Int Aquat Res (2018) 10:145–152 https://doi.org/10.1007/s40071-018-0195-4 OR IGINAL RESEARCH Aeromonas salmonicida isolated from wild and farmed ﬁsh and invertebrates in Oman . . Aliya Alghabshi Brian Austin Margaret Crumlish Received: 6 November 2017 / Accepted: 14 May 2018 / Published online: 1 June 2018 The Author(s) 2018 Abstract Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit ﬁsh and tilapia, and an invertebrate (abalone) in Oman during December 2011–May 2012. The cyto- toxic enterotoxin ast gene was found widely distributed among the isolates; aerolysin-like protein (act) and the ﬂagellin structural gene lafA less so; and the nuclease gene (nuc) not at all. However, there was not any evidence of pathogenicity among the isolates when tested in laboratory-based experiments using rainbow trout and Nile tilapia. Therefore, the risk of the pathogen to ﬁsh in Oman is unclear. Keywords Aeromonas salmonicida Fish Invertebrates Aquaculture Oman Introduction Aeromonas salmonicida is the aetiological agent of furunculosis in salmonids and causes ulcer disease in cyprinids and marine ﬂatﬁsh (Austin and Austin 2016). Traditionally, the organism is regarded as an obligate ﬁsh pathogen (Schubert 1974) being only recoverable from clinically diseased ﬁsh. In part, the restricted ecology reﬂected the difﬁculty of recovering viable and culturable cells from ﬁsh in the absence of clinical disease or from environmental samples (Austin and Austin 2016). Pathogenesis of Aeromonas infections may be correlated with stress of the susceptible ﬁsh and the production of cell-associated and extracellular viru- lence determinants (Austin and Austin 2016). Although numerous virulence factors, such as surface polysaccharides (capsule, lipopolysaccharide, and glucan), iron-binding systems, exotoxins and extracellular enzymes, secretion systems, ﬁmbriae and ﬂagella, contribute to pathogenesis of ﬁsh and human diseases caused by Aeromonas spp., none of the factors alone are responsible for all of the clinical signs of disease presented during an infection (Ali et al. 1996). Variations in the distribution of potential virulence genes between Aeromonas isolates may well contribute to their degree of pathogenicity (Albert et al. 2000). Sha et al. (2002) reported the presence and expression of three enterotoxin genes (alt, ast and act genes) in Aeromonas spp. that led to a 100% reduction in ﬂuid secretion in a mouse model. Conversely, Sen and Rodgers (2004) reported that the mere presence of these toxins may not be sufﬁcient for virulence. Therefore, A. Alghabshi (&) Microbiology Section, Fish Quality Control Centre, Ministry of Agriculture and Fisheries Wealth, Muscat, Sultanate of Oman e-mail: firstname.lastname@example.org B. Austin M. Crumlish Institute of Aquaculture, University of Stirling, Stirling, UK 123 146 Int Aquat Res (2018) 10:145–152 there is a need to continuously assess the presence of several accepted virulence factors in Aeromonas isolates for better understanding of the overall pathogenesis of infections (Sen and Rodgers 2004). The present study was undertaken to investigate the pathogenicity of strains of A. salmonicida recovered from a range of aquatic animals in Oman. Materials and methods Bacterial isolates In this study, 9 isolates of A. salmonicida were recovered from skin, gill and kidney of 417 ﬁsh representing 4 wild ﬁsh, including red spot emperor (Lethrinus lentjan), king soldier bream (Argyrops spinifer), white-spotted rabbit ﬁsh (Siganus canaliculatus), abalone (Haliotis mariae) and one farmed tilapia (Oreochromis niloticus). The ﬁsh were collected from 3 areas (Muscat, Mudhaibi and Salalah) considered suitable for aquaculture in Oman based on the Atlas of suitable sites for aquaculture in Oman. The animals were mostly healthy, as judged visually, except for one tilapia that demonstrated abnormal behaviour. The abalone was from a population that had a history of high mortality albeit without any clinical signs of disease. From freshly dead ﬁsh and muscle from the abalone, samples were taken by means of inoculating loops from skin, gill and kidney, and were directly streaked onto the surface of tryptone soya agar plates (TSA; CM0131, Oxoid) with incubation at 28 C for 48 h. From each plate with dense growth, colonies representing the most commonly occurring morphological types were aseptically selected and subcultured for purity. Smears were prepared for the Gram-staining reaction and micro-morphology. Catalase production and the determination of motility followed the methods of Frerichs and Millar (1993) and Martin-Carnahan and TM Joseph (2005), respectively. Oxidase production was determined using oxidase strips (OxiStrips , Oxoid). The API 20E rapid identiﬁcation system (BioMerieux, France) was used following the manufacturer’s instructions, except that the inoculated strips were incubated at 28 C and the results read at 48 h (Crumlish et al. 2002). The demonstration of haemolysin activity was assessed from the presence of complete (b- haemolysis) or incomplete (a-haemolysin) zones of clearing around colonies on 5% sheep blood agar fol- lowing incubation at 28 C for 48 h. The susceptibility to the vibriostatic agent (O/129) (150- and 10-lg discs; Oxoid) was achieved by the disc method of Kirby and Bauer on TSA agar plates (Buller 2004). After 24-h incubation at 28 C, zones of clearing of \ 7 and [ 7 mm were recorded as indicative of resistance and sensitivity, respectively (Whitman 2004). Serology was determined by indirect agglutination reactions using the MONO-As kit (BIONOR AS, Skien, Norway) for A. salmonicida, following the manufacturer’s instruc- tions. This kit consists of antibody-coated latex beads, and is designed for speciﬁc identiﬁcation of all A. salmonicida. Growth at 5, 20, 22, 28, 30 and 37 C and the ability to produce brown diffusible pigment were evaluated on TSA (Austin et al. 1998). Here, inoculated media were examined every 24 h for up to 5 days to determine the presence of colony growth, and up to 14 days to assess for brown diffusible pigment production. For each test performed, a positive control, i.e. A. salmonicida subsp. salmonicida (NCIMB 1102 ), was included. Molecular identiﬁcation Genotyping of the suspected A. salmonicida used 16S rRNA sequencing (Borrell et al. 1997) and rDNA-RFLP (Borrell et al. 1997; Figueras et al. 2000). Species identiﬁcation was conﬁrmed by comparing the DNA sequence obtained in this study with the NCBI GenBank database using BLAST [basic local alignment search tool, standard nucleotide comparison (http://www.ncbi.nlm.nih.gov/BLAST/)] (Altschul et al. 1990). The sequences were aligned using ClustalW for multiple sequence alignment with the DNASTAR computer program and phylogenetic analyses (maximum likelihood method) were conducted by the MEGA software version 6.0 (Tamura et al. 2013) to provide a phylogenetic tree. Standard errors were obtained with 1000 bootstrap replicates. 123 Int Aquat Res (2018) 10:145–152 147 Preparation of extracellular products (ECP) ECP was prepared using the cellophane overlay method of Gudmundsdottir (1996). The bacteria were grown in tryptone soya broth (TSB; Oxoid) for 24 h at 28 C, and centrifuged at 10,0009g at 4 C for 30 min. The supernatant containing the ECP fractions was sterilized by ﬁltration through 0.22-lm ﬁlters (Millipore). To conﬁrm the absence of bacterial colonies, 0.2 ml volume of the ﬁltered supernatants was streaked over TSA plates and incubated for 48 h at 28 C. The protein concentration of the ECP was determined by the method of Bradford (1976) using a protein determination kit (Bio-Rad, USA) according to the manufacturer’s instruc- tions with bovine serum albumin (BSA; Sigma-Aldrich, UK) as the standard. The ECP extractions were then subsequently stored at - 20 C until required. Determination of putative virulence characteristics Bacteria from 24-h-old cultures on TSA and ﬁltered supernatants obtained from the ECP preparations were used to assess protease activity (Gudmundsdo ´ ttir 1996), haemolytic activity (Brender and Janda 1987), DNase activity (Buller 2004), Congo red dye uptake (Crump and Kay 2008) and the production of A-layer as visualized on Coomassie brilliant blue agar (CBB; Cipriano and Bertolini 1988). Positivity for A-layer was indicated by the presence of dark-red colonies on Congo red agar and dark-blue colonies on CBB agar, whereas negativity was indicated by the presence of pale colonies on these media. Detection of virulence genes The presence of genes encoding the virulence factors aerolysin (aer) (Pollard et al. 1990), aerolysin-like proteins (act) (Sen and Rodgers 2004), cytotoxic enterotoxins (ast, alt) (Aguilera-Arreola et al. 2005), glycerophos- pholipid cholesterol acyltransferase (gcat) (In-Young and Kiseong 2007), structural gene ﬂagellin (lafA, lafB) (Aguilera-Arreola et al. 2005) (In-Young and Kiseong 2007) and serine protease (In-Young and Kiseong 2007) was determined by use of the polymerase chain reaction (PCR) using primers and conditions already published. Determination of pathogenicity Cells from 24-h cultures in TSB were harvested by centrifugation, washed in 0.85% (w/v) saline and the optical density (OD ) was adjusted to 1. Viable colony counts were performed following the Miles and Misra (1938) method and tenfold serial dilutions prepared for challenge studies in tilapia and rainbow trout. The ﬁrst challenge was performed in the Fishery Quality Control Centre in Oman using farmed tilapia stocks of 30 g average weight. The ﬁsh were fed with commercial pelleted ﬁsh diet for the 5-day period of each experiment with quarantine for 14 days prior to administration of 0.1 ml volume of bacterial suspension 8 -1 containing l0 cells ﬁsh by intraperitoneal (i.p) and intramuscular (i.m) injections. Control ﬁsh received 0.1 ml volume of sterile 0.85% (w/v) saline by i.p. and i.m. injections. A second experiment was performed at the University of Stirling using the same concentration of bacterial cells in an in-house tilapia population of 30 g average weight and farmed rainbow trout of 50 g average weight. The ﬁsh at Stirling were health checked prior to use, where tissue samples were aseptically taken from a subsamples of the group and ﬁxed in 10% neutral buffered formalin before processing into wax-embedded tissue sections. These were trimmed and 5-lm sections cut, stained with haemotoxylin and eosin and viewed for health check prior to any experi- mentation. Additional ﬁsh experiments sought to determine the effect of ECPs, which were injected i.m. and -1 i.p. in 0.1 ml volume (0.1 mg of protein ml ). Control ﬁsh received 0.1 ml volume of sterile 0.85% (w/v) saline. All ﬁsh were maintained in freshwater at 26 ± 2 and 15 ± 1 C for tilapia and rainbow trout, respectively, and examined daily for 5 days. Any dead or clinically diseased ﬁsh were sampled microbio- logically in which loopfuls of samples from kidney and spleen were streaked onto TSA plates with incubation at 28 C for 24 h, and the resulting colonies identiﬁed as previously described. Also, spleen and kidney tissues were ﬁxed in 10% (v/v) neutral buffered formalin for 24 h before embedding in parafﬁn following routine tissue processing. Thus, 5-lm-thick sections were cut and stained with haematoxylin and eosin (H&E) (Oliveira Ribeiro et al. 1981) and Gram stained for histological examination. All slides were examined at a magniﬁcation of 4009 using a Carl Zeiss microscope. 123 148 Int Aquat Res (2018) 10:145–152 Results and discussion Nine isolates of A. salmonicida were recovered and identiﬁed from skin, gill and kidney from four ﬁsh species, i.e. red spot emperor, king soldier bream, white-spotted rabbit ﬁsh, tilapia and one of abalone, all of which are considered as commercially important for aquaculture development in Oman. These isolates were all tentatively equated with A. salmonicida (Martin-Carnahan and Joseph 2005), insofar as they produced smooth convex colonies on TSA, and comprised Gram-negative rod-shaped bacteria that grew at 5–30 C, produced catalase, b-galactosidase, gelatinase, indole and oxidase, but not tryptophan deaminase or urease, fermented glucose, mannose, rhamnose, melibiose and sucrose but not inositol, they did not utilize sodium citrate and were positive for the Voges–Proskauer reaction. All biochemical test results using the API 20E rapid identiﬁcation kit were in agreement with those of the NCIMB reference strain (Table 1). Unlike descriptions of virulent A. salmonicida subsp. salmonicida as recovered from furunculosis in salmonids (McCarthy and Roberts 1980), the Omani strains did not produce brown diffusible pigment around the colonies on TSA, and so the isolates correspond to the description of ‘atypical’ A. salmonicida (Austin et al. 1998; Martin-Carnahan and Joseph 2005). Sequencing of the 16S rRNA gene revealed that three isolates, 291MS, 293MS and 295MS, had the highest (99%) similarity with A. salmonicida spp., sequences deposited in GenBank, whereas isolates 373MG and 388MS demonstrated 98% homology, and 395M, 340M, 26 MS2 and 16MG revealed 95–92% identity levels, respectively (Fig. 1). It is emphasized that reliance only on sequence analysis of the 16S rRNA gene to identify A. salmonicida has limited value, although the technique is useful for conﬁrmation of membership of the genus Aeromonas (Figueras et al. 2011; Han et al. 2011). Extracellular nuclease and haemolysin activities are considered to be virulence-associated factors belonging to many Aeromonas species (Gonza ´lez-Rodrı ´guez et al. 2002; Rahman et al. 2002). Haemolytic activity was present in 67% of the isolates, whereas extracellular protease occurred in 44% of the cultures from Oman. Only a minority of the isolates demonstrated uptake of Congo red (22%) and CBB (33%). Table 1 Differential phenotypic characteristics of the isolates Phenotypic characteristics Isolate no. A. 16MG 26MS2 291MS 293MS 295MS 340M 373MG 388MS 395M salmonicida Colonial colour on TSA N B BBBBA B B B Motility V - ????- ? ? ? Pigment production V - ----- - - - Autoagglutination in saline V - -?--- - - - Growth at: 5 CV - -???- - ? - 37 CV ? ????? ? ? ? Arginine dihydrolase V ? ????? ? ? ? Lysine decarboxylase V ? ??--? ? ? ? Ornithine decarboxylase V - ?---- ? - - H S production V - ----- - - - Fermentation of sorbitol V ? -?--? ? -- - Fermentation of rhamnose _ ? ----? - - - Fermentation of melibiose _ ? ----? - - - Fermentation of amygdalin V ? -??-? ? ? ? Fermentation of arabinose V ? -???? ? - - Susceptibility to the R/R R/R S/S R/R R/R R/R S/R S/R S/S R/R vibriostatic agent O/129:150 lg/10 lg A, light creamy colonies; B, yellowish colonies; ?, positive; -, negative; V, variable result; N, no data; R, resistant; S, sensitive Data are from the following references: Abbott et al. (1992), Austin et al. (1989), Carnahan and Altwegg (1996), Huys et al. (1996), Grifﬁths et al. (1953), Schubert (1974), McCarthy and Roberts (1980), Pavan et al. (2000) and Yamada et al. (2000) 123 Int Aquat Res (2018) 10:145–152 149 Fig. 1 The phylogenetic tree based on 16S rRNA fragment sequences, showing relationship of the A. salmonicida cultures (constructed by maximum likelihood method using MEGA6 software); scale bar 0.01 represents sequence divergence Haemolytic and proteolytic activities were not seen in any of the ECPs. The distribution of the putative virulence genes has been presented in Table 2. Thus, the ast gene was distributed among 67% of the isolates; slightly less (56%) contained aerolysin-like proteins (act). The genes for lafA (33% of isolates) were less common. Also, aerA, alt, gcat and ser were present in only 22% of the cultures, lafB occurred in a single isolate; and nuc not at all. In this study, mortalities were not recorded in any of the challenge experiments. Indeed, pathological changes were not observed in any of the tissues examined by histology (Fig. 2), although i.p. injection of isolate 340M in rainbow trout led to the development of pale liver and darkened kidneys. Moreover, only ﬁsh injected with 16MG and 340M contained culturable cells at the end of the experiments. The discrepancy in the presence of aerolysin (aerA) and aerolysin-like protein (act) genes among the Omani cultures suggested that the isolates may possess but not express these genes (Wang et al. 2003) under the situations described. Enterotoxin genes alt, ast and act were not expressed in any of the isolates. Two cultures harboured both act and ast genes, which is unusual as this combination has been only rarely reported among environmental isolates (Albert et al. 2000; Chang et al. 2008). Yet, pathogenicity was not recorded among these isolates. Some studies reported a correlation between the higher number of virulence genes harboured in Aeromonas spp. and their potential for causing disease (Albert et al. 2000; Chang et al. 2008). These workers mentioned that the number of isolates positive for both the alt and ast genes was signiﬁcantly higher in children with diarrhoea than for healthy controls. In this study, there was not any such correlation. An explanation could be that the experimental conditions used in this study inﬂuenced the expression of the genes involved in pathogenicity. Also, the level of virulence has inevitably been correlated with the amount of enzymes and toxins produced (Kozin ´ ska 1996). Another possibility is that their presence in A. salmonicida does not infer that disease is inevitable reﬂecting the susceptibility of the host, immune state and actual number of bacterial cells in and around the host (Ottaviani et al. 2011). Notwithstanding, some isolates did lead to the development of small haemorrhages in/on the internal organs, as reported previously (Austin and Adams 1996). In this respect, it is worthwhile to consider the comments of Austin and Austin (1993) and Austin (2011), who considered that loss of virulence might well reﬂect the effects of storage, i.e. the transition to what are effectively laboratory cultures, and the inability to replicate conditions of the initial disease, which led to the recovery of the cultures. The recovery of A. salmonicida from Omani ﬁsh and abalone in the absence of clinical signs of disease contradicts the commonly held view that the organism is an obligate ﬁsh pathogen. However, this may reﬂect that scientists have focused on recovery only from diseased ﬁsh, namely salmonids, cyprinids and marine ﬂatﬁsh, rather than other groups of aquatic animals and environmental samples. All the isolates recovered in this study had similar morphologies and lacked diffusible brown pigment production. Also, there was not any direct relationship found between pathogenicity and the presence of putative virulence factors. Again, it is questionable whether this reﬂects the loss of activity during storage. Clearly, further research is needed to extend the knowledge of this group of organisms, particularly in an emerging aquaculture industry. 123 150 Int Aquat Res (2018) 10:145–152 Table 2 Results of infectivity studies, and presence of different virulence factors expression among the isolates Isolates Recovered Location Clinical signs Genetic method Phenotypic method no. from Areo- ACT AST ALT GCAT SER NUC LafB LafA Haemolysin Congo Protease DNase gene red/CBB 16MG Tilapia Mudhaibi Weakness, swimming on one -- 1 -- - - -- c - 1 - side 26MS2 Tilapia Mudhaibi NC - 11 -- - - --? b - 11 291MS White-spotted Muscat NC - 1 -- 11 - 11 ? b -- 1 rabbit ﬁsh 293MS White-spotted Muscat NC -- 1 -- - - -- c -- 1 rabbit ﬁsh 295MG King soldier Muscat NC 11 1 - 11 -- 1 a -- 1 bream 340M Abalone Salalah History of high mortality with no -- 1 -- - - - 1 c -- - clear clinical sign 373MG Red spot Muscat NC 1 - 1 -- - - --? b -- - emperor 388MS Tilapia Mudhaibi NC - 1 - 1 - - - --? b - 11 395M Abalone Salalah NC - 1 - 1 - - - --? b - 11 M, muscle; MG, mucus of gill; NC, no clinical signs of disease; a, a-haemolysis; b, b-haemolysis; c, c-haemolysis; ?, presence; -, absence Int Aquat Res (2018) 10:145–152 151 Fig. 2 Light microscopic appearance of the (a) spleen and (b) kidney of tilapia injected with extracellular products (ECP) of A. salmonicida isolates (H&E, scale bar 20 lm) Acknowledgements The authors acknowledge ﬁnancial support from the Agricultural and Fishery Development Fund-1/3/30) AFDF), Sultanate of Oman. 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