Identification of twelve Aeromonas spp. species with monoclonal antibody-based immunoassay in water and fish samples

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Identification of twelve Aeromonas spp. species with monoclonal antibodybased immunoassay in water and fish samples Wenbin Wanga,b,c,∗, Dandan Liua,b,c, Yunshan Gaoa,b,c, Yunong Sanga,b,c, Xiaxia Lianga,b,c, Jianxin Liua,b,c, Jinyan Shid, Lei Guoa,b,c, Saikun Pana,b,c a

Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, Jiangsu, PR China Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, Jiangsu, PR China c Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, Jiangsu, PR China d Laboratory of Tuberculosis, The Fourth People's Hospital of Lianyungang, Lianyungang, Jiangsu, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Hemorrhagic septicemia Aeromonas Monoclonal antibody Detection Immunoassay

Aeromonas spp. are the causative agents of motile Aeromonas septicemia (MAS) in fish, as well as gastroenteritis and infections in humans. The antigen diversity between Aeromonas spp. and serotypes greatly challenges the identification of this zoonotic pathogen in environmental, food, and clinical samples. In the present study, six conserved peptides in the outer membrane of Aeromonas were selected and used as immunogens after conjugation to produce monoclonal antibodies (mAbs). Peptides 1 and 4 with the amino sequences of “VYDKDGTTFD” and “GGFKGKLSYQTND”, effectively elicited cross-reactive antibodies against Aeromonas in mice. Based on the mAbs 1B1 and 14H5, the developed sandwich immunoassay detected twelve Aeromonas spp. species, including A. hydrophila (8/8), A. veronii (7/7), A. caviae, A. sobria, A. dhakensis, A. media, A. popoffii, A. punctata subsp. caviae, A. bivalvium, A. lacus, A. jandaei, A. bestiarum and A. salmonicida (1/2). The detection limit of these strains primarily ranged from 1.69 × 104 to 4.57 × 105 CFU mL−1. No cross-reactivity was observed with the other tested strains. The sensitivity and specificity of this method was 96% (26/27) and 100% (15/15), respectively. Furthermore, real sample analyses detected river water samples containing 105 CFU mL−1 of A. hydrophila or A. caviae, and detected Carassius auratus samples showing hemorrhagic septicemia after enrichment in buffered peptone water. The established immunoassay may be promising as an effective screening method for monitoring Aeromonas spp. in environmental and food samples.

1. Introduction Aeromonas spp. is the main causative agent of motile Aeromonas septicemia (MAS) in fish, which is characterized by hyperemia on the body and an inflated abdomen (Longyant et al., 2010). Aeromonads are primarily prevalent in freshwater fish species and occasionally in marine fish species. Other aquatic animals, including prawn, crabs, mussels, eel, and frogs are also susceptible (Vivekanandhan et al., 2005; Yano et al., 2015). The mechanisms of septicemia in fish are complex and related to multiple factors, such as the synergistic effects of other bacteria, deterioration of the living environment, and virulence factors (e.g., aerolysin or hemolysin of Aeromonas spp.) (Beazhidalgo and Figueras, 2013; Pang et al., 2015; Ran et al., 2018). Aeromonas. hydrophila is believed to primarily cause MAS in aquatic animals (Nielsen et al., 2001; Vivekanandhan et al., 2005; Zhang et al., 2014). Some epidemiological investigations, however, have found that other species

∗

of Aeromonas spp. (e.g., A. veronii, A. sobria, A. caviae, and A. salmonicida) also lead to infections (Abd-El-Malek, 2017; Bin Kingombe et al., 2010; Coscelli et al., 2017; Saleh et al., 2011). A recent study by Ran et al., in 2018 revealed that A. veronii rather than A. hydrophila accounted for cyprinid fish septicemia outbreaks in four southern Chinese provinces from 2009 to 2014 (Ran et al., 2018). More importantly, the transmission of Aeromonas spp. from environment to human through water and aquatic food lead to increasing infections in recent years, including acute diarrhea, as well as skin and soft tissue infection (Batra et al., 2016; Senderovich et al., 2012; Soltan et al., 2016). Currently, Aeromonas spp. strains have been increasingly highlighted as foodborne pathogens. The predominant strains in the clinical samples are A. hydrophila, A. veronii, and A. caviae, as well as the newly isolated A. dhakensis (Chen et al., 2016; Teunis and Figueras, 2016). Besides, like other Gram-negative bacteria, Aeromonas spp. has a broad diversity of O antigen (44 serogroups according to Sakazaki and

Corresponding author. Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, Jiangsu, PR China. E-mail address: [email protected] (W. Wang).

https://doi.org/10.1016/j.aquaculture.2019.734646 Received 30 January 2019; Received in revised form 22 October 2019; Accepted 26 October 2019 Available online 02 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Wenbin Wang, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734646

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Hospital of Lianyungang.

Shimada schedule) (Shimada and Kosako, 1991). The prevalent serogroups, including O:11, O:16, O:34, O:19, typically vary regarding the area and host (fish or human) (Esteve et al., 1994; Sendra et al., 1997). The versatile nature of different species among Aeromonas spp. has urged and challenged the identification of this pathogen at the genus level in environmental, food, and clinical samples. Although traditional culture-based methods remain the gold standard of Aeromonas spp. detection, it takes at least three days, is a timeconsuming process, and is labor-intensive. Moreover, the molecular biology-based detection methods (e.g., polymerase chain reaction [PCR]) rely on thoroughly examined primers and target gene, which are very limited for Aeromonas spp. to our knowledge (Arora et al., 2006). Previous PCR methods were mainly used to identify the virulent gene of Aeromonas spp. (Bin Kingombe et al., 2010; Xiong et al., 2019). Also, they rely on sophisticated instruments and professional staff (Chu and Lu, 2005; Tacão et al., 2005). Alternatively, immunoassays based on a generic hapten design and cross-reactive antibodies are simple, rapid, and accurate for both pathogen and antibiotic detection (Kong et al., 2017; Scharinger et al., 2016; Wang et al., 2017a; Wang et al, 2016). Aeromonas antibodies in the previous study were mainly produced by whole-cell antigens and the specificity was typically limited due to the broad antigen diversity of Aeromonas spp. (Longyant et al., 2010; Saleh et al., 2011; Sendra et al., 1997). It has been reported that most of the previous immunoassays for Aeromonas spp. have a high detection limit and low specificity and are generally not reliable (Batra et al., 2016). An effective immunoassay for the accurate and sensitive detection of Aeromonas spp. is urgently required for environmental, food, and clinical analysis. The outer membrane protein (Omp)F of A. hydrophila, similar to the OmpF of E. coli, is an important osmoporin on the outer membrane and help to transport nutrients and adapt to osmotic pressure (Amit et al., 2010; Mahendran et al., 2010). Recent studies by Yadav et al., in 2018 have shown that OmpF shares high homology among the Aeromonas genus and is promising for the development of an Aeromonas vaccine for fish (Yadav et al., 2014; Yadav et al., 2018). Thus, it appears possible that OmpF is a useful immune target to prepare genus-specific diagnostic antibodies. Previous studies, however, have demonstrated that this protein is exclusively expressed in inclusion bodies (Yadav et al., 2014; Yadav et al., 2018), which has also been confirmed by the results in our laboratory (Fig. S1). The inclusion bodies differed from the native protein in both structure and antigenicity and the renaturation was unsatisfactory. Compared with the protein antigen, a deliberately selected peptide antigen can precisely control the antibody specificity and is relatively easy to synthesize (Ali and Islam, 2015; Patro et al., 2016; Wang et al., 2017b). The present study aimed to select conserved peptides of OmpF with a high surface probability using bioinformatics tools, produce mAbs with artificial peptide immunogens, and develop a sandwich immunoassay for the rapid and accurate identification of Aeromonas spp. in environmental and aquatic food samples.

2.2. Instruments and reagents Bovine serum albumin (BSA), 3,3′,5,5′-tetramethylbenzidine (TMB) Liquid Substrate System, horseradish peroxidase (HRP), as well as complete and incomplete Freund's adjuvant were purchased from Sigma-Aldrich (St. Louis, MO, USA). We acquired 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC sodium) from J&K Chemicals (Shanghai, China). Peroxidase-conjugated goat anti-mouse IgG was obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA). A mouse monoclonal antibody isotyping kit was provided by Proteintech Co., Ltd (Wuhan, China). Skimmed milk powder was purchased from Becton, Dickinson and Company (Lake Franklin, NJ, USA). All other chemicals were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). BALB/c mice were provided by the College of Veterinary Medicine, Yangzhou University. A 96-well microplate was obtained from Wuxi GuoSheng Bio-Engineering Co., Ltd. (WuXi, China). Millipore-Amicon (cut-off MW, 3000), was provided by Merck (Darmstadt, Germany). A Multiskan FC microplate reader was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene fluoride (PVDF) membranes were obtained from GE Healthcare (Fairfield, CT, USA). 2.3. Peptide selection and immunogen synthesis The amino acid sequence of the outer membrane protein F from A. hydrophila (GI: 415432849) was obtained from the protein database of the National Center for Biotechnology Information. The level of sequence homology with other A. hydrophila, Aeromonas spp. strains and other common strains were assessed with the Basic Local Alignment Search Tool (BLAST). The peptides that were conserved and specific to Aeromonas spp. were sorted and subjected to analysis using bioinformatics software. After comprehensive comparison, candidate peptides were selected and synthesized by GL Biochem (Shanghai) Ltd. N-terminal cysteine-modified peptides were conjugated with BSA by the bifunctional linker Sulfo-SMCC sodium as described (Wang et al, 2016). Briefly, 8 mg carrier protein BSA in 0.1 M phosphate-buffered saline (PBS, pH 7.2) was conjugated with 3.6 mg Sulfo-SMCC by constant stirring with a magnet for 1 h at room temperature. The excess linker was separated and discarded using three rounds of ultrafiltration. The SMCC-coupled BSA was further reacted with 8 mg of the candidate peptide in 0.1 M PBS by stirring with a magnet for 2–6 h at room temperature. The conjugate was dialyzed with 0.01 M PBS and characterized by denatured protein electrophoresis. The concentration of the stacking and separating gels was 5% and 10%, respectively. 2.4. Immunization and mAb preparation The peptide and BSA conjugates were emulsified with Freund's adjuvant using an equivalent volume and used as an immunogen. BALB/c mice aged 6–8 weeks old were subcutaneously injected with 80 μg, 80 μg, or 40 μg of peptide-BSA conjugate at three-week intervals. One week after the second and third immunization, an indirect ELISA was used to assess the titer and cross-reactivity against Aeromonas spp. The coating concentration of each bacteria was 108 CFU mL−1 in carbonate buffer (pH 9.6). The mouse with the broadest cross-reactivity and highest specificity against Aeromonas spp. was sacrificed. The spleen cells were fused with Sp2/0 myeloma cells as described by Kohler and Milstein with modification (Köhler et al., 1976). The positive cells were first selected against A. hydrophila (CICC 10500) and its cross-reactivity with Aeromonas spp. strains and E. coli were evaluated by an indirect ELISA. The cell lines with broad cross-reactivity and specificity for Aeromonas spp. were subcloned three times and preserved in liquid nitrogen. BALB/c mice pre-injected with paraffin oil were injected with

2. Materials and methods 2.1. Bacterial and culture conditions All the bacterial strains used in this study are listed in Table 1. The bacteria from the genus Aeromonas were cultured overnight at 28 °C in nutrient broth (1% peptone, 0.3% beef extract, with 0.5% sodium chloride and 0.1% glucose, Beijing Land Bridge Technology CO., LTD.) whereas A. salmonicida and A. bestiarum required an additional 2% of sodium chloride. V. parahaemolyticus and V. vulnificus were cultured overnight at 28 °C in nutrient broth supplemented with 3% sodium chloride. Other strains were cultured overnight at 37 °C in LB (1% tryptone, 0.5% yeast extract, 1% sodium chloride). The isolated strain of Mycobacterium tuberculosis was autoclaved and provided by doctor Jinyan Shi from the laboratory of tuberculosis in the Fourth People's 2

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Table 1 Bacteria strains used in this study. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Bacterial species Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas veronii Aeromonas dhakensis Aeromonas caviae Aeromonas punctata subsp. caviae Aeromonas popoffii Aeromonas sobria Aeromonas bivalvium Aeromonas media Aeromonas lacus Aeromonas bestiarum Aeromonas jandaei Aeromonas salmonicida Aeromonas salmonicida g Proteus vulgaris Proteus vulgaris Proteus mirabilis Psychrobacter sp. Edwardsiella tarda Pseudoalteromonas sp. Vibrio cholerae Vibrio anguillarum Vibrio harveyi Vibrio parahaemolyticus Vibrio vulnificus Escherichia coli DH5α E. coli O157:H7 Streptococcus mutans Mycobacterium Tuberculosis

Strain number a

CICC 10500 CICC 10868 MCCC b 1A00007 MCCC 1A00190 NAU e ML-19 NAU ML-21 NAU JH-18 NAU JH-79 MCCC 1K03237 MCCC 1A00180 NAU- XH-19 NAU XH-53 NAU XX-10 NAU XX-12 NAU XX-17 MCCC 1A12235 MCCC 1A12231 CGMCC c 1.1960 CGMCC 1.9063 NAU XH-21 MCCC 1A02126 MCCC 1A12437 MCCC 1K03235 CICC 23940 ATCCd 49568 JOU isolated D5-7 CICC 23565 NAU 11 NAU 9 NAU 5 NAU 7 JOU f isolated JOU f isolated JOU f isolated JOU f isolated JOU f isolated CICC 21619 CGMCC 1.8674 CGMCC 1.12873 CICC 10907 CICC 10438 Isolated strains

Source and origin

Detection by ELISA

Unknown Unknown Unknown Unknown Kidney of Bream Liver of Bream Liver of Bream Gill of Bream Unknown Unknown Carassius auratus Gill of Black carp Gill of Chub Gill of Chub Water of fishpond Unknown Unknown Guinea pigs Algal water Water of fishpond Unknown Unknown Unknown Soil of Tarim river feces sample Sea water Unknown unknown unknown unknown unknown Sea water Sea water Sea water Sea water Sea water Unknown Unknown Unknown Unknown Unknown Tuberculosis patients

+++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + + +++ – – – – – – – – – – – – – – – –

+++, Strong positive result; ++, Moderate positive result; +, Weak positive result. -, Negative result. a China Center of Industrial Culture Collection (CICC). b Marine Culture Collection of China (MCCC). c China General Microbiological Culture Collection Center (CGMCC). d American type culture collection (ATCC). e College of Veterinary Medicine, Nanjing Agricultural University (NAU). f College of Marine Life and Fisheries, Jiangsu Ocean University (JOU). g Aeromonas Sp. from CICC and was further classified as A. salmonicida by 16SRNA sequencing.

37 °C for 2 h. The Aeromonas spp. mAbs, 1B1, and 14H5, were diluted 1:4000 in antibody dilution buffer, separately incubated with the PVDF membrane, and allowed to react overnight at 4 °C. After washing, the HRP-conjugated goat anti-mouse antibody diluted 1:4000 in antibody dilution buffer was added and incubated at 37 °C for 1 h. The PVDF membrane was washed and TMB liquid substrate was added. After reacting at room temperature for 10 min, the membrane was imaged. The affinities (Ka) of these mAbs against A. hydrophila (CICC 10500) were determined using an indirect ELISA. Briefly, the microplate was coated with 108 CFU mL−1, 5✕107 CFU mL−1 and 2.5✕107 CFU mL−1 of bacteria cells in Carbonate-Bicarbonate (CB) buffer and blocked with 0.2% gelatin in CB buffer. mAb was diluted from 1000 times with a 3fold gradient in antibody dilution buffer. The diluted antibody was added to the microplate (100 μL/well) and allowed to react for 30 min at 37 °C, the following steps were the same with conventional indirect ELISA. The Sigmoid curves of antibody concentration and absorbance at 450 nm were obtained by Origin 8.5. The antibody concentrations corresponding to half of the maximum absorbance of each

the selected hybridoma cells to produce ascites containing mAbs. Ascites were then collected and purified using the octanoic acid and saturated ammonium sulfate method. 2.5. Antibody specificity, affinity, subtypes, and pairwise study The subtypes of the obtained mAbs were identified with an antibody isotyping kit following the manufacturer's instructions. The specificity of the mAbs was assessed with an indirect ELISA and Western blot (WB). The recombinant OmpF protein of A. hydrophila was expressed in E. coli BL 21 and purified in our laboratory. The indirect ELISA was conducted as described in 2.4, except the mAbs were serially diluted from 1000 times dilution with a three-fold gradient. The bacterial proteins were prepared by the ultrasonic disruption in ice water before performing the WB. The bacterial proteins were separated on a polyacrylamide gel and transferred to a PVDF membrane with a TE 70 semi-dry transfer unit at 30 mA for 40 min. The PVDF membrane was then blocked in 5% skimmed milk in 0.01 M PBS at 3

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and after diluting them three times to determine if there was a matrix effect. Carassius auratus with an average weight of 150 g were purchased from the local market and divided into three groups containing 10 fish per group. After a temporary culture for one week, the two groups of the fish were intraperitoneally injected with 107 CFU of live A. hydrophila (CICC 10500) and A. veronii (MCCC 1K03237), respectively. The final group was not injected and used as a negative control. Following the injection, the fishes showing symptoms (e.g., hyperemia on the body, inflated abdomen, and anal swelling) were picked up and dissected. Samples of ascites and internal organs were obtained and transferred to buffered peptone water (BPW) with inoculation needle under sterile operation. After enriching for 12 h, and 24 h, the samples were boiled for 10 min and analyzed. Moreover, the spleen and kidney were harvested under sterile conditions. The specimens were mixed with 2 mL of PBST and individually homogenized using a mortar. Then the liquid samples were stored at 4 °C for 20 min and the supernatants were collected and boiled for 10 min before being tested directly or after diluting 1:3 or 1:9 in PBS.

curve were recorded as [Ab]t and [Ab’]t. Then each two antibody concentrations were used to calculate the antibody affinity (Ka). n−1 Ka = 2 × n ([Ab′] t − [Ab] t ) , n was the ratio of the coating concentration for the two curves. Three Ka values were obtained and the mean value represented the affinity of mAb. Purified mAbs were conjugated with HRP via the sodium periodate method as previously described (Kuang et al., 2013). The conjugates were examined using a direct ELISA with A. hydrophila as the coating antigen. Both mAbs and HRP-conjugated mAbs were subjected to a pairwise analysis with a sandwich ELISA. Briefly, each mAbs was coated on one column of the microplate. After blocking, standard and negative control were respectively added to the neighboring row of the plate and allowed to react for 1 h at 37 °C. After washing, each HPR labeled mAb was added to two neighboring rows of the plate to be paired with each coated mAb. Then the plate was allowed to react for 1 h at 37 °C. After washing, coloring and termination steps, the absorbance of the plate was measured by a microplate reader. The ratios of absorbance from positive control and negative control (P/ N) of each antibody pair were obtained. The concentration of the coating and HRP-conjugated antibodies was 4 μg/mL and 2 μg/mL, respectively. Aeromonas. hydrophila (MCCC 1A0007) was denatured by boiling for 15 min and used as the standard (107 CFU.mL-1). 10 mM PBS was used as the negative control.

3. Results and discussion 3.1. Peptide analysis and immunogen characterization

2.6. Bioinformatic analysis of the two protein epitopes

The positive pairs that produced a high P/N ratio (> 20) were further compared by testing more Aeromonas spp. standard strains at 107 CFU mL−1 with a sandwich ELISA. Pairs with balanced detection limit were thoroughly evaluated. The detection limit and cross-reactivity against standard and isolated strains of A. hydrophila, Aeromonas spp., and other non-Aeromonas bacteria listed in Table 1 were studied. The bacterial were denatured by boiling and serially diluted from 109 CFU mL−1 to 5645 CFU mL−1 in 0.01 M PBS with a three-fold gradient and tested by the sandwich ELISA. The antibody pair with the broadest cross-reactivity, highest detection limit against Aeromonas spp., and the lowest reactivity with the other non-Aeromonas bacteria was used to establish the sandwich ELISA for Aeromonas spp.

As a common porin protein, OmpF together with OmpC are regulated by EnvZ-OmpR, an important two-component system of Gramnegative bacteria used to adapt to osmolarity pressure changes in the environment (Maffeo et al., 2012; Masi and Pagã¨S, 2013). A BLAST search of the amino acid sequence of OmpF from A. hydrophila (GI: 415432849) with Aeromonas spp. and other common bacteria (e.g., E. coli and Salmonella) revealed that this protein has more than 70% homology among the Aeromonas genus (Fig. 1A) and less than 30% homology with E. coli and Salmonella. The gray or discontinuous areas are sequences that share low homology and the red area denotes a higher homology. It should be noted that two-to-four amino acid displacement was not shown. After sequence comparisons, 15 peptides with a homology of more than 90% in Aeromonas spp. were obtained. Further bioinformatic predictions of these peptides revealed that six of the 15 peptides had high hydrophilicity, antigen index, and surface probability, which indicated that these peptides were both conserved, accessible, and antigenic. Thus, they were selected as the candidate peptides for immunogen preparation. The position of the six candidate peptides was also marked (Fig. 1B). The amino acid (AA) sequences and locations of the six candidate peptides are listed in Table 2. The sequences are all short peptides ranging from 10 to 13 AA with a molecular weight of 1,263 to 1,694 Da. To conjugate with the carrier protein, the peptides were all Cys modified on the N-terminus. As shown in Fig. 2, the BSA protein band moved to a higher position after reacting with Sulfo-SMCC (lane 2) and was further increased after reacting with the peptides (lanes 3–8), which indicated the successful conjugation of each step. The diffusion bands of the product were caused by the inhomogeneous and random reaction of SMCC and peptides with the carrier protein.

2.8. Aquatic and fish sample detection

3.2. Serum titer and specificity of the six peptide immunogens

The river water was collected to prepare artificial Aeromonas-contaminated water samples. First, 200 mL of the water sample was filtered through a membrane with 0.45 μm pores to remove the influence of the possible presence of Aeromonas. Secondly, the river water was mixed with E. coli and V. vulnificus at a concentration of 108 CFU mL−1. Then, the freshly cultured A. hydrophila (CICC 10500) and A. caviae (MCCC 1A12231) were added to the river water with concentrations of 107, 106, and 105 CFU mL−1, respectively. Both the positive and control samples were boiled for 10 min and subjected to the immunoassay after they had cooled down. The water samples were analyzed both directly

After the third immunization with the six peptide antigens, the serum (3 × diluted) of the mice immunized with P1-BSA and P4-BSA conjugates generally exhibited stronger binding with A. hydrophila (CICC 10500) cells (Fig. 3A). Besides, although P2-BSA also elicited antibodies that reacted with the cell body, the absorbance decreased significantly with further dilution. One of the six mice immunized with P3-BSA produced antibodies binding with A. hydrophila cells, which was largely consistent with the previous finding that the 66–80 AA of OmpF with the fusion protein elicited cross-reactive antibodies against Aeromonas spp. (Sharma and Dixit, 2015). The significant difference

The selected mAbs were targeted with the peptides P1 (VYDKDGTTFD) and P4 (GGFKGKLSYQTND) of A. hydrophila OmpF, which still do not have a clear protein structure obtained by X-ray crystallography. To reveal the location of the two peptides P1 and P4, the structure of the A. hydrophila OmpF was modeled using the SWISS-MODEL online bioinformatic tool. The typical amino acid location of peptides P1 and P4 was captured. The BLAST results of the A. hydrophila OmpF amino acid sequence revealed that the conserved domain of this protein belonged to the Gram-negative bacteria porins protein (cd00342), which was also a porin protein with trimer subunits. By comparing the amino acids, the locations of six A. hydrophila candidate peptides were further predicted. 2.7. Establishment of an Aeromonas spp. sandwich immunoassay

4

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Fig. 1. Bioinformatic analysis of the OmpF of Aeromonas spp. A, sequence alignment of the A. hydrophila OmpF with other Aeromonas strains. B, the second structure, hydrophilicity, antigen index, surface probability of OmpF protein sequence.

No obvious reaction was observed with the other tested strains (Fig. 3B). Other strategies to prepare broad-spectrum mAbs of bacteria include using recombinant conserved Omp protein in whole or partial form, and artificial antigen conjugated with whole lipopolysaccharide (LPS) or synthesized oligosaccharides. Cross-reactive mAbs against Listeria spp. and Salmonella were prepared with the conserved P60 protein (Omp) and LPS conjugated BSA, respectively as an immunogen

between mice in the same group, however, revealed that the extracellular accessibility of peptide 3 was limited. In contrast, the P5-BSA and P6-BSA conjugates did not elicit effective antibodies against the A. hydrophila cells. Further study validated that the P1-BSA and P4-BSA immunogens elicited antibodies with high titers (81K–729K) after the fourth immunization and broadly cross-reacted with all the tested Aeromonas spp. strains (i.e., A. hydrophila, A. popoffii, A. punctata subsp. caviae, A. bivalvium, and A. media) except A. salmonicida CICC 23565. 5

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Table 2 Amino acid sequences of the six candidate peptides. Code name

Amino acid sequences

Range

Modification

MW (kDa)

Peptide Peptide Peptide Peptide Peptide Peptide

VYDKDGTTFD YGRVQANYYGD KTEWQVAAENS GGFKGKLSYQTND YKGEGRGYELAA FTSKLKAYTEYKI

2–11 13–23 64–74 140–152 245–256 298–310

N-terminal-Cys N-terminal-Cys N-terminal-Cys N-terminal-Cys N-terminal-Cys N-terminal-Cys

1263 1408 1365 1517 1416 1694

1 2 3 4 5 6

(P1) (P2) (P3) (P4) (P5) (P6)

Fig. 2. Characterization of the conjugation progress of peptides and BSA by SDS-PAGE. Fig. 3. Titers and cross reactivity of serums from peptide antigens. A, Titers of serum (diluted 3000) against A. hydrophila in indirect ELISA. B, Cross reactivity of serums (diluted 9000) obtained with peptide antigens P1-BSA and P4-BSA.

(Wang et al, 2017a; Wang et al, 2016). Liu et al. generated broadspectrum mAbs against potyviruses with soluble recombinant conserved 121-amino-acid core regions of the capsid proteins as immunogen; the C4 mAb detected at least 14 potyviruses by indirect ELISA and WB (Liu et al., 2015).

mutans (1K–3K). Also, mAb 2D7 and 14H5 exhibited a minimal reaction with all the non-Aeromonas strains (1K–3K). This may be because slightly different amino acids were identified with each of the mAbs, exhibiting insignificant homology with the other non-Aeromonas strains. In addition to the whole-cell binding test, the actual target proteins of tested strains that these mAbs reacted were further studied using the WB. Ultrasonication was used to extract the whole-cell proteins from the samples. The results revealed that the mAb 1B1 reacted with the purified recombinant OmpF protein and whole-cell proteins from all the Aeromonas spp. strains but not with the samples from the other strains, which exhibited excellent specificity (Fig. 4A). In contrast, mAb 14H5 reacted with OmpF protein and whole-cell proteins from both the Aeromonas spp. and the non-Aeromonas spp. strains (Fig. 4B). These results were in line with the former results of a whole cellbased indirect ELISA, except the indiscriminate binding with A. bestiarum and A. salmonicida CICC 23565 using WB, which was of interest. We further tested an isolated strain of A. salmonicida D5-7 and found mAb 1B1 and 14H5 reacted with both the denatured intact cell in ELISA (Fig. 6) and whole-cell protein sample in WB (Fig. S3). Consequently, it can't be concluded that the mAbs don't recognize all the A. salmionicida cells. The reason of low binding ability with A. salmonicida (CICC 23565) may because of the steric hindrance from the surface antigens such as capsule, lipopolysaccharide, and pili on this strain. In addition, we found that there was one aminol acid displacement in peptides 1 and 4 for some A. salmonicida strains (Fig. S4), which may also lead to the slight change of protein structure, and will affect the accessibility of the epitope on the outer membrane and result in low binding ability with intact cell. In contrast, for WB, the Omps could be unfolded after ultrasonic extraction, which made the epitopes more accessible. Our

3.3. Antibody specificity, affinity, and subtypes After cell fusion and selection, nine stable cell lines against the A. hydrophila OmpF peptides were obtained. The mAb 1B1, 2D7, 3D1, 5D5, 7E8, 8B12, and 9A4 were prepared against peptide 1 and the other two mAbs, including 11B8 and 14H5, against peptide 4. As shown in Table 3, the mAbs were all IgG1 or IgG2 subtypes with a kappa light chain, which indicated the antibodies were mature and stable. Furthermore, the affinities of these antibodies against A. hydrophila (CICC 10500) were all above 109 and reached 2.02 × 1010 for mAb 1B1. The affinity values (Ka) of mouse mAbs ranges from 106 L mol−1 to 1012 L mol−1, the higher value of Ka indicates higher affinity and lower detection limit in immunoassay, immunoaffinity chromatography (IAC) and immunoblotting. Especially, mAb with Ka more than 109 L mol−1 can be used to develop IAC. Accordingly, the mAbs produced in this study exhibited a high binding capacity. The mAb titers were evaluated with inactivated Aeromonas spp. cells by an indirect ELISA. The results illustrated that, for all the nine mAbs, high titers ranging from 6561K to 243K were obtained with the Aeromonas spp. strains as listed in Table 1. In contrast, low titers ranging from 27K to 81K were observed with A. bestiarum. In addition, the negligible titers of 3K with A. salmonicida CICC 23565 were obtained (Fig. S2). The specificity study showed that mAb 1B1 did not react with the other non-Aeromonas strains, including E. tarda, Pseudoalteromonas sp., V. cholerae, V. anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, E. coli DH5α, E. coli O157:H7, S. mutans, whereas 3D1, 5D5, 7E8, 8B12, 9A4, and 11B8 moderately reacted with E. coli DH5α and S. 6

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Table 3 The antibody subtype, light chain type, and antibody affinity of the mAbs.

Antibody subtype Antibody light chain Antibody affinity

1B1

2D7

3D1

5D5

7 E8

8B12

9A4

11B8

14H5

IgG1 Kappa 2.02✕1010

IgG1 Kappa 6.08✕109

IgG2b Kappa 1.36✕109

IgG1 Kappa 2.86✕109

IgG2a Kappa 1.21✕109

IgG2a Kappa 3.91✕109

IgG1 Kappa 4.89✕109

IgG1 Kappa 2.46✕109

IgG1 Kappa 8.02✕109

contrast, the location of peptides 1, 4, and 6 on the hydrophilic side of the porin indicated that they were extracellularly exposed and accessible to specific antibodies. The predicted location of six peptides obtained by similar Klebsiella Osmoporin and simulated structure with E. coli OmpF as a model were accordant. Besides, these results were generally in agreement with the antigen immunization results that peptides 1 and 4 elicited a high antibody titer against the denatured cell body of Aeromonas spp. The less optimal binding of peptide 6-specific antibodies may be due to the formation of a stable conformational epitope in this area. The extracellular location together with the conserved sequence and high antigenic index explained the broad crossreactivity and high specificity of mAb 1B1 against peptide 1 and mAb 14H5 against peptide 4. Recently, bioinformatics tools have demonstrated wide applications in various areas of immunology (e.g., pathogen vaccine design and cancer immunotherapy) (Garner et al., 2018; Monterrubiolópez et al., 2015; Soriaguerra et al., 2014). Our results also revealed that using bioinformatic tools and databases were useful for conserved antigen selection, B cell epitope prediction, and epitope location for developing excellent mAbs against foodborne pathogens. mAbs with a clear epitope sequence and homology have controlled specificity and ultimately lay the foundation of dependable rapid immunoassays for a diverse range of pathogens.

study also found that the detection limit of the boiled extracted bacterial protein of A. bestiarum in the sandwich ELISA was improved (equivalent with 3.7 × 107 CFU mL−1) compared with the boiled bacterial cell (1.1 × 108 CFU mL−1). The detailed reason still relies on the further study. Thus, our results illustrated that the mAbs prepared by the selected peptide antigen identified Aeromonas spp. with broad crossreactivity and a high titer.

3.4. Location of the epitopes on 3D-modeled A. hydrophila OmpF proteins The simulated model of A. hydrophila OmpF by a SWISS-MODEL showed that it was a β-barrel protein with a homo-trimer (Fig. S5). A BLAST analysis of the OmpF amino acid sequence found that it had a conserved domain similar to Gram-negative porin cd00342, a Klebsiella Osmoporin trimer (Fig. 5A). These results indicated that the Aeromonas hydrophila OmpF was also a porin with a trimer subunit, which provided aqueous channels for the transfer of small hydrophilic molecules (e.g., glucose) through the outer membrane (Mahendran et al., 2010; Tran et al., 2013). The locations of the six peptides on the OmpF porin were both examined with the conserved domain of similar porin cd00342 by Cn3D (Fig. 5A) and with simulated OmpF structure obtained by SWISSMODEL (Fig. 5B). The pictures were mainly obtained from the side view of the structure. The hydrophilic side on the top was the extracellular side. Fig. 5A and B showed that the peptides 2, 3, and 5 were primarily located on the hydrophobic side of OmpF, which indicated an inner location and was not easily accessible for antibody reactions. In

Fig. 4. Western blotting of the mAb 1B1 (A) and mAb 14H5 (B) against the whole cell lysates of Aeromonas and other tested strains. M, Protein marker; 1–4, A. hydrophila, CICC 10500, MCCC 1A00007, MCCC 1A00190, CICC 10868; 5–9, A. bivalvium, A. media, A. popoffii, A. punctata subsp. caviae, A. bestiarum; 10, Recombinant OmpF protein; 11, A. veronii; 12, A. caviae; 13, A. salmonicida CICC 23565; 14, E. coli DH5α; 15, Vibrio cholerae; 16, V. anguillarum; 17, V. parahaemolyticus. 7

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Fig. 5. The location of peptides on the conserved domain family Gram-negative porin protein cd00342 (A); The location of peptides on the Aeromonas OmpF protein model simulated by SWISS-MODEL (B).

the sandwich ELISAs based on 1B1 and 1B1-HRP or 14H5 and 1B1-HRP all positively responded to an increasing concentration of boiled recombinant OmpF (inclusion body), the detection limit of two methods was 123 ng. mL−1 (Fig. S6). These results indicated that the self-paired phenomenon may be attributed to the homo-trimer structure of the OmpF protein. Aeromonas spp. have dynamic characteristics and are difficult to properly classify (Awan et al., 2018). Similarly, due to the great variability of the Aeromonas spp. regarding surface antigens and homologies, it is challenging to identify all of them homogenously in a single immunoassay. In the present study, the sandwich immunoassay based on mAb 1B1 and 14H5 exhibited broad cross-reactivity among the Aeromonas spp. The heat-killed liquid culture of reference and isolated strains (classified by 16SRNA) of Aeromonas spp. were tested to obtain the standard curves in Sandwich ELISA. The detection limit was defined as the concentration corresponding to 2.1 times of the background. As shown in Fig. 6A, the absorbance of the 26 strains positively

3.5. Pairwise study and performance of the established sandwich immunoassay The conjugation of mAbs with HRP was confirmed by direct ELISA. Next, both the mAbs and HRP-conjugated mAbs were subjected to a pairwise study by a sandwich ELISA. The tested strain was A. hydrophila (MCCC 1A0007) with a concentration of 107 CFU mL−1 in PBS. Table 4 represented the ratio of absorbance from positive control and negative control (P/N); higher P/N values indicated a lower detection limit. The results showed that when the 1B1-HRP conjugate was paired with the other mAbs, substantially higher P/N ratios were obtained, especially when paired with mAb 1B1 and 14H5. The subsequent evaluation confirmed the superior performance of both pairs. The mAb 14H5 as capture antibody and 1B1-HRP as the detection antibody were eventually used to establish a sandwich immunoassay for Aeromonas spp. due to greater homogeneous cross-reactivity and lower detection limit. It is also important to note that each mAb in this study could be paired with the other mAbs, including itself. Further study revealed that 8

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Fig. 6. Standard curves of the sandwich ELISA against 16 standard strains and 11 isolated strains of Aeromonas spp. (A). Cross-reactivity of this immunoassay with Aeromonas spp. and other non-Aeromonas spp. strains (B).

jandaei was obtained and the detection limit was 1.11 × 108 CFU mL−1. Besides, there was no reaction with A. salmonicida CICC 23565, although the isolated strain A. salmonicida (D5-7) could be detected at 1.52 × 105 CFU mL−1. Consequently, the overall sensitivity of this method for tested strains was 96% (26/27 strains). Aeromonas. salmonicida primarily causes furunculosis in cold water fish (e.g., salmonid). Identification of A. salmonicida will enable the wide application of this immunoassay in both Salmonidae samples in the cold region and the tropic and subtropic regions where other Aeromonas spp. are popular. To improve the detection limit, future study will focus on whether surface antigens such as capsule, lipopolysaccharide, and pili exist on A.

responded with an increase in cell concentration. The detection limits were 1.69 × 104 CFU mL−1 for A. hydrophila (CICC10500, NAU ML21), and A. veronii NAU XX-12; 5.08 × 104 CFU mL−1 for A. punctata subsp. caviae, A. hydrophila (CICC 10868, NAU JH-18, and NAU JH-79) and A. dhakensis; 1.52 × 105 CFU mL−1 for A. hydrophila (MCCC 1A0007 and MCCC 1A00190), A. veronii (NAU XH-19, NAU XX-10, and NAU XX-17), A. media, A. bivalvium, and A. salmonicida (D5-7); 4.57 × 105 CFU mL−1 for A. veronii (MCCC 1A00180 and NAU XH-53), A. caviae, A. popoffii, and A. lacus; 1.37 × 106 CFU mL−1 for A. veronii MCCC 1K03237 and A. sobria NAU XH-21; 4.11 × 106 CFU mL−1 for A. hydrophila NAU ML-19. A weak reaction with A. bestiarum and A. 9

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antibody-based nanoparticle agglutination method developed by Saleh et al. was specific to all the A. salmonicida strains but not with A. hydrophila (Saleh et al., 2011). A recent report in 2018 conducted by Ballyaya et al. also found similar results that a flow-through immunogold assay, based on mAb B8E3 prepared with A. hydrophila as the immunogen, was specific to different A. hydrophila strains and did not react with other species (e.g., A. caviae, A. sobria, and A. veronii) (Ballyaya et al., 2018). To our knowledge, the sandwich ELISA developed in our study was the most superior immunoassay for Aeromonas spp. with regards to both cross-reactivity and specificity.

Table 4 Pairwise study of the mAbs against peptide 1 (mAb 1B1, 2D7, 3D1, 5D5, 7E8, 8B12 and 9A4) and peptide 4 (mAb 11B8 and 14H5) with boiled A. hydrophila (MCCC 1A0007) as standard. Standard concentration was 107 CFU mL−1 in PBS. Capture mAb Detect mAb

1B1

2D7

3D1

5D5

7E8

8B12

9A4

11B8

14H5

1B1-HRP 2D7-HRP 3D1-HRP 5D5-HRP 7E8-HRP 8B12-HRP 9A4-HRP 11B8-HRP 14H5-HRP

30.3 19.1 18.6 20.2 20.0 28.6 22.1 22.4 23.8

32.1 18.4 25.7 19.6 19.2 11.6 20.8 20.4 15.9

28.8 12.8 14.9 14.9 17.3 22.0 15.3 14.0 20.8

24.4 20.2 21.2 16.2 20.6 21.2 19.7 11.7 20.7

28.4 16.8 15.6 16.7 20.1 21.5 16.2 13.7 17.5

28.8 16.7 13.8 15.6 19.8 23.5 17.5 12.5 19.1

28.7 18.1 22.0 14.3 20.3 25.1 19.4 12.2 22.7

11.5 12.9 11.8 11.8 12.7 15.1 12.4 8.1 17.8

31.3 26.7 27.0 22.7 23.4 23.2 23.0 23.8 24.0

3.6. Results of the aquatic and fish sample detection River water samples were artificially contaminated with A. hydrophila (CICC 10500) and A. caviae (MCCC 1A12231), respectively. The collected water samples were all filtered and added with E. coli and V. vulnificus (108 CFU mL−1) to eliminate the effect of possible Aeromonas cells and simulate the presence of non-targeted bacteria. The backgrounds of the original and spiked water samples in the sandwich ELISA were similar (0.0947 ± 0.007). The analysis of these samples found that the matrix effect in the river water was significant. To solve the matrix effect, we diluted the river water samples three times before the test and found that the absorbance was not substantially decreased. As a result, the detection limit in the river water samples was improved by 10-fold. As shown in Table 5, the detection limits of A. hydrophila and A. caviae in the river water with this ELISA were all at 105 CFU mL−1; Our analyses of the enrichment culture from the Carassius auratus artificially infected with A. hydrophila and A. veronii revealed that the results were all strong positive after 24 h enrichment in BPW for fish exhibiting septicemia. In contrast, neither the 12 h enrichment samples or direct analysis of the kidney and spleen samples of diseased fish failed to result in any positive results (data not shown). This suggested that a 16 h or 18 h enrichment may be suitable for rapid detection. Yano et al. found that the estimated density of Aeromonas spp. in a normal inland shrimp pond in Thailand ranged from 4,667 to 1,500,000 CFU/g (Yano et al., 2015), which indicated that a base level of Aeromonas spp. existed in aquaculture ponds; however, the higher concentration of Aeromonas spp. were typically positively related with the prevalence of septicemia in aquatic animals (Abd-El-Malek, 2017; Nielsen et al., 2001) and threatened the safety of the aquatic foods (Chen et al., 2016; Nawaz et al., 2010). As a consequence, the monitoring of Aeromonas spp. in these samples and taking corresponding measures was an important means by which to avoid the outbreak of septicemia in fish and gastrointestinal disease in human. Our present findings in analyzing A. hydrophila and A. veronii contaminated river water and fish samples suggest that this immunoassay may be useful for the rapid detection of Aeromonas spp.

salmonicida CICC 23565 and affect the binding of antigen and antibody. We also evaluated the specificity of this immunoassay with strains other than Aeromonas spp. The results presented in Fig. 6B revealed that the absorbances of non-Aeromonas strains were all below 0.15 at 109 CFU mL−1 and the absorbance of positive result should be above 0.17. Accordingly, there was no cross-reactivity with five strains of Vibrio spp., Edwardsiella tarda, two strains of Proteus vulgaris, Proteus mirabilis, Psychrobacter sp., Pseudoalteromonas sp., E. coli DH5α, E. coli O157:H7, Streptococcus mutans, and M. tuberculosis. The specificity of this method was 100% (15/15). Specifically, our evaluation of an isolated M. tuberculosis strain with indirect ELISA, and Western blotting (Fig. S7) illustrated the two mAbs did not react with the whole-cells and proteins of M. tuberculosis, which existed in the Complete Freund's adjuvant used in this study. In the past, antibodies and different immunoassays (e.g., Dot and indirect ELISAs, and flow-through immunogold assay) have been developed. These antibodies were all prepared with bacterial cells as immunogens and extremely limited specificity, which usually leads to poor accuracy of the method. The rabbit antibodies were prepared by Sendra and coauthors, with heat-killed cells specifically reacting with A. hydrophila serogroup O:19 but not with the other serogroups (Sendra et al., 1997). Delamare and coauthors prepared mAb 5F3 with A. hydrophila cells and found that it specifically reacted with all the 12 A. hydrophila strains but did not react with other species, including A. sobria, A. salmonicida, and A. trota (Delamare et al., 2002). Longyant et al. used a combination of eight A. hydrophila isolates as the immunogen and prepared 10 groups of mAbs, the majority of which were highly specific to one or two A. hydrophila strains; some of these isolates exhibited limited cross-reactivity with other species and only one mAb, AH-396, bound to all isolates of A. hydrophila, A. sobria, A. caviae, and A. veronii in a dot blot assay (Longyant et al., 2010). The rabbit

Table 5 The detection results of spiked water and fish samples containing A. hydrophila, A. caviae and A. veronii by the Sandwich immunoassay. All the water samples contained E. coli and V. vulnificus at 108 CFU mL−1. All the data was based on 6 parallel tests. +++, Strong positive result; ++, Moderate positive result; +, Weak positive result. -, Negative result. Hemorrhagic septicemia fish

Spiked river water Tested strains

Spiked (CFU.mL−1)

Absorbance (450 nm)

CV (%)

Result

Sample number

Absorbance (450 nm)

CV (%)

Result

A. hydrophila

107 106 105 107 106 105

1.841 0.859 0.421 1.038 0.379 0.234

0.027 0.013 0.007 0.019 0.026 0.023

1.48 1.54 1.69 1.81 6.81 9.97

+++ ++ + ++ + +

1 2 3 1 2 3

1.738 1.979 1.992 0.493 1.737 2.165

0.012 0.021 0.031 0.042 0.054 0.051

0.69 1.07 1.55 8.59 3.13 2.35

+++ +++ +++ + +++ +++

Blank

0.089 ± 0.008

8.99

–

Blank

0.086 ± 0.008

9.92

–

A. caviae (Water) A. veronii (Fish)

± ± ± ± ± ±

10

± ± ± ± ± ±

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Aeromonas sobria, an outbreak of natural infection in cultured silver catfish Rhamdia quelen (Quoy & Gaimard, 1824) in Argentina. J. Fish Dis. 40, 1929–1933. Delamare, A.P.L., Echeverrigaray, S., Duarte, K.R., Gomes, L.H., Costa, S.O.P., 2002. Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification. J. Appl. Microbiol. 92, 936–940. Esteve, C., Amaro, C., Toranzo, A.E., 1994. O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels. FEMS Microbiol. Lett. 117, 85–90. Garner, L.I., Hartland, A., Breuning, J., Brown, M.H., 2018. CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action. Immunology 155, 273–282. Köhler, G., Howe, S.C., Milstein, C., 1976. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur. J. Immunol. 6, 292–295. Kong, D., Xie, Z., Liu, L., Song, S., Kuang, H., Xu, C., 2017. 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4. Conclusion Various pathogenic strains in Aeromonas spp. pose a threat to both the aquaculture industry and the safety of aquatic foods for human health. Currently, a rapid and simple immunoassay for Aeromonas spp. is strongly urged but remains hindered by the poor cross-reactivity of the antibodies against the genus of Aeromonas. In the present study, surface accessible and conserved peptides from the Omp of A. hydrophila were selected using bioinformatic tools. The artificial peptide antigens, P1-BSA and P4-BSA, were successfully used to prepare Aeromonas spp.-specific mAbs, 1B1 and 14H5. Based on these findings, a sandwich immunoassay for the rapid identification of Aeromonas spp. was established. Our results revealed that 12 species of Aeromonas spp. (26/27 strains) were detected using this method, including popular species, such as A. hydrophila, A. veronii, A. caviae, A. sobria, and A. dhakensis. The detection limits primarily ranged from 1.69 × 104 to 4.57 × 105 CFU mL−1. Moreover, cross-reactivity was not observed with Vibrio spp. strains, E. tarda, Proteus vulgaris, Psychrobacter sp., Pseudoalteromonas sp., M. tuberculosis, and E. coli. These results indicated the broad cross-reactivity, acceptable detection limits, and excellent specificity for detecting Aeromonas spp. using this immunoassay. Moreover, the presence of A. hydrophila, A. caviae, and A. veronii in the water or fish samples were sensitively and stably detected. Thus, the mAbs based immunoassay may be promising for the rapid monitoring of Aeromonas spp. in both water and aquatic food samples. Furthermore, the bioinformatical strategy of peptide antigen preparation provides an effective and innovative means of preparing bacterial mAbs for diagnostic use. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work is supported by National Natural Science Foundation of China (Grant No. 31701680), Natural Science Foundation of Jiangsu Province of China (Grant No. SBK2017041308) and also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We appreciate the help of professor Yongjie Liu of Nanjing Agriculture University and professor Liantai Li in Jiangsu Ocean University for the isolated Aeromonas strains. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734646. References Masi, M., Pagã¨S, J.M., 2013. Structure, function and regulation of outer membrane proteins involved in drug transport in Enterobactericeae: the OmpF/C - TolC case. Open Microbiol. J. 7, 22–33. Abd-El-Malek, A.M., 2017. Incidence and virulence characteristics of Aeromonas spp. in fish. Vet. World 10, 34–37. Ali, M.T., Islam, M.O., 2015. A highly conserved GEQYQQLR epitope has been identified in the nucleoprotein of Ebola virus by using an in silico approach. Adv. Bioinf. 278197 2015. Amit, K., Eric, H., Paolo, R., Matteo, C., 2010. Structural and dynamical properties of the porins OmpF and OmpC: insights from molecular simulations. J. Phys. Condens. Matter 22, 454125. Arora, S., Agarwal, R.K., Bist, B., 2006. Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin. Int. J. Food Microbiol. 106, 177–183. Awan, F., Dong, Y., Liu, J., Wang, N., Mushtaq, M.H., Lu, C., Liu, Y., 2018. Comparative genome analysis provides deep insights into Aeromonas hydrophila taxonomy and virulence-related factors. BMC Genomics 19, 712. Ballyaya, A.P., Mondal, M., Kalkuli, S.M., Purayil, S.B.P., 2018. Development of a simple

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