Received: 16 May 2017 | Revised: 20 June 2017 | Accepted: 21 June 2017

DOI: 10.1111/jfd.12686

ORIGINAL ARTICLE

Recovery of Bacillus mycoides, B. pseudomycoides and

Aeromonas hydrophila from common carp (Cyprinus carpio) and
rainbow trout (Oncorhynchus mykiss) with gill disease

P Orozova1 | I Sirakov2 | D A Austin3 | B Austin4

1
National Reference Laboratory for Fish,
Molluscs and Crustacean Diseases, National Abstract
Diagnostic Science and Research Veterinary During a 3-month period from June to the end of August 2016, ~5% mortalities
Medical Institute, Sofia, Bulgaria
2 were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and
Department of Microbiology, Medical
University - Sofia, Sofia, Bulgaria one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was mani-
3
School of Life Sciences, Heriot-Watt fested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was
University, Riccarton, Edinburgh, Scotland,
UK isolated from the internal organs of all the diseased fish. Bacillus mycoides or
4
Institute of Aquaculture, University of B. pseudomycoides were recovered from the gill lesions on diseased carp and rain-
Stirling, Stirling, Scotland, UK
bow trout, respectively, with identification achieved by conventional phenotyping
Correspondence and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all
Brian Austin, Institute of Aquaculture,
three organisms were pathogenic to rainbow trout.
School of Natural Sciences, University of
Stirling, Stirling, Scotland, UK.
Email: baustin5851@gmail.com KEYWORDS
Aeromonas hydrophila, B. pseudomycoides, Bacillus mycoides, co-infection, gill disease

1 | INTRODUCTION 30 g average weight in Bulgaria when the water temperature

Bacterial gill disease (BGD) is a common external infection of hatchery
reared salmonids and occasionally of warm water species reared under
intensive conditions (Austin & Austin, 2016). Generally, the aetiologi-
cal agents are considered to be Flavobacterium (Wakabayashi, Huh, &
Kimura, 1989), with acute or chronic forms of the disease leading to
daily mortality rates approaching 20% (Warren, 1981). BGD is particu-
larly prevalent in salmonids during the spring and early summer
months, which coincides with the period when young fish are most
abundant, feeding rates are increasing, and crowding develops (Austin
& Austin, 2016). Another gill condition, that is gill necrosis, is wide-
spread in farmed common carp particularly in eastern Europe. Farkas,
Olah, and Magyar (1986) investigated the development of gill necrosis
and the relationship between environmental stress and F. columnare.
Typically, gill disease occurred in conjunction with or as a result of pre-
disposing factors, which foster disease progression and severity (e.g.,
Bullock et al., 1994). Host and environmental predisposing factors
considered to be important include overcrowding, low dissolved oxy-
gen, reduced water flows, elevated unionized ammonia (NH3) and tur-
bidity in water (Austin & Austin, 2016; Swain et al., 2007).
During June 2016, mortalities started among a population of
232,000 farmed rainbow trout (Oncorhynchus mykiss Walbaum) of
increased suddenly from 9–10 to 18°C in 3 days. During this period,
the oxygen concentration in the water was ~8.5–9.5 mg/ml. The fish
became lethargic, dark in colour and died unexpectedly with signs of
asphyxiation, with total mortalities estimated at 5% over a 1-month
period. Tobacco-yellow coloured ulcers were noted on the gills. Sub-
sequently at the end of August 2016, mortalities totalling~5% over a
1-month period occurred among farmed 1-year-old common carp
(Cyprinus carpio L.) of 45 g average weight reared under intensive
conditions when the temperature of water was 20–22°C. Pale, swol-
len gills with excess of mucus and gill necrosis, exophthalmia and
ascites were noted. In both cases, parasites and water quality prob-
lems were ruled out as likely causes of the disease. Gill scrapings
from diseased rainbow trout and common carp revealed dense popu-
lation of long chains of Gram-positive endospore-forming bacteria.
2 | MATERIALS AND METHODS
2.1 | Bacterial isolation and characterization
Material was collected from gills (ulcerated and non-ulcerated areas),
brain, kidney and liver of moribund fish by means of sterile cotton-
tipped swabs and inoculated onto 5% (v/v) sheep blood in blood

J Fish Dis. 2018;41:125–129. wileyonlinelibrary.com/journal/jfd © 2017 John Wiley & Sons Ltd | 125
126 | OROZOVA
agar base (BA, Oxoid, Basingstoke, UK), MacConkey agar (Oxoid)
and tryptone soya agar (TSA; Oxoid) with incubation aerobically at
28°C for 2 days whereupon extremely dense growth developed, and
was used to obtain pure cultures by streaking and restreaking on
fresh media. The cultures were examined for key phenotypic traits
(after Barrow & Feltham, 2004; Logan & DeVos, 2009) and by use
of rapid identification systems, namely the Micronaut bacteria identi-
fication system (Panel type RPO and NF; Merlin, Berlin, Germany),
API 50CH (bioMe 
rieux, Marcy-l’Etoile, France) and BIOLOG GEN III
(Biolog, Hayward, CA, USA). Antimicrobial susceptibility testing was
determined with the Merlin Micronaut semiautomated system
(Panel type R5, H3 and H4) and by disc diffusion method (Bauer,
Kirby, Sherris, & Turck, 1966). The antibiotics included erythromycin
15 lg, flumequine 30 lg, florfenicol 30 lg, cotrimoxazole 25 lg,
oxolinic acid 2 lg, oxytetracycline 30 lg and tetracycline 30 lg
(Hi-media, Mumbai, India).
2.2 | Sequencing of the 16S rRNA gene
Cultures were inoculated into 10 ml volumes of tryptone soya broth
(TSB; Oxoid) and incubated aerobically overnight at 25°C with shak-
ing at 100 r.p.m. DNA-Sorb-B (AmpliSens, Bratislava, Slovak Repub-
lic) kit was used for DNA extraction. The PCR for 16S rRNA gene
has been performed with 10 pmol primers 27F 50 - AGA GTT TGA
0 0
TCM TGG CTC AG -3 (Lane, 1991) and 1492r 5 - GGT TAC CTT
GTT ATG ACT T -30 (Harris, Kelley, & Pace, 2004) using 25 ll My
Taq PCR Mix (Bioline, London, UK), 3 ll DNA and PCR water (Bio-
line) to 50 ll. The following thermal profile has been used for ampli-
fication: initial denaturation - 95°C – 2 min; cycles – 30;
denaturation - 95°C – 1 min; annealing - 56°C – 1 min; elongation -
72°C – 2 min; final extension – 72°C - 7 min. PCR products were
purified by columns and agarose gel DNA Extraction Kit (Geneshun
Biotech, Guangzhou, China), according to the instructions of the
manufacturer. The quality and quantity of the extracted DNA and
PCR products were checked by 2% agarose gel (SeaKemâ LE Agar-
ose, Lonza, USA) and electrophoresis at 120 V, 80 mA for 40 min at
room temperature. DNA Ladder 100 bp (New England BioLabs,
Hitchin, UK) and HyperLadderTM 1 kb (Bioline) were used. The puri-
fied products and primers 27F and 1492R with concentration of
10 pmol were sent for sequencing at Macrogen Europe (Amsterdam,
The Netherlands). Obtained sequences were processed by MUSCLE
(Edgar, 2004). MEGA7 software (Kumar, Stecher, & Tamura, 2016)
was used for multiple alignments and construction of phylogenetic
trees based on nucleotide and amino acid sequences. One thousand
bootstrap replications were used to build phylogenetic trees. The
outcome was that 1330 nucleotides from the 16S rRNA gene
sequences of the Bacillus isolates (B1 Rt and B2 carp) were used to
enable multiple alignments with the evolutionary history inferred
using the maximum likelihood method based on the Tamura-Nei
model (Tamura & Nei, 1993). The initial trees for the heuristic search
were obtained automatically by applying Neighbor-Joining and BioNJ
algorithms to a matrix of pairwise distances estimated using the
maximum composite likelihood (MCL) approach, and then selecting
ET AL.
the topology with superior log likelihood value. The tree was drawn
to scale, with branch lengths measured in the number of substitu-
tions per site. Sequences were analysed for close homology using
the basic local alignment search tool (BLAST) available at the
National Center for Biotechnology Information (NCBI) (http//:www.
ncbi.nlm.nih.gov/BLAST). Partial sequences of 16S rRNA (1,330 bp
and 1,380 bp) were deposited in the GenBank data base under
Accession no MF 029646 (B1 Rt) and MF 029661 (B2 carp), respec-
tively.
2.3 | Pathogenicity determination
Rainbow trout of 15 g average weight were obtained from a com-
mercial fish farm in Scotland and maintained in free-flowing fresh
water at 12°C. The fish were held under quarantine conditions for
10 days, and the health certified after Austin and Austin (2016). Bac-
terial cultures (B1 Rt, B2 carp; AH 220816) were grown overnight at
22°C in TSB, washed and resuspended in 0.9% (w/v) saline to
108 cells/ml as determined using a haemocytometer slide (improved
Neubauer type, Sigma-Aldrich, Irvine, UK) at a magnification of
9400 on a Euromex BioBlue microscope (Arnhem, the Netherlands).
Groups of five rainbow trout were injected intraperitoneally (i.p.) or
intradermally (i.d.) with 0.1 ml volumes containing 106 cells fish1.
Control groups (n = 5) received 0.1 ml of sterile 0.9% (w/v) saline by
i.p. injection. The fish were monitored for up to 14 days, and dead
or moribund fish, and the survivors examined microbiologically to
determine the presence of the bacterial culture. Thus, kidney and
spleen swabs were inoculated onto TSA with incubation at 22°C for
48 hr. Authenticity was confirmed according to the original descrip-
tion of the cultures before use in subsequent challenge experiments.
3 | RESULTS AND DISCUSSION
Rainbow trout became lethargic, darkened in colour and died unex-
pectedly. Tobacco-yellow coloured ulcers with fluffy-like protrusions
were noted on the gills (Figure 1). Scrapings revealed the presence
exclusively of long chains of Gram-positive endospore-forming bac-
teria. In addition, there was evidence of swollen abdomen and
bloody ascites. The rainbow trout died suddenly and unexpectedly
with signs of asphyxiation. Common carp developed swollen, pale
gills with high mucus secretion. Fusion of adjacent lamellae was
extensive. These were followed by a gill necrosis and separation of
the epithelium from the gill lamellae. Fish displayed exophthalmia
and swollen abdomen with ascites.
From gill ulcers on rainbow trout and diseased gill tissue from
common carp, dense pure growth of cream rhizoidal colonies, which
displayed filamentous swirling patterns, were obtained on BA and
TSA. These colonies comprised long chains of Gram-positive, cata-
lase-positive oxidase-negative, ß-haemolytic endospore-forming rods
(Figure 2). Also, all the diseased non-ulcerated rainbow trout and
carp gills revealed the presence on BA, MacConkey agar and TSA of
dense growth of Gram-negative, catalase-positive oxidase-positive
OROZOVA ET AL. | 127

T A B L E 1 Characteristics of Bacillus isolates from diseased fisha

Bacillus from:

Rainbow Common
Character trout (B1 Rt) carp (B2 carp)
Motility  
Endospore formation + +
Aerobiosis + +
Catalase production + +
Voges Proskauer reaction + +
Acid production from:
D-glucose + +
Glycogen + +
D-mannose  
Methyl-ß-xyloside  
Salicin + +
Starch + +
D-xylose  
FIGURE 1 Diseased rainbow trout displaying tobacco-yellow Utilization of citrate  
ulceration Degradation/hydrolysis of:
ß-haemolytic rods. From the internal organs of all the diseased fish, Gelatin + +
dense pure culture growth of ß-haemolytic, Gram-negative, catalase- Starch + +
positive, oxidase-positive rods were obtained, but not Gram-positive Tyrosine + +
cells. Growth at pH 5.0  
The phenotypic characteristics of the Gram-positive bacteria, Growth at pH 6.0  
that is, B1 Rt and B2 carp have been included in Table 1. By com- Growth at 5°C  
parison with the characteristics in Logan and DeVos (2009) and by
Growth at 30°C + +
use of BIOLOG GEN III, the isolates were identified as B. mycoides.
Growth at 40°C  
In comparison, use of API 50 CH and Micronaut RPO plates led to
a
an identification of B. cereus (Table 2). However, in contrast to the These characteristics matched those of B. mycoides, which was indistin-
guishable from B. pseudomycoides (Logan & DeVos, 2009).
species description of B. cereus in Logan and DeVos (2009), the iso-
lates differed in terms of motility, growth at 40°C (both positive for
B. cereus) and colonial morphology. The cultures were sensitive to Sequencing of the 16S rRNA gene revealed that the Gram-posi-
erythromycin, oxolinic acid, florfenicol and flumequine. tive bacteria from common carp and rainbow trout belonged to dif-
The Gram-negative bacteria, of which the representative culture ferent groups and were distinguished from the other members of
was AH 220816, matched the description of Aeromonas hydrophila the genus. The outcome was that the common carp (B2 carp) and
(Austin & Austin, 2016; Whitman & MacNair, 2004). This was con- rainbow trout isolates (B1 Rt) matched entries in the database for
firmed by use of Micronaut and the API 50CH rapid identification B. mycoides (homology = 100%) and B. pseudomycoides (homol-
systems. The cultures were sensitive to florfenicol, oxolinic acid and ogy = 99%), respectively (Table 3; Figure 3). Moreover, it should be
flumequine. emphasized by B. mycoides and B. pseudomycoides are

F I G U R E 2 Growth of Bacillus mycoides

(left) and B. pseudomycoides (right)
displaying rhizoidal colonies of 5–6 mm
diameter on tryptone soya agar (TSA) with
incubation at room temperature overnight
128 | OROZOVA ET AL.

T A B L E 2 Identification of Bacillus isolates from rainbow trout (B1 Rt) and common carp (B2 carp)
Identification by:

Ref. no. API 50CH Micronaut BIOLOG 16S rRNA gene sequencing
B1 Rt B. cereus B. cereus B. mycoides B. pseudomycoides (GenBank Acces. No MF 029646)
B2 carp B. cereus B. cereus B. mycoides B. mycoides (GenBank Acces. No MF 029661)

T A B L E 3 Comparison of the 16S rRNA sequences of Bacillus B1 indistinguishable phenotypically and could not be differentiated by
Rt and B2 carp with entries in GenBank means of the tests used in this study (Logan & DeVos, 2009; Naka-
Homology mura, 1998).
with closest The experimental challenges with Bacillus B1 Rt or B2 carp or
Ref no. Name in GenBank neighbour
Aeromonas AH 220816 did not result in any mortalities, but clinical
MF 029646 (B1 Rt) Bacillus disease developed. Thus, following i.d. injection with 106 cells fish1,
KY312800 B. pseudomycoides 99% the Bacillus isolate from rainbow trout (B1 Rt) led to the develop-
HF678922 Bacillus sp. 99% ment of severe ulceration and necrosis at the injection site, swollen
KY316437 B. pseudomycoides 99% spleen, softened kidney and anal discharge. In contrast, the Bacillus
KY774367 Bacillus sp. 99% from common carp (B2 carp) led only to ulceration and necrosis
KU986671 Bacillus sp. 99% around the i.d. injection site. Administration of the Aeromonas from
MF 029661 (B2 carp) Bacillus rainbow trout (AH 220816) by i.p. injection led to the development

KY887028 B. mycoides 100% of swollen spleen and softened kidney. The Aeromonas from com-
mon carp caused swollen spleen, softened kidney, and protruded
CP020743 B. mycoides 100%
anus with discharge. The isolates were recovered from diseased tis-
KY124218 B. mycoides 100%
sues on TSA with incubation at room temperature for 2 days.
KX767102 B. mycoides 100%
The association of motile Aeromonas with fish diseases is well
KX035066 B. mycoides 100%
established (Austin & Austin, 2016), but the interaction with Bacillus

F I G U R E 3 Molecular phylogenetic
analysis by maximum likelihood method of
25 nucleotide sequences of the 16S rRNA
gene with length of 1330 nt. Bacillus
isolates from trout MF 029646 and carp
MF 029661 are bold
OROZOVA ET AL.
is unusual. It is unclear whether Aeromonas and Bacillus represent
examples of co-infections, microbial population successions, or the
bacilli being secondary invaders of already damaged tissue. The shar-
ply increasing water temperatures were most likely an important
trigger for the onset of disease. Certainly, there have been some
previous reports of Bacillus as fish pathogens, causing generalized
necrotizing dermatitis (Oladosu, Ayinla, & Ajiboye, 1994), bacillary
necrosis (Ferguson et al., 2001) and gill disease. Thus, B. cereus and
B. subtilis were associated with branchionecrosis in common carp
(Pychynski, Malanowska, & Kozlowski, 1981) and striped bass (Baya,
Lupiani, & Hetrick, 1992). In addition, B. mycoides was associated
with an epizootic in channel catfish (Goodwin, Roy, Grizzle, &
Golsby, 1994), with the characteristics of the culture matching those
of this study. However, this is the first mention of B. pseudomycoides
with fish disease. Normally, B. mycoides and B. pseudomycoides have
been regarded as soil borne (Logan & DeVos, 2009) rather than
aquatic inhabitants, although there may be input of organisms from
soil to freshwater. Fortunately, control of the disease in common
carp was achieved by reducing the stocking density and by the ces-
sation of feeding whereas for rainbow trout erythromycin was used
successfully in the fish farm.
ACKNOWLEDGEMENTS
We thank the leader of the Department “Aquaculture and Aquatic
Animals and Bee Diseases” and the Director of NDRVMI, Sofia.
Dr Ekaterina Mileva, a Ph.D. student of the National Reference
Laboratory of Fish, Molluscs and Crustacean Diseases NDRVMI,
Sofia, Bulgaria, is acknowledged for technical support.
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How to cite this article: Orozova P, Sirakov I, Austin DA,
Austin B. Recovery of Bacillus mycoides, B. pseudomycoides
and Aeromonas hydrophila from common carp (Cyprinus carpio)
and rainbow trout (Oncorhynchus mykiss) with gill disease.
J Fish Dis. 2018;41:125–129. https://doi.org/10.1111/
jfd.12686