Aeromonas jandaei and Aeromonas veronii caused ...

doi:10.1111/jfd.12617

Journal of Fish Diseases 2017

Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia, Oreochromis niloticus (L.) H T Dong1 , C Techatanakitarnan1,2, P Jindakittikul1, A Thaiprayoon1, S Taengphu2, W Charoensapsri2,3, P Khunrae1, T Rattanarojpong1 and S Senapin2,3 1 Department Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok, Thailand 2 Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand 3 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand

Abstract

Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non-A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport-induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 9 106 CFU fish 1) and A. veronii (8.9 9 106 CFU fish 1) killed 100% of experimental fish within 24 h, while a 10-fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100-fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to

Correspondence S Senapin and T Rattanarojpong (e-mails: [email protected] and [email protected]) Ó 2017 John Wiley & Sons Ltd

1

either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation. Keywords: Aeromonas jandaei, Aeromonas veronii, virulence, Nile tilapia.

Introduction

Tilapia is a fast-growing, well-adapted fish species which has been commercially farmed in over 100 countries in the tropical and subtropical regions (El-Sayed 2006). Like other aquaculture fish species, under stress conditions of intensive farming, tilapia is also susceptible to various bacterial diseases such as streptococcosis caused by Streptococcus agalactiae, Streptococcus iniae or Lactococcus garviae (Suanyuk et al. 2008; Anshary et al. 2014; Kayansamruaj et al. 2014); francisellosis caused by Francisella noatunensis subsp. orientalis (Soto et al. 2009; Lin et al. 2015; Nguyen et al. 2015); columnaris caused by Flavobacterium columnare (Figueiredo et al. 2005; Dong et al. 2016); haemorrhagic septicaemia caused by Aeromonas spp. (Li & Cai 2011; Eissa et al. 2015); or miscellaneous disease caused by multiple infections (Cutuli et al. 2015; Dong et al. 2015; Assis et al. 2016). In addition, some pathogenic bacteria were occasionally reported in farmed tilapia systems such as Aerococcus viridans (Ke et al. 2012) and Hahella chejuensis (Senapin et al. 2016).


Infectious disease caused by motile aeromonads is one of the most common problems in aquaculture freshwater fish, and a significant body of knowledge usually links to Aeromonas hydrophila infection (Austin & Austin 2012). Aeromonads are highly diverse and probably different among fish hosts. A significant number of aeromonads associated with disease in freshwater fish have been taxonomically identified, but proof of pathogenicity is still lacking, at least in the host from which the bacteria were recovered (Austin & Austin 2012). In tilapia, apart from A. hydrophila, several aeromonads associated with disease have been reported, including Aeromonas sobria (Li & Cai 2011), Aeromonas dhakensis (A. hydrophila subsp. dhakensis) (Soto-Rodriguez et al. 2013), Aeromonas veronii (synonyms are Aeromonas ichthiosmia, Aeromonas culicicola and A. allosaccharophila) (Huys, Cnockaert & Swings 2005; Nhung et al. 2007; Dong et al. 2015; Eissa et al. 2015). Up to date, there is no report of Aeromonas jandaei caused disease in tilapia. The objectives of this study were to (i) identify non-A. hydrophila species associated with mortality of a batch of Nile tilapia after exposed to transportinduced stress and (ii) determine their ability to cause disease in experimental infection without stress inducer.

Materials and methods

Diseased fish A batch of apparently healthy Nile tilapia Oreochromis niloticus (L.) juveniles (approximately 40 g body weight) was obtained from a commercial farm in Pathum Thani Province, Thailand. Fish were transported to Centex Shrimp laboratory in Bangkok and stocked in two plastic tanks containing 50 L dechlorinatetap waterwithnon-stopaeration. Afterexposedto transport-induced stress, fish started dying from day 1 and mortality increased gradually and reached 68.4% at day 22 post-stocking. Clinically sick fish exhibited symptoms resembling infectious disease including pale body surface, fin rot, exophthalmia, cloudy eyes andseverehaemorrhage inliverand brain(pictures not shown). Bacterial isolates and phenotypic tests At day 5 post-fish stocking, bacterial isolation was performed from two clinically sick fish Ó 2017 John Wiley & Sons Ltd

2

H T Dong et al. A. jandaei and A. veronii killed Nile tilapia

described above. Skin, kidney, brain and gills were directly streaked onto tryptic soy agar (TSA) and incubated overnight at 30 °C. Based on bacterial colony morphology, two distinctive bacterial isolates designated as NT-01 and NT03 exhibiting the major population among the obtained bacteria were subjected to further purification and detailed investigation in this study. Phenotypic tests were performed using the Gram-staining method and an API 20E kit following the manufacturer’s instructions (BioMerieux). For long-term preservation, bacterial cells were prepared in tryptic soy broth containing 20% glycerol and kept in 80 °C until used. 16S rDNA amplification, DNA sequencing and phylogenetic analysis Genomic DNA of the bacterial isolates was prepared as previously described by Dong et al. (2015). Amplification of 16S rDNA was performed with two bacterial isolates using universal primers Uni-Bact-F/AGA GTT TGA TCM TGG CTC AG and Uni-Bact-R/ACG GHT ACC TTG TTA CGA CTT (Weisburg et al. 1991). Each 25 lL of PCR mixture consisted of 0.5 lM of each primer pair, 0.2 mM of dNTPs, 0.25 lM of MgCl2, 1 unit of Taq polymerase (Invitrogen), 100 ng of bacterial genomic DNA and 19 reaction buffer. The conditions for thermocycling were set as follows: 94 °C for 5 min, 35 cycles of 94 °C for 40 s, 50 °C for 40 s, 72 °C for 1.5 min and final extension at 72 °C for 7 min. PCR products were ethidium bromide-stained and visualized after gel electrophoresis and then purified using a Favogen Gel/PCR Purification Kit following the manufacturer’s instructions. The 16S rDNA fragments were then cloned into pGEM-T Easy vector (Promega), and recombinant plasmids were subjected to Sanger sequencing (1st BASE Pte Ltd.) using T7 and SP6 primers. Sequence assembly was carried out with both forward and reverse directions using ContigExpress software. The sequences were blasted against available sequences in the GenBank database. A phylogenetic (neighbour-joining) tree was generated by MEGA 6 software following multiple alignments (Clustal W) of the 16S rDNA sequences in this study and their closely related species retrieved from GenBank.


Challenge test Prior to the challenge test, two bacterial isolates A. jandaei NT-01 and A. veronii NT-03 were recovered from glycerol stocks. Bacteria were streaked directly onto TSA plates and incubated at 30 °C for 18 h. A pure single colony of each isolate was then cultured in 5 mL of TSB overnight. Then, 1% of the bacterial suspension was transferred to 20 mL of TSB and incubated at the same temperature with shaking at 250 rpm for 2 h. Bacterial suspension was adjusted to OD600 = 0.8 and diluted 10-, 100- and 1000-fold before injection. Infection doses were retrospectively known by conventional plate count as 3.7 9 107 CFU fish 1 for A. jandaei NT-01 and 8.9 9 107 CFU fish 1 for A. veronii NT-03, equivalent to OD600 of 0.8. Healthy Nile tilapia juveniles (n = 85, mean weight 36.7 4.1 g) used for the experimental challenge were obtained from a commercial tilapia hatchery in Samut Songkhram Province. Fish were acclimatized in normal laboratory conditions for 17 days, and all appeared to be healthy as no abnormal symptoms or mortality was observed. Prior to the challenge tests, five fish were randomly subjected to bacterial isolation and found to be free of Aeromonas spp. Fish were then divided into eight groups with 10 individuals each. The first three treatment groups received 0.1 mL of three different doses of A. jandaei NT-01 by intraperitoneal injection, while three other groups were exposed to three different doses of A. veronii NT-03. One control group was injected with 0.1 mL of TSB sterile medium without bacteria, and the other noninjected group also served as a control group. After injection, fish were returned and cultured in 50-L water tanks with non-stop gentle aeration and fed daily with tilapia commercial feed (3% body weight). Water temperature and pH during experiments were 24.0 1.0 °C and 7–7.5, respectively. Clinical signs and mortality were observed every 6 h for 14 days. Representatives of freshly dead or moribund fish from each treatment were subjected to bacterial isolation and histological study. Rechallenge test To investigate whether surviving fish after primary infection were able to resist secondary infection, the survivors from the lowest dose of each bacterial isolate were adjusted to eight fish per group and maintained for rechallenge test. Fish from Ó 2017 John Wiley & Sons Ltd

3


two control groups were pooled and randomly divided into two groups with eight individuals each. Bacterial suspension was prepared as described above. At 27 days post-primary injection, each group of respective surviving fish individually received A. jandaei (4.7 9 105 CFU fish 1) or A. veronii (7.6 9 105 CFU fish 1) by intraperitoneal injection. Two control groups were challenged with the bacteria in the same manner. The secondary infection experiment was monitored for 13 days, and mortality was recorded daily. Histological examination Internal organs (liver, spleen, brain and intestine) collected from moribund fish during the experimental challenge were preserved in buffer formalin 10% for histological study. Those specimens were routinely processed with a conventional histological method and stained with haematoxylin and eosin (H&E) and examined under a light microscope.

Results

Isolation of A. jandaei and A. veronii from clinically sick tilapia Two bacterial isolates NT-01 and NT-03 representing the major population of bacteria recovered from naturally diseased Nile tilapia were Gram negative, rod-shaped bacteria. Biochemical results using API 20E kit indicated that two isolates were phenotypically identical except the Voges–Proskauer reaction (Table 1). Based on Abbott, Cheung & Janda (2003) and Esteve et al. (2003), the isolates NT-01 and NT-03 had biochemical characteristics resembling A. jandaei and A. veronii bv. sobria, respectively (Table 1). Amplification of 16S rDNA yielded ~1.5-kb amplicons from extracted DNA of the two bacterial isolates (figure not shown). After cloning and DNA sequencing analysis, BLAST results revealed that the 16S rDNA fragment of isolate NT-01 is 99.9% identical to the reference strain A. jandaei ATCC 49568 (GenBank accession no. X60413) while that of the isolate NT-03 had a 99.9% nucleotide match to A. veronii B565 (GenBank accession no. CP002607). Based on a combination of phenotypic characteristics and homology of 16S rDNA sequences, two bacterial isolates



Table 1 Biochemical characteristics of Aeromonas jandaei and Aeromonas veronii A. veroniia

Characteristics

NT-01

A. jandaeia

NT-03

A. veronii bv sobria

A. veronii bv veronii

Gram Bacterial morphology Motility ONPG Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization H2S production Urease TDA Indole production Voges–Proskauer Gelatin Acid production D-glucose D-mannitol Inositol D-sorbitol L-rhamnose D-sucrose D-melibiose Amygdalin L-arabinose NO2 production

Negative Rod + + + +




Negative Rod + +

+

+

+

V ND

V ND

+ + +

+ + +

+

ND +

+

+

ND + + +

+ +

+ +

+ +

+ +

+ +

+

+ V

+

+

+ +

+

+

Data obtained from Abbott et al. (2003) and Esteve et al. (2003); +, positive;

a

NT-01 and NT-03 obtained in this study were identified as A. jandaei and A. veronii, respectively. The phylogenetic tree constructed, based on 16S rDNA sequences of the two bacterial isolates and the reference strains of Aeromonas species, strongly supported our identification results (Fig. 1). These sequences were deposited under GenBank accession numbers KX714287 and KX714288, respectively. Virulence of A. jandaei and A. veronii to Nile tilapia Initially, a batch of healthy-looking Nile tilapia obtained from a farm in Pathum Thani Province was transported to our laboratory in Bangkok. However, upon transportation and acclimation in the laboratory, the fish exhibited diseased symptoms and died gradually, and cumulative mortality reached 68.4% at day 22 post-stocking (Fig. 2a). Subsequently, A. jandaei NT-01 and A. veronii NT-03 representing the dominant bacterial population were isolated from diseased fish as mentioned above. To investigate pathogenicity of A. jandaei NT-01 and A. veronii NT-03, Ó 2017 John Wiley & Sons Ltd

4

+ +

+

+

+

, negative; V, variable.

challenge tests were then performed in Nile tilapia. The results showed that both tested Aeromonas species were pathogenic to tilapia juveniles and mortality rate was dose dependent. The high dose of A. jandaei (3.7 9 106 CFU fish 1) and A. veronii (8.9 9 106 CFU fish 1) killed 100% of fish within 24 h (Fig. 2b) without remarkable clinical signs. When the challenge dose was reduced 10- and 100-fold, cumulative mortality in the groups administrated with A. jandaei decreased to 70% and 20%, respectively, while in those groups which received A. veronii, it was 50% and 10%, respectively (Fig. 2b). Clinically sick fish which were administered two lower doses of each bacteria exhibited dark bodies, abnormal swimming and loss of appetite. Before death, sick fish usually suspended on the water surface and sunk to the bottom of the tank. Internally, diseased fish showed severe haemorrhage and blood congestion notably in the liver (Fig. 3), and food emptied from the gut displayed a significant amount of yellowish liquid (Fig. 3). Pure bacterial colonies resembling Aeromonas spp. were recovered from the liver, kidney and brain of the dead and moribund fish.



Aeromonas caviae ATCC 15467 (X60409) Aeromonas caviae ATCC 15468 (NR118947) 94 Aeromonas dhakensis LMG 19562T (AJ508765) 81 Aeromonas hydrophila ATCC 7966 (NR118947) Aeromonas sobria ATCC 43979 (NR119044) 85 85 Aeromonas veronii B565 (CP002607) Aeromonas veronii NT-03 Aeromonas veronii bv. veronii ATCC 35624 (X60414) 77 99 Aeromonas veronii bv. sobria ATCC 9071 (AF410949) 93 Aeromonas veronii DSM 6393 (X71120) Aeromonas jandaei NT-01 79 Aeromonas jandaei ATCC 49568 (X60413) Plesiomonas shigelloides NCIMB9242 (X60418) 65 96

0.01

Figure 1 Phylogenetic tree was constructed based on 16S rDNA of the bacterial isolates in this study (NT-01 and NT-03) and their closely related species. Plesiomonas shigelloides was selected as an out-group. Percentage bootstrap values (1000 replicates) are shown at each branch point. The scale bar represents 0.01-nucleotide change per nucleotide position.

Figure 2 Daily cumulative per cent mortality of naturally diseased Nile tilapia (a) and experimental challenged fish (b). Mortality of a batch of Nile tilapia was observed for 22 days. Experimental challenged fish received three different doses of Aeromonas jandaei and Aeromonas veronii by intraperitoneal injection. Number in brackets represents dose of bacterial cells. Two control groups received either TSB medium or no injection. There were 10 fish in each group, and experiments were monitored for 14 days.

Resistance of surviving fish to rechallenge of A. jandaei and A. veronii Two groups of surviving fish from the primary infection subjected to secondary infection with Ó 2017 John Wiley & Sons Ltd

5

A. jandaei NT-01 or A. veronii NT-03 resulted in 12.5% (one of eight) and 0% (0 of eight) mortality, respectively (Fig. 4). With the same infection dose, 37.5% (three of eight) fish mortality was observed in the two control groups (Fig. 4), which



corresponded to the mortality during the primary infection at similar dose levels (3.7 9 105 CFU fish 1 for A. jandaei and 8.9 9 105 CFU fish 1 for A. veronii) (Fig. 2b). Histological manifestation of moribund fish A histological investigation was conducted from fish specimens upon primary infections with A. jandaei NT-01 and A. veronii NT-03. A similar histological manifestation was revealed in the two Aeromonas-infected groups. The remarkable changes could be found in the brain, liver, spleen and intestine of the moribund fish. The brain and liver showed severe blood congestion, but more severe signs were found in the liver (Fig. 5). The liver of the infected fish also exhibited tissue degeneration and accumulation of hemosiderin around the vessels and liver cells (Fig. 5d). Hyperaemia and haemorrhage were observed in the spleen, while severe sloughing and necrosis occurred in epithelial cells of the fish intestine (Fig. 5e,f). No abnormal changes were observed in the control groups (figures not shown). Discussion

Infections caused by motile aeromonads are probably the most important bacterial diseases of freshwater fish (Cipriano, Bullock & Pyle 1984; Noga 2010). However, aeromonad infections in fish were mostly reported to be associated with A. hydrophila. In fact, evidence of natural infections caused by A. hydrophila with reliable taxonomic identification is relatively rare in tilapia. Motile aeromonads are very diverse, and not only A. hydrophila but other Aeromonas species have also been reported as threats to aquaculture freshwater fish (Dong et al. 2015; Eissa et al. 2015; Peepim et al. 2016; Zhu et al. 2016). With advances in

(a)

molecular biological tools in taxonomy, a significant number of other motile aeromonads associated with diseased fish have been recently revealed but still lack experimental evidence for their pathogenicity in the host that the bacteria were recovered (Huys et al. 2005; Beaz-Hidalgo et al. 2009; Janda & Abbott 2010; Soto-Rodriguez et al. 2013). In the present study, the number of dead fish increased steadily (Fig. 2a) and two non-A. hydrophila species (A. jandaei and A. veronii) were subsequently isolated from clinically sick tilapia. This suggests that the batch of initially healthy-looking fish might already carry both of the identified bacterial species and stress during transport and transfer is likely to trigger occurrence of disease and mortality. So far, a few reports indicated that A. jandaei is pathogenic to aquaculture fish such as European eels, Anguilla anguilla (L.) (Esteve, Biosca & Amaro 1993), and striped catfish, Pangasianodon hypophthalmus (Sauvage) (Kumar et al. 2015). By contrast, a relatively higher number of fish hosts such as Chinese longsnout catfish, Leiocassis longirostris (G€ unther) (Cai et al. 2012), loach, Misgurnus anguillicaudatus (Cantor) (Zhu et al. 2016), oscar, Astronotus ocellatus (Agassiz) (Sreedharan, Philip & Singh 2011), and tilapia, Oreochromis sp. (Dong et al. 2015; Eissa et al. 2015; Peepim et al. 2016), have been reported to be infected by A. veronii. The challenge experiments were performed to prove whether the two identified bacterial species could induce disease in healthy fish. Artificial infection using individual bacterial species revealed that these isolates killed Nile tilapia without the same kind of stress inducer and produced severe diseased symptoms even at varied dose levels of infection (Fig. 2b). The findings suggest that both A. veronii and A. jandaei could pose a risk to fish health and should be considered as potential threats in farmed tilapia. Tilapia producers,

(b) Figure 3 Fish infected with Aeromonas jandaei NT-01 (a) and Aeromonas veronii NT-03 (b) exhibited similar internal clinical signs. Moribund fish showed severe haemorrhagic liver (asterisk) and yellow liquid accumulation in the swollen intestine (arrows).

Ó 2017 John Wiley & Sons Ltd

6



Figure 4 Daily cumulative per cent mortality of surviving fish from the primary infection upon secondary infection. Na€ıve control fish and survivors of the primary infection were re-infected with respective bacteria at an indicated dose. There were eight fish in each group, and experiments were monitored for 13 days.

Figure 5 Histological changes of the fish infected with Aeromonas jandaei NT-01 were similar to the fish infected with Aeromonas veronii NT-03. Severe blood congestion occurred in the brain (a, b) and liver (c, d). The spleen exhibited hyperaemia and haemorrhage (e). Fish intestine showed epithelial cell damage and sloughing into gut lumen (f).

(a)

(b)

(c)

(d)

(e)

(f)

therefore, should be aware of the presence of these bacteria in tilapia aquaculture systems, especially when the fish exposed to stressors such as after being transferred from hatchery to rearing ponds or from rearing ponds to cultured cage system. The current work first proved that A. jandaei was pathogenic and caused mortality in Nile tilapia. Additionally, A. veronii was confirmed as an important threat of tilapia as previously reported in our recent studies (Dong et al. 2015; Peepim et al. 2016). Furthermore, this study first described histological changes in experimentally infected fish caused by A. jandaei and A. veronii. Ó 2017 John Wiley & Sons Ltd

7

Severe blood congestion and haemorrhage observed in multiple organs of infected fish as well as enteritis might suggest involvement of bacterial toxins. In this study, three challenge dose levels were preferable than a single challenge dose with replicates. Experimental infection, using individual bacterial isolate, exhibited very acute mortality (Fig. 2). Similarly, acute mortality was also found in the experimental challenge of red tilapia (Oreochromis sp.) with A. veronii in our previous study (Dong et al. 2015). The disease progression and death pattern of artificial infection were much


quicker than those of that occurs naturally. This suggests that A. veronii or A. jandaei alone at low density and no stress inducer may not kill the host. More likely, disease manifestation in fish needs the contribution of stressful factors (i.e. environmental stress, handling stress or synergistic effect of co-infections). In naturally diseased outbreaks, multiple pathogens (F. columnare, S. agalactiae, Plesiomonas shigelloides, Vibrio cholerae and Iridovirus) that accompanied A. veronii in famed tilapia were previously addressed (Dong et al. 2015). This study also found co-infection of A. veronii and A. jandaei was associated with mortality in tilapia but an artificial co-challenge of two bacteria has not yet been investigated. Instead, individual bacterial species with varied doses were used for the challenge experiment, and the results showed that percentage mortality of experimental fish was dose dependent. The isolate A. jandaei NT-01 seems to be more virulent than A. veronii NT-03 because even the dose approximately two times lower still induced higher mortality (Fig. 2b). Interestingly, surviving fish from lowest dose were able to resist second infection while the control group remained their susceptibility. The result suggests that fish exposed to a sublethal dose may be able to trigger an adaptive immune response which saved the fish from secondary infection. Further investigation of the adaptive immune response and bacterial clearance mechanism of the resistant host will be useful for the development of vaccine-based disease prevention. In conclusion, this study reported two nonA. hydrophila species (A. jandaei and A. veronii) associated with disease and mortality of Nile tilapia after exposed to transport-induced stress. The experimental infection without the same kind of stress inducer proved that both species were capable of causing disease and mortality in tilapia and suggested that both A. jandaei and A. veronii could pose a risk to fish heath. Thus, tilapia producers should be aware that mortality from nonA. hydrophila species constitutes a probable component of disease caused by motile aeromonads that may have been previously assumed as A. hydrophila. Acknowledgements H.T. Dong has been supported by the postdoctoral research grant from King Mongkut’s University of Technology Thonburi (KMUTT). The Ó 2017 John Wiley & Sons Ltd

8


authors would like to thank Panadda Meenium for technical assistance.

References Abbott S.L., Cheung W.K. & Janda J.M. (2003) The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. Journal of Clinical Microbiology 41, 2348–2357. Anshary H., Kurniawan R.A., Sriwulan S., Ramli R. & Baxa D.V. (2014) Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia (Oreochromis niloticus) cultured in net cages in Lake Sentani, Papua, Indonesia. Springerplus 3, 627. Assis G.B.N., Tavares G.C., Pereira F.L., Figueiredo H.C.P. & Leal C.G. (2016) Natural coinfection by Streptococcus agalactiae and Francisella noatunensis subsp. orientalis in farmed Nile tilapia (Oreochromis niloticus L.). Journal of Fish Diseases. doi:10.1111/jfd.12493. Austin B. & Austin D.A. (2012) Bacterial Fish Pathogens. Diseases of Farmed and Wild Fish. 5th edn. Springer, Netherlands. Beaz-Hidalgo R., Alperi A., Figueras M.J. & Romalde J.L. (2009) Aeromonas piscicola sp. nov., isolated from diseased fish. Systematic and Applied Microbiology 32, 471–479. Cai S.H., Wu Z.H., Jian J.C., Lu Y.S. & Tang J.F. (2012) Characterization of pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome from Chinese longsnout catfish (Leiocassis longirostris Gunther). Brazilian Journal of Microbiology 43, 382–388. Cipriano R.C., Bullock G.L. & Pyle S.W. (1984) Aeromonas hydrophila and motile aeromonad septicemias of fish. US Fish & Wildlife Publications. Paper 134, http://digitalc ommons.unl.edu/usfwspubs/134. Cutuli M.T., Gibello A., Rodriguez-Bertos A., Blanco M.M., Villarroel M., Giraldo A. & Guarro J. (2015) Skin and subcutaneous mycoses in tilapia (Oreochromis niloticus) caused by Fusarium oxysporum in coinfection with Aeromonas hydrophila. Medical Mycology Case Reports 9, 7–11. Dong H.T., Nguyen V.V., Le H.D., Sangsuriya P., Jitrakorn S., Saksmerprome V., Senapin S. & Rodkhum C. (2015) Naturally concurrent infections of bacterial and viral pathogens in disease outbreaks in cultured Nile tilapia (Oreochromis niloticus) farms. Aquaculture 448, 427–435. Dong H.T., Senapin S., Lafrentz B. & Rodkhum C. (2016) Virulence assay of rhizoid and non-rhizoid morphotypes of Flavobacterium columnare in red tilapia, Oreochromis sp., fry. Journal of Fish Diseases 39, 649–655. Eissa I.A.M., Maather E., Mona S., Desuky E., Mona Z. & Bakry M. (2015) Aeromonas veronii biovar sobria a causative agent of mass mortalities in cultured Nile Tilapia in ElSharkia governorate, Egypt. Life Science Journal 12, 90–97. El-Sayed A-FM (2006) Tilapia Culture. CABI Publishing, Cambridge, MA. Esteve C., Biosca E.G. & Amaro C. (1993) Virulence of Aeromonas-hydrophila and some other bacteria isolated


from European eels Anguilla-anguilla reared in fresh-water. Diseases of Aquatic Organisms 16, 15–20. Esteve C., Valera L., Gutierrez C. & Ventosa A. (2003) Taxonomic study of sucrose-positive Aeromonas jandaei-like isolates from faeces, water and eels: emendation of A. jandaei Carnahan et al. 1992. International Journal of Systemaic Evolutionary Microbiology 53, 1411–1419. Figueiredo H.C., Klesius P.H., Arias C.R., Evans J., Shoemaker C.A., Pereira D.J. Jr & Peixoto M.T. (2005) Isolation and characterization of strains of Flavobacterium columnare from Brazil. Journal of Fish Diseases 28, 199–204. Huys G., Cnockaert M. & Swings J. (2005) Aeromonas culicicola Pidiyar et al. 2002 is a later subjective synonym of Aeromonas veronii Hickman-Brenner et al. 1987. Systematic and Applied Microbiology 28, 604–609. Janda J.M. & Abbott S.L. (2010) The genus Aeromonas: taxonomy, pathogenicity, and infection. Clinical Microbiology Reviews 23, 35–73. Kayansamruaj P., Pirarat N., Katagiri T., Hirono I. & Rodkhum C. (2014) Molecular characterization and virulence gene profiling of pathogenic Streptococcus agalactiae populations from tilapia (Oreochromis sp.) farms in Thailand. Journal of Veterinary Diagnostic Investigation 26, 488–495. Ke X., Lu M., Ye X., Gao F., Zhu H. & Huang Z. (2012) Recovery and pathogenicity analysis of Aerococcus viridans isolated from tilapia (Oreochromis niloticus) cultured in southwest of China. Aquaculture 342–343, 18–23. Kumar K., Prasad K.P., Tripathi G., Raman R.P., Kumar S., Tembhurne M. & Purushothaman C.S. (2015) Isolation, identification, and pathogenicity of a virulent Aeromonas jandaei associated with mortality of farmed Pangasianodon hypophthalmus, in India. Israeli Journal of AquacultureBamidgeh 67, 1–7. Li Y. & Cai S.H. (2011) Identification and pathogenicity of Aeromonas sobria on tail-rot disease in juvenile tilapia Oreochromis niloticus. Current Microbiology 62, 623–627. Lin Q., Li N., Fu X., Hu Q., Chang O., Liu L., Zhang D., Wang G., San G. & Wu S. (2015) An outbreak of granulomatous inflammation associated with Francisella noatunensis subsp. orientalis in farmed tilapia (Oreochromis niloticus 9 O. aureus) in China. Chinese Journal of Oceanology and Limnology 34, 460–466. Nguyen V.V., Dong H.T., Senapin S., Pirarat N. & Rodkhum C. (2016) Francisella noatunensis subsp. orientalis, an emerging bacterial pathogen affecting cultured red tilapia (Oreochromis sp.) in Thailand. Aquaculture Research 47, 3697–3702. Nhung P.H., Hata H., Ohkusu K., Noda M., Shah M.M., Goto K. & Ezaki T. (2007) Use of the novel phylogenetic

Ó 2017 John Wiley & Sons Ltd

9


marker dnaJ and DNA-DNA hybridization to clarify interrelationships within the genus Aeromonas. International Journal of Systematic and Evolutionary Microbiology 57, 1232–1237. Noga E.J. (2010) Fish Disease: Diagnosis and Treatment. 2nd edn. Wiley-Blackwell, Ames, Iowa. Peepim T., Dong H.T., Senapin S., Khunrae P. & Rattanarojpong T. (2016) Epr3 is a conserved immunogenic protein among Aeromonas species and able to induce antibody response in Nile tilapia. Aquaculture 464, 399–409. Senapin S., Dong H.T., Meemetta W., Siriphongphaew A., Charoensapsri W., Santimanawong W., Turner W.A., Rodkhum C., Withyachumnarnkul B. & Vanichviriyakit R. (2016) Hahella chejuensis is the etiological agent of a novel red egg disease in tilapia (Oreochromis spp.) hatcheries in Thailand. Aquaculture 454, 1–7. Soto E., Hawke J.P., Fernandez D. & Morales J.A. (2009) Francisella sp., an emerging pathogen of tilapia, Oreochromis niloticus (L.), in Costa Rica. Journal of Fish Diseases 32, 713–722. Soto-Rodriguez S.A., Cabanillas-Ramos J., Alcaraz U., GomezGil B. & Romalde J.L. (2013) Identification and virulence of Aeromonas dhakensis, Pseudomonas mosselii and Microbacterium paraoxydans isolated from Nile tilapia, Oreochromis niloticus, cultivated in Mexico. Journal of Applied Microbiology 115, 654–662. Sreedharan K., Philip R. & Singh I.S. (2011) Isolation and characterization of virulent Aeromonas veronii from ascitic fluid of oscar Astronotus ocellatus showing signs of infectious dropsy. Diseases of Aquatic Organisms 94, 29–39. Suanyuk N., Kong F., Ko D., Gilbert G.L. & Supamattaya K. (2008) Occurrence of rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp. and Nile tilapia O. niloticus in Thailand—relationship to human isolates? Aquaculture 284, 35–40. Weisburg W.G., Barns S.M., Pelletier D.A. & Lane D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173, 697–703. Zhu M., Wang X.R., Li J., Li G.Y., Liu Z.P. & Mo Z.L. (2016) Identification and virulence properties of Aeromonas veronii bv. sobria isolates causing an ulcerative syndrome of loach Misgurnus anguillicaudatus. Journal of Fish Diseases 39, 777–781. Received: 20 October 2016 Revision received: 4 January 2017 Accepted: 4 January 2017