201 MAS 251 210 214 122 211 221 217
2WCL101 MAS 251 210 214 122 211 221 217
ST78-3-3 Stigmatosis 252 211 143 211 212 222 218

References
Abbott, S.L., Cheung, W.K.W., Janda, J.M., 2003. The genus Aeromonas: biochemical charac-
teristics, atypical reactions, and phenotypic identication schemes. J. Clin. Microbiol.
41, 23482357.
Aguilera-Arreola, M.G., Hernandez-Rodriguez, C., Zuniga, G., Figueras, M.J., Castro-
Escarpulli, G., 2005. Aeromonas hydrophila clinical and environmental ecotypes
as revealed by genetic diversity and virulence genes. FEMS Microbiol. Lett. 242,
231240.
Aguilera-Arreola, M.G., Hernandez-Rodriguez, C., Zuniga, G., Figueras, M.J., Garduno, R.A.,
Castro-Escarpulli, G., 2007. Virulence potential and genetic diversity of Aeromonas
caviae, Aeromonas veronii and Aeromonas hydrophila clinical isolates from Mexico
and Spain: a comparative study. Can. J. Microbiol. 53, 877887.
Akinbowale, A.L., Peng, H., Grant, P., Barton, M.D., 2007. Antibiotic and heavy metal resis-
tance in motile aeromonads and pseudomonads from rainbow trout (Oncorhynchus
mykiss) farms in Australia. Int. J. Antimicrob. Agents 30, 177182.
Ard, L., Yin, G., Xu, P., Vradi, L., Szigeti, G., Jeney, Z., Jeney, G., 2008. Chinese herbs
(Astragalus membranaceus and Lonicera japonica) and boron enhance the non-
specic immune response of Nile tilapia (Oreochromis niloticus) and resistance
against Aeromonas hydrophila. Aquaculture 275, 2633.
Austin, B., Adams, C., 1996. Fish pathogens. In: Austin, B., Altwegg, M., Gosling, P.J., Joseph,
S. (Eds.), The Genus Aeromonas. Wiley, J, Chichester, pp. 197243.
Cipriano, R.C., 2001. Aeromonas hydrophila and motile aeromonad septicemias of sh. Fish
Dis. Lea. 68.
da Silva, B.C., Mourino, J.L.P., Vieira, F.N., Jatoba, A., Seiffert, W.Q., Martins, M.L.,
2012. Haemorrhagic septicaemia in the hybrid surubim (Pseudoplatystoma
corruscans Pseudoplatystoma fasciatum) caused by Aeromonas hydrophila.
Aquac. Res. 43, 908916.
Gopalakannan, A., Arul, V., 2006. Immunomodulatory effects of dietary intake of chitin,
chitosan and levamisole on the immune system of Cyprinus carpio and control of
Aeromonas hydrophila infection in ponds. Aquaculture 255, 179187.
Harikrishnan, R., Balasundaram, C., 2005. Modern trends in Aeromonas hydrophila disease
management with sh. Rev. Fish. Sci. 13, 281320.
Hishamunda, N., Subasinghe, R., 2003. Aquaculture development in China: the role of
public sector policies. FAO Fisheries Technical Paper, 427. FAO, Rome, pp. 68.
Huys, G., Kampfer, P., Albert, M.J., Kuhn, I., Denys, R., Swings, J., 2002. Aeromonas
hydrophila subsp dhakensis subsp nov., isolated from children with diarrhoea in
Bangladesh, and extended description of Aeromonas hydrophila subsp hydrophila
(Chester 1901) Stanier 1943 (Approved Lists 1980). Int. J. Syst. Evol. Microbiol. 52,
705712.
Huys, G., Pearson, M., Kampfer, P., Denys, R., Cnockaert, M., Inglis, V., Swings, J., 2003.
Aeromonas hydrophila subsp ranae subsp nov., isolated from septicaemic farmed
frogs in Thailand. Int. J. Syst. Evol. Microbiol. 53, 885891.
Janda, J.M., Abbott, S.L., 1998. Evolving concepts regarding the genus Aeromonas: an
expanding panorama of species, disease presentations, and unanswered questions.
Clin. Infect. Dis. 2, 332344.
Janda, J.M., Abbott, S.L., 2010. The genus Aeromonas: taxonomy, pathogenicity, and infec-
tion. Clin. Microbiol. Rev. 23, 3573.
Jolley, K.A., Maiden, M., 2010. BIGSdb: scalable analysis of bacterial genome variation at
the population level. BMC Bioinforma. 11, 595.
Karunasagar, I., Karunasagar, I., Rosalind, G., 1991. Immunological response of the Indian
major carps to Aeromonas hydrophila vaccine. J. Fish Dis. 14, 413417.
Lee, S., Kim, S., Oh, Y., Lee, Y., 2000. Characterization of Aeromonas hydrophila isolated
from rainbow trouts in Korea. J. Microbiol. 38, 17.
Lee, C., Cho, J.C., Lee, S.H., Lee, D.G., Kim, S.J., 2002. Distribution of Aeromonas spp. as iden-
tied by 16S rDNA restriction fragment length polymorphism analysis in a trout farm.
J. Appl. Microbiol. 93, 976985.
Li, X.P., Li, J.R., Wang, Y.B., Fu, L.L., Fu, Y.Y., Li, B.Q., Jiao, B.H., 2011. Aquaculture industry in
China: current state, challenges, and outlook. Rev. Fish. Sci. 19, 187200.
Liu, X.Z., Zou, X.L., Mo, X.Y., 1993. Characteristics on pathogen of caudal n rot disease of
Tilapia (Oreochromis niloticus) in mariculture. Trop. Oceanol. 12, 100103 (in Chinese
with English abstract).
Liu, J.Y., Yang, W.M., Li, A.H., He, G.W., 2008. Preliminary study on the etiology of channel
catsh intussusception disease. Acta Hydrobiol. Sin. 32, 824831 (in Chinese with En-
glish abstract).
Lu, C.P., 1992. Pathogenic Aeromonas hydrophila and the sh diseases caused by it. J. Fish.
China 16, 282288 (in Chinese).
Martinez-Murcia, A.J., Monera, A., Saavedra, M.J., Oncina, R., Lopez-Alvarez, M., Lara, E.,
Figueras, M.J., 2011. Multilocus phylogenetic analysis of the genus Aeromonas. Syst.
Appl. Microbiol. 34, 189199.
Martino, M.E., Fasolato, L., Montemurro, F., Rosteghin, M., Manfrin, A., Patarnello, T.,
Novelli, E., Cardazzo, B., 2011. Determination of microbial diversity of Aeromonas
strains on the basis of multilocus sequence typing, phenotype, and presence of puta-
tive virulence genes. Appl. Environ. Microbiol. 77, 49865000.
National Bureau of Statistics of China, 2009. China Statistical Yearbook 2009 [in Chinese].
China Statistics Press, Beijing.
Nielsen, M.E., Hoi, L., Schmidt, A.S., Qian, D., Shimada, T., Shen, J.Y., Larsen, J.L., 2001. Is
Aeromonas hydrophila the dominant motile Aeromonas species that causes disease
outbreaks in aquaculture production in the Zhejiang Province of China? Dis. Aquat.
Org. 46, 2329.
Qian, D., Chen, Y., Shen, J., Shen, Z., 1997. Studies on the pathogen of sh bacterial septi-
cemia in Zhejiang Province during 19891992: biochemical characteristics, virulence
and serogroups of Aeromonas hydrophila. In: Yingqi, Z., Fuyuan, H., Hongqi, Z., He, C.,
Chaoqi, Y., Fuhui, D., Yi, L. (Eds.), Proceedings of Fourth Asian Fisheries Forum, Beijing
1620 Oct 1995. China Ocean Press, Beijing, pp. 242245.
Rahman, M.H., Suzuki, S., Kawai, K., 2001. The effect of temperature on Aeromonas
hydrophila infection in goldsh, Carassius auratus. J. Appl. Ichthyol. 17, 282285.
Roberts, R., 1997. Epizootic ulcerative syndrome (EUS): progress since 1985. In: Flegel, T.
W., MacRae, I.H. (Eds.), Diseases in Asian Aquaculture III. Asian Fisheries Society,
Manila, pp. 125128.
Sechi, L.A., Deriu, A., Falchi, M.P., Fadda, G., Zanetti, S., 2002. Distribution of virulence
genes in Aeromonas spp. isolated from Sardinian waters and from patients with diar-
rhoea. J. Appl. Microbiol. 92, 221227.
Stevenson, R.M.W., 1988. Fish vaccination. In: Ellis, A.E. (Ed.), Vaccination Against
Aeromonas hydrophila. Academic Press Ltd, London, pp. 112123.
Sun, Q.H., Sun, P.F., Jin, L.H., Wu, J.L., 1991. On the pathogenic bacteria of the hemolytic
ascitesosis of allogynogenetic crucian carp. J. Fish. China 15, 130139 (in Chinese
with English abstract).
Szczuka, E., Kaznowski, A., 2004. Typing of clinical and environmental Aeromonas sp
strains by random amplied polymorphic DNA PCR, repetitive extragenic palindrom-
ic PCR, and enterobacterial repetitive intergenic consensus sequence PCR. J. Clin.
Microbiol. 42, 220228.
Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 15961599.
6 X. Zhang et al. / Aquaculture 432 (2014) 16
Thomas, L.V., Gross, R.J., Cheasty, T., Rowe, B., 1990. Extended serogrouping scheme for
motile, mesophilic Aeromonas species. J. Clin. Microbiol. 28, 980984.
Thomas, P.C., Divya, P.R., Chandrika, V., Paul-ton, M.P., 2009. Genetic characterization of
Aeromonas hydrophila using protein proling and RAPD PCR. Asian Fish. Sci. 22,
763771.
Urwin, R., Maiden, M.C.J., 2003. Multi-locus sequence typing: a tool for global epidemiol-
ogy. Trends Microbiol. 11, 479487.
Xiao, D., Cao, H.P., Hu, K., Yang, X.L., 2011. ERIC-PCR genotyping and drug resistant anal-
ysis of pathogenic Aeromonas hydrophila from freshwater animals. J. Fish. Sci. China
18, 10921099.
Xu, B.H., Ge, R.F., Xiong, M.L., 1980. Studies on the stigmatosis of silver carp
(Hypophthalmichthys molitrix) and bighead (Aristichthys nobilis). Oceanol. Limnol.
Sin. 11, 8593 (in Chinese with English abstract).
Xu, B.H., Yin, Z., Wu, Y.S., Cai, T.Z., 1993. Studies on the taxonomy of pathogenic bacteria of
the bacterial hemorrhagic septicemia in cultured shes in freshwater. Acta Hydrobiol.
Sin. 17, 259266 (in Chinese with English abstract).
Yez, M.A., Cataln, V., Apriz, D., Figueras, M.J., Martinez-Murcia, A.J., 2003. Phylogenet-
ic analysis of members of the genus Aeromonas based on gyrB gene sequences. Int. J.
Syst. Evol. Microbiol. 53, 875883.
Yin, G.J., Ard, L., Thompson, K.D., Adams, A., Jeney, Z., Jeney, G., 2009. Chinese herbs
(Astragalus radix and Ganoderma lucidum) enhance immune response of carp,
Cyprinus carpio, and protection against Aeromonas hydrophila. Fish Shellsh Immunol.
26, 140145.
Zhang, J.W., Jostein, R., 2004. Aquaculture in China. Innovation NorwayBeijing Ofce,
Beijing pp. 1120.
Zhang, X.J., Yang, W.M., Li, T.T., Li, A.H., 2013a. Research on the genetic diversity and vir-
ulence characteristics of Aeromonas hydrophila isolated from shponds with disease
outbreaks in Hubei province. Acta Hydrobiol. Sin. 3, 458466 (in Chinese with English
abstract).
Zhang, X.J., Yang, W.M., Zhang, D.F., Li, T.T., Gong, X.N., Li, A.H., 2013b. Does the gastroin-
testinal tract serve as the infectious route of Aeromonas hydrophila in crucian carp
(Carassius carassius)? Aquac. Res. http://dx.doi.org/10.1111/are.12168.