Introduction

Misgurnus anguillicaudatus occupies a significant position in freshwater fish

culture enterprises in China both for food and commercial purposes (Zaxia et al.
2007). In 2010, the total cultured output of mud loach in China mainland was 204552
tons (Bureau of Fisheries MoA 2011). Since 1950s mud loach are consumed as a
delicious fish food with high nutritional value and as traditional Chinese medicine. M.
anguillicaudatus was widely cultured in the Jiangsu province of China, making a
great profit on economics in recent years (Zhang et al. 2012). The loach, for a long
time, had been employed as traditional Chinese medicine in folk remedies for
treatment of hepatitis, osteomyelitis, carbuncles, inflammations and cancers, as well
as for restoration of health in debilities caused by various pathogens and aging (Qin et
al. 2002); some active substances obtained from loach were found to be of high
medical value (Kimura et al.1994; Park et al.1997; Dong et al. 2002; Zhang & Huang
2005; Zhang & Huang 2006). The frequent outbreaks of diseases have caused
decreased production and economic losses. Bacterial infection is a common and
serious economic problem in cultivation of M. anguillicaudatus. Mortality has been
reported on M. anguillicaudatus at different habitats for many decades. A serious
mortality of cultured loach M. anguillicaudatus occurred in farms of Donghai County,
Jiangsu Province in October 2008 (Bing et al. 2009) and 2010 (Zhang et al. 2012).
Currently, the most serious problem in both loach hatcheries and farms is bleeding in
head, opercula and lower jaw, swollen muscle, anus, liver, and spleen, and empty
intestines (Bing et al. 2009).
Aeromonas spp. are omnipresent inhabitants of aquatic ecosystems such as
freshwater, coastal water, and sewage (Mollby, Khun & Katouli 1993). They are
progressively being reported as important pathogens for humans and for lower
vertebrates, including amphibians, reptiles, and fish (Janda & Abbott 1998).
Aeromonas veronii is ubiquitous in fresh water and is found in association with a
variety of vertebrates and invertebrates with both beneficial and pathogenic outcomes
(Hanninen, Salmi, Mattila, Taipalinen & Siitonen 1995; Janda, Abbott 1998 & 2011;

Silver, Rabinowitz, Kueffer, Graf 2007). This species has been divided into two
biovars, Aermonas veronii bv. veronii and Aeromonas veronii bv. sobria, with the
latter being considered more virulent [Janda & Abbott 2011]. The taxonomic position
of a closely related species, Aeromonas allosaccharophila, has been debated [Huys,
Kampfer, Swings 2001; Nhung , Hata, Ohkusu, Noda, Shah, et al. 2007], with some
investigators considering them to be synonymous with A. veronii and others as
separate species (Alperi, Martinez-Murcia, Ko, Monera, Saavedra, et al. 2010). In
order to investigate the cause of the disease, the pathogenic microorganisms were
isolated, purified and identified with 16S rDNA sequencing. And some effective drugs
were screened for preventing and controlling based on antimicrobial susceptibility
testing in vitro. This work is aimed at providing ways for stopping the death of
M.anguillicaudatus caused by Aeromonas veronii and reducing the economic loss of
the farmers.
Materials and methods
Fish
Diseased loaches were collected from a fish farm in Wuhan City. The typical
disease signs were external haemorrhages and ulcers, mainly located on the head and
ventral part of the body. Prior to infection, loaches were kept for 7 days in aquaria
with aeration. They were fed with commercial forage once daily. Water was replaced
daily and maintained at 25oC.
Bacterial isolation
For bacterial isolation, samples taken from gill, liver, spleen and body ulcers of
diseased loaches were plated on nutrient agar. After incubation at 37 oC for 24h,
bacterial colonies were picked and plated on fresh nutrient agar plates until pure
cultures were obtained.
Biochemical characteristics of the isolates
Biochemical characteristics of the isolates were confirmed by microbial
biochemical identification tubes (Guangdong Huankai Microorganism Reagent Co.,
Ltd) with reference to Bergerys Manual of Determinative Bacteriology (Holt et al.
1994).

16S rRNA sequence analysis

Bacteria were incubated on the agar medium at 28oC for 24 hours. The bacterial
suspension was prepared by diluted in sterile double-distilled water and centrifuge at
12,000 rpm in 5 min at 4C. Genomic DNA was extracted using ammonium acetate
precipitation technique as described previously (Bruford, Hanotte, Brookfield &
Burke 1998) with slight modification. The quality of genomic DNA was evaluated
using electrophoresis in 1% agarose gels. The quantity of genomic DNA samples was
measured by using a Nanodrop 2000 (Thermo Scientific, USA). DNA was stored at
-20oC until use. Polymerase Chain Reaction (PCR) was performed on all strains. The
universal bacterial primers were used to amplify the 16S ribosomal RNA genes of the
isolates (Xiao et al. 2009). Primers were synthesized by Shanghai Sangon
Biotechnology Co., Ltd. PCR was performed in 0.2-mL PCR tubes. The reaction
mixture had a final volume of 50L and contained 2.0 L DNA template, 5.0 L 10X
Taq buffer (Mg2+ plus), 2.0 L dNTP mixture (10 mm each), 1.0 L each primer (10
mm), 2.0 L Taq polymerase and 37 L distilled water. The PCR program comprised
an initial denaturation step of 94C for 5 min, 30 cycles of 94 oC for 30 s, 58oC for 45s,
72oC for 1.5 min, followed by a final extension of 10 min at 72 oC. Successful
amplification was confirmed by electrophoresis of 5 L of PCR products on a 1%
agarose gel. The gel image was visualized through a UV gel image acquisition
camera. PCR products were purified using a PCR purification kit (Takara), according
to the manufacturers instruction. Purified products were directly sequenced by
Shanghai Sangon Biotechnology Co. Ltd. Sequence analysis was completed by using
BLAST network services, Clustal X 2.0 and MEGA 5.2. The Basic Local Alignment
Search Tool (BLAST) was used to search the Entrez database for homologous
sequences, and 6 of them were chosen for the phylogenetic analysis. The unrooted
evolutionary tree was inferred using the neighbour-joining method; Kimura- 2
parameter, 1000 bootstrap replicates.
Bacterial Tolerance Test
Effect of pH.

The pH range of sterile nutrient broth was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5
and 9.0. For each PH, ten tubes filled with 2 mL sterile nutrient broth were prepared,
inoculated with 25L of fresh bacterial suspension and incubated at 30C. After 24 h,
all the cultures were sampled, and OD610 values were determined.
Effect of salinity.
The salinity range of sterile nutrient broth was adjusted to 0%, 0.5%, 1%, 2%,
3%, 4%, 5%, 6% and 7% NaCl w/v. For each salinity, ten tubes filled with 2 mL
sterile nutrient broth were prepared, and 25L of fresh bacterial suspension was
inoculated into them. They were statically incubated at 30C, and after 24 h, all the
cultures were sampled and OD610 values were determined.
Real-time PCR assays for quantifying virulence gene-containing Aeromonas
veronii.
The existing haemolysin, aerolysin and lipase nucleotide sequences of
Aeromonas were retrieved from the NCBI GenBank (Hem gene:Accession No.
AY157998, X65048, X65046, AF410466, CP002607; Aer gene: M84709, M16495,
X65043, X65044, X65045; Lip gene : U63543, AB206038, DQ131226, DQ131227,
DQ131221). These sequences were aligned in Clustal X 2.0, and haemolysin gene
primers (Table .1) were designed using IDT primer quest based on the conserved
region. Each reaction was performed in a total volume of 25 L, with Quanti- Tect
SYBR Green PCR Master Mix (QIAGEN Inc., Valencia, CA), 600 nM forward and
reverse primers, and 5 L of DNA templates. The PCR thermal cycle for both genes
of A. veronii was 50C for 2 min and 95C for 15 min, followed by 36 cycles of 95 C
for 30 s, optimum annealing temperature (57C for Aer gene and 62C for Hem and
Lip gene) for 45 s, and 72C for 30 s. PCR amplification and detection were
performed by using an real-time PCR detection system (BioRad). The cycle threshold
(Ct) value was determined automatically by computer software. All concentrated and
unconcentrated DNA samples were divided into three sets.
Antibiotic susceptibility
The susceptibility pattern of bacterial isolates to 20 antimicrobial agents (Oxoid)
including amoxilin (10 g), florfenicol (75 g), levofloxacin (5 g), tetracylin (30

g), neomycin (30 g), novobiocin (30 g), doxycylin (30 g), gentamicin (10 g),
norfloxacin (10 g), aztreonan (30 g), fortum (30 g), kanamycin (30 g),
ceftriaxone sodium (30 g), ofloxacin (5 g), piperacillin (100 g), clindamycin (2
g), rifampicin (5 g), imipenem (10 g), erythromycin (15 g) and chloramphenicol
(30 g) was tested and determined using the standard KirbyBauer method (Bauer,
Kirby, Sherris & Turck 1966) on Mueller-Hinton agar and incubated for 24 h at 28oC.
Results were interpreted as susceptible, medium susceptible or resistant based on zone
diameters of inhibition, including the diameter of the disc (mm) (Liu et al., 2009).

Results:
Morphologic, phenotypic and biochemical characteristics:
All three isolates were phenotypically homogeneous, short rod-shaped, occurring
in singly in pairs or as short chains. The biochemical characteristics of isolates are
summarized in Table 2. All our isolates yielded identical biochemical test results to
those of A. veronii. They were Gram negative, oxidase positive, motile, fermentative
and resistant to the vibriostatic compound. No gas was produced from glucose by all
isolates. All isolates were positive for indole production, MR, hydrolysis of starch,
sucrose, lactose, arabinose and mannitol. Negative reaction was shown for
fermentation of catalase, sucrose and esculine hydrolysis in all tested strains.
Antibiotic-susceptibility test
The effects of antibiotic susceptibility on A. veronii were quite variable. Ceftriaxone
sodium exhibited the strongest effect with the inhibitory zone values (45mm), and A.
sobria C26 was the most susceptible strain when 0.8 lg mL 1 of CP incorporated in
the medium. The MIC values of OTC
16S rRNA sequence analysis of the isolates
The 16S rRNA sequences of the isolates were analysed via BLAST network
services. The 16S rRNA genes of WHMA1 and WHMA3 showed a high similarity
(97% identity) to that of A. veronii, The phylogenetic tree (Fig. 1) constructed from
16S rRNA sequences of the isolates and 26 homologous sequences, using the
neighbour-joining method, clustered the isolates with A. veronii WHMA2 within the

same group.

Discussion: