Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains l-perosamine (4-amino-4,6-dideoxy-l-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established: O-PS 1: →2)-α-l-Rhap4NAc-(1→; O-PS 2: →2)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→. Western blotting and an enzyme-linked immunosorbent assay (ELISA) showed that the cross-reactivity between the LPS of A. veronii bv. sobria K557 and the A. hydrophila JCM 3968 O6 antiserum, and vice versa, is caused by the occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides, whereas an additional →4)-α-d-GalpNAc-associated epitope defines the specificity of the O6 reference antiserum. Investigations of the serological and structural similarities and differences in the O-antigens provide knowledge of the immunospecificity of Aeromonas bacteria and are relevant in epidemiological studies and for the elucidation of the routes of transmission and relationships with pathogenicity.


Introduction
The genus Aeromonas, which belongs to the family Aeromonadaceae along with four other genera Telumonas, Oceanimonas, Oceanisphaera and Zobellella, is composed of a large number of species classified PGO1, and PGO2 dominated among both carp and trout isolates. In turn, motile aeromonas septicemia (MAS) incidences in rainbow trout have been related to the strains of serogroups O11, O16, O34, and O14. Moreover, pathogenic isolates of two species: Aeromonas veronii bv. sobria and Aeromonas sobria were mainly classified within the O6 serogroup.
The A. veronii species, originally described by Hickman-Brenner et al. [30] as a novel member of the genus, is commonly associated with diarrhea. Nevertheless, this species, which consists of two biovars, A. veronii bv. sobria, which is negative for aesculin hydrolysis and ornithine decarboxylase, and A. veronii bv. veronii, which is positive for these reactions [15,31], is commonly known as a fish pathogen associated mainly with ulcerative syndrome [32,33]. It is worth emphasizing that A. veroni bv. sobria was one of the dominant species among carp isolates collected during 5 years in Polish culture facilities, and 76% of these isolates were classified as virulent [28]. In the light of the increased Aeromonas infection incidence rate and the economic importance of these diseases in cultured fish, it is essential to characterize virulence factors associated with the pathogenesis of this species, particularly including LPS, which is the major surface glycoconjugate of Gram-negative bacteria. Recently, two new structures of O-antigens were established for the species A. veronii bv. sobria and A. sobria [34,35].
Here we report immunochemical investigation of LPS, especially the O-specific polysaccharide of A. veronii bv. sobria strain K557, which was isolated from the common carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm [28,36]. The structural characterization revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). Serological studies using Western blotting and an enzyme-linked immunosorbent assay (ELISA) with intact and adsorbed O-antisera showed that the O-antigen of A. veronii bv. sobria strain K557 is related but not identical to that of A. hydrophila JCM 3968 O6, which is a reference strain for Aeromonas serogroup O6 [37].

Bacterial Cultivation, Isolation of LPS, and SDS-PAGE Study
Cells of Aeromonas veronii bv. sobria strain K557 were extracted with hot aqueous 45% phenol [38], and LPS species were harvested from the phenol phase in a yield of 3.9% of the bacterial cell mass. SDS-PAGE analysis of the LPS followed by silver staining showed a pattern typical for LPS isolated from smooth bacterial cells (Figure 1) with the content of both slow-migrating high-molecular-weight (HMW) S-LPS species and fast-migrating low-molecular-weight R-LPS glycoforms. Moreover, the electrophoregram indicated that the studied S-LPS contained molecules where the core oligosaccharides were substituted with shorter O-chains in comparison to the LPS species of A. hydrophila JCM 3968, O6.
Mar. Drugs 2019, 17,399 3 of 21 PGO1, and PGO2 dominated among both carp and trout isolates. In turn, motile aeromonas septicemia (MAS) incidences in rainbow trout have been related to the strains of serogroups O11, O16, O34, and O14. Moreover, pathogenic isolates of two species: Aeromonas veronii bv. sobria and Aeromonas sobria were mainly classified within the O6 serogroup. The A. veronii species, originally described by Hickman-Brenner et al. [30] as a novel member of the genus, is commonly associated with diarrhea. Nevertheless, this species, which consists of two biovars, A. veronii bv. sobria, which is negative for aesculin hydrolysis and ornithine decarboxylase, and A. veronii bv. veronii, which is positive for these reactions [15,31], is commonly known as a fish pathogen associated mainly with ulcerative syndrome [32,33]. It is worth emphasizing that A. veroni bv. sobria was one of the dominant species among carp isolates collected during 5 years in Polish culture facilities, and 76% of these isolates were classified as virulent [28]. In the light of the increased Aeromonas infection incidence rate and the economic importance of these diseases in cultured fish, it is essential to characterize virulence factors associated with the pathogenesis of this species, particularly including LPS, which is the major surface glycoconjugate of Gram-negative bacteria. Recently, two new structures of O-antigens were established for the species A. veronii bv. sobria and A. sobria [34,35].
Here we report immunochemical investigation of LPS, especially the O-specific polysaccharide of A. veronii bv. sobria strain K557, which was isolated from the common carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm [28,36]. The structural characterization revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-L-mannose (N-acetyl-Lperosamine, L-Rhap4NAc). Serological studies using Western blotting and an enzyme-linked immunosorbent assay (ELISA) with intact and adsorbed O-antisera showed that the O-antigen of A. veronii bv. sobria strain K557 is related but not identical to that of A. hydrophila JCM 3968 O6, which is a reference strain for Aeromonas serogroup O6 [37].

Bacterial Cultivation, Isolation of LPS, and SDS-PAGE Study
Cells of Aeromonas veronii bv. sobria strain K557 were extracted with hot aqueous 45% phenol [38], and LPS species were harvested from the phenol phase in a yield of 3.9% of the bacterial cell mass. SDS-PAGE analysis of the LPS followed by silver staining showed a pattern typical for LPS isolated from smooth bacterial cells (Figure 1) with the content of both slow-migrating highmolecular-weight (HMW) S-LPS species and fast-migrating low-molecular-weight R-LPS glycoforms. Moreover, the electrophoregram indicated that the studied S-LPS contained molecules where the core oligosaccharides were substituted with shorter O-chains in comparison to the LPS species of A. hydrophila JCM 3968, O6.

Serological Studies of the A. veronii bv. sobria K557 O-antigen
A. veronii bv. sobria strain K557 was serologically typed by agglutination tests using heat-inactivated bacteria and antisera for 44 defined Aeromonas O-serogroups (NIH system) and 20 provisional serogroups (PGO1-PGO20) for selected Polish isolates and classified as a representative of the Aeromonas serogroup O6 [28,36].
The LPS preparations from A. veronii bv. sobria strain K557 and A. hydrophila JCM 3968, which is the reference strain to serogroup O6, were studied by Western blotting and ELISA with intact and adsorbed polyclonal rabbit O-antisera.
In Western blot, the reference antiserum O6 recognized electrophoretically separated LPS molecules of both strains (Figure 2a). Strong reaction to slow-migrating bands corresponding to the O-PS containing LPS species was observed and suggested that the O-antigens shared common epitopes. Additionally, the stained bands of fast-migrating R-LPS molecules may indicate similarities in the core region of the studied strains. In turn, the other Western blot (Figure 2b)
The LPS preparations from A. veronii bv. sobria strain K557 and A. hydrophila JCM 3968, which is the reference strain to serogroup O6, were studied by Western blotting and ELISA with intact and adsorbed polyclonal rabbit O-antisera. In Western blot, the reference antiserum O6 recognized electrophoretically separated LPS molecules of both strains (Figure 2a). Strong reaction to slow-migrating bands corresponding to the O-PS containing LPS species was observed and suggested that the O-antigens shared common epitopes. Additionally, the stained bands of fast-migrating R-LPS molecules may indicate similarities in the core region of the studied strains. In turn, the other Western blot (Figure 2b)   Accordingly, in ELISA (Table 1), the rabbit polyclonal O antiserum specific for A. hydrophila JCM 3968 O6 reacted strongly with the homologous LPS, and cross-reaction was observed with the LPS of A. veronii bv. sobria strain K557 to the lower titer of 1:64,000. In turn, the polyclonal O-antiserum against A. veronii bv. sobria strain K557 revealed reactions at the same level as both the homologous and heterologous LPS samples. The O6 reference antiserum revealed stronger reactivity than the K557-specific one, as demonstrated by the Western blotting results.
Adsorption of the antiserum specific for the A. veronii bv. sobria K557 O-antigen with A. hydrophila strain JCM 3968 O6 cells totally abolished the reactivity of this antiserum with both LPS preparations. The opposite reaction, i.e., adsorption of the reference O6 antiserum with the A. veronii bv. sobria K557 cells, only decreased its reactivity in the homologous system, whereas there was no reaction of the adsorbed O6 antiserum with A. veronii bv. sobria K557 LPS. The latter findings indicated that the adsorption process was complete and resulted in the removal of anti-K557 antibodies from the reference O6 antiserum. The remaining antibodies, strongly reacting with the homologous O6 LPS, were most probably specific to an additional epitope characteristic for the A. hydrophila JCM 3968 O6 antigen. These data indicated that the reference O6 antiserum and the A. veronii bv. sobria K557 O-antiserum shared common antibodies but the reference one contained additional immunoglobulins recognizing structural determinants that were not present in the A. veronii bv. sobria K557 O-antigen.
Therefore, detailed chemical analyses were performed to establish the structure of the A. veronii bv. sobria strain K557 O-PS.

Chemical and Mass Spectrometry Analyses of LPS
Compositional analysis of the degraded polysaccharide (dgPS) liberated from the phenol-soluble LPS was performed using GLC-MS of alditol acetates. It showed the presence of d-glucose (d-Glc), The negative ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum of the A. veronii bv. sobria K557 lipopolysaccharide ( Figure 3) showed the most intensive signals in the m/z range 1600-2000, which were attributable to a lipid A and a core oligosaccharide species (Y-and C-type fragment ions) arising from an in-source fragmentation at the glycosidic bond between the Kdo and the lipid A [41]. The ions at m/z 1768.17, 1796.19, and 1824.22 originated from hexaacylated lipid A species (Y-fragment ions) [37]. The ion at m/z 1824.22 represented a variant of lipid A, where the diglucosaminyl backbone bisphosphorylated at O-1 and O-4' was substituted by four 3-hydroxytetradecanoic acids 14:0(3-OH) and two tetradecanoic acids 14:0, instead of two dodecanoic acids 12:0, compared with the ion at m/z 1768.17. In turn, the ion at m/z 1796.19 contained a sugar backbone acylated by three, instead of four, 3-hydroxytetradecanoic acids, one 3-hydroxyisopentadecanoic acid, and two dodecanoic acids. The composition of the ions is shown in Table 2.
The ions at m/z 1954.5 and 1874.5 (C-fragment ions) were assigned to the core oligosaccharide with the following composition: Hep 6 Hex 2 HexN 1 Kdo. The mass difference between the ions of 80 amu corresponded to species containing phosphorylated and dephosphorylated variants of the core oligosaccharide, respectively.

Structural Studies of O-polysaccharide (O-PS)
The O-PS was released from phenol-soluble LPS by mild-acid degradation followed by gel-permeation-chromatography (GPC) on Sephadex G-50 fine to give a high-molecular-weight O-polysaccharide with the yield of 33% of the LPS mass. GLC-MS sugar analysis of alditol acetates obtained after full acid hydrolysis of the O-PS showed the presence of rhamnose and 4-amino-4,6-dideoxymannose (Rha4N) in a peak area ratio~1: 28.4. Other compounds detected in a small amount (below 10 %) in the GLC chromatogram of the A. veronii bv. sobria K557 O-PS, i.e., Glc, Gal and two heptose isomers (d,d-Hep and l,d-Hep), represented the core oligosaccharide sugars Determination of the absolute configurations of the monosaccharides by GLC of the acetylated (S)-and (SR)-2-octyl glycosides [42] showed that Rha4N had the l configuration. Samples from the O-polysaccharide of Citrobacter gillenii O9a,9b [43]   The low-field region of the 1 H NMR spectrum of the O-polysaccharide ( Figure 5) contained one major and three minor signals for anomeric protons at δ 5.18, 5.04, 5.00, and 4.97. The high-field region of the spectrum included signals for N-acetyl groups at δ 2.07, CH3-C groups of 6-deoxy sugars in the range of δ 1.21-1. 24  The low-field region of the 1 H NMR spectrum of the O-polysaccharide ( Figure 5) contained one major and three minor signals for anomeric protons at δ 5.18, 5.04, 5.00, and 4.97. The high-field region of the spectrum included signals for N-acetyl groups at δ 2.07, CH 3 -C groups of 6-deoxy sugars in the range of δ 1.21-1. 24. The 1 H and 13 C resonances of the O-PS of A. veronii bv. sobria K557 were assigned using 2D homonuclear 1 H, 1 H DQF-COSY, TOCSY, NOESY, heteronuclear 1 H, 13 C HSQC, and HMBC experiments. The 1 H and 13 C NMR data are collected in Table 3.  Table 3. NAc-N-acetyl groups, IS-acetone as an internal standard (δH 2.225), asterisk-free acetic acid.

Sugar Residue
Chemical Shifts (δ, ppm)   Table 3. NAc-N-acetyl groups, IS-acetone as an internal standard (δ H 2.225), asterisk-free acetic acid. The 1 H, 1 H TOCSY, and DQF-COSY spectra revealed one major and three minor spin systems for monosaccharide residues, which were labelled A-D in the order of the decreasing chemical shifts of their anomeric protons. The high-field positions of H-6 (δ 1.21-1.24) and C-6 (δ 18.2) resonances and the values of vicinal coupling constants 3 J 1,2 (~2 Hz), 3 Table 3.
In the TOCSY spectrum, at the H-1 coordinate, the cross-peaks with H-2-H-5 were visible for Rha4NAc A, but only one cross-peak with H-2 for Rha4NAc B, and two ones with H-2 and H-3 for Rha4NAc C and D. In turn, starting from the H-2 proton signal, cross-peaks with H-3-H-6 were visible for all spin systems; however, some signals overlapped. The 1 H, 1 H COSY spectrum allowed unambiguous differentiation between protons within the spin system A and only partly resolved the cross-peaks for the Rha4NAc B, C and D. The difficulties in the assignment of the H-3, H-4, and H-5 of Rha4NAc B, C and D were overcome in the 1 H, 13 C HMBC and 1 H, 13 C HSQC experiments.
The 13 C NMR resonances of e.g., Rha4NAc C were assigned by the long-range H-6/C-4 and H-6/C-5 correlations at δ 1.24/53.2 and 1.24/69.5, and then the C-4/H-3 and C-4/H-2 correlations at δ 53.2/3.93 and 53.2/3.87, respectively, in the 1 H, 13 C HMBC spectrum. In the 1 H, 13 C HSQC spectrum, the cross-peak of the proton at the nitrogen-bearing carbon to the corresponding carbon at δ 4.00/53.2 was assigned to the H-4/C-4 correlation of Rha4NAc C. Moreover, in the 1 H, 13 C HMBC spectrum, correlations of the anomeric proton with carbons C-2 and C-5 were found, and then the proton resonances were assigned from the 1 H, 13   In the TOCSY spectrum, at the H-1 coordinate, the cross-peaks with H-2-H-5 were visible for Rha4NAc A, but only one cross-peak with H-2 for Rha4NAc B, and two ones with H-2 and H-3 for Rha4NAc C and D. In turn, starting from the H-2 proton signal, cross-peaks with H-3-H-6 were visible for all spin systems; however, some signals overlapped. The 1 H, 1 H COSY spectrum allowed unambiguous differentiation between protons within the spin system A and only partly resolved the cross-peaks for the Rha4NAc B, C and D. The difficulties in the assignment of the H-3, H-4, and H-5 of Rha4NAc B, C and D were overcome in the 1 H, 13 C HMBC and 1 H, 13 C HSQC experiments.
The 13 C NMR resonances of e.g., Rha4NAc C were assigned by the long-range H-6/C-4 and H-6/C-5 correlations at δ 1.24/53.2 and 1.24/69.5, and then the C-4/H-3 and C-4/H-2 correlations at δ 53.2/3.93 and 53.2/3.87, respectively, in the 1 H, 13 C HMBC spectrum. In the 1 H, 13 C HSQC spectrum, the cross-peak of the proton at the nitrogen-bearing carbon to the corresponding carbon at δ 4.00/53.2 was assigned to the H-4/C-4 correlation of Rha4NAc C. Moreover, in the 1 H, 13 C HMBC spectrum, correlations of the anomeric proton with carbons C-2 and C-5 were found, and then the proton resonances were assigned from the 1 H, 13 C HSQC spectrum. Similar long-range correlations were searched during identification of H-3, H-4 and H-5 proton signals of Rha4NAc B and D. The chemical shifts of the C-2 signal of Rha4NAc B and the C-3 signals of Rha4NAc C and D were confirmed after consideration of the methylation analysis data.
The α-configuration of all Rha4NAc residues was inferred from the relatively high-field position of the C-5 signals at δ 69.5-69.7 for the residues in the O-polysaccharides, compared with δ 68.6 and δ 72.4 for α-Rha4NAc and β-Rha4NAc, respectively [44,45].
The correlation signals between H-4 of all the Rha4NAc residues and carbonyl group signals at δ C 176.0 and between the latter and methyl proton signals at δ H 2.07, which were observed in the 1 H, 13 C HMBC spectrum, confirmed that the residues building the O-PS were N-acetylated.
In the NOESY spectrum (Figure 7), intraresidue H-1/H-2 and interresidue H-1/H-5 correlations at δ 5.18/4.16 and δ 5.18/3.85, respectively, typical of α-(1→2)-linked sugars with the manno configuration, indicated that the Rha4NAc A residues constitute the main O-polysaccharide (O-PS 1) being the homopolymer [47]. In turn, in the spectrum inter-residue NOE contacts were observed for protons of residues B→C, C→D, and D→B. shifts of the C-2 signal of Rha4NAc B and the C-3 signals of Rha4NAc C and D were confirmed after consideration of the methylation analysis data. The α-configuration of all Rha4NAc residues was inferred from the relatively high-field position of the C-5 signals at δ 69.5-69.7 for the residues in the O-polysaccharides, compared with δ 68.6 and δ 72.4 for α-Rha4NAc and β-Rha4NAc, respectively [44,45].  Table 3.
The correlation signals between H-4 of all the Rha4NAc residues and carbonyl group signals at δC 176.0 and between the latter and methyl proton signals at δΗ 2.07, which were observed in the 1 H, 13 C HMBC spectrum, confirmed that the residues building the O-PS were N-acetylated.
The sequence of monosaccharides in the repeating unit of O-PS 2 was confirmed, in the 1 H, 13 C HMBC spectrum (   Table 3. The sequence of monosaccharides in the repeating unit of O-PS 2 was confirmed, in the 1 H, 13   The serological results (Western blotting and ELISA) allowed recognition of structural determinants that are most probably responsible for antibody binding (putative epitopes). The occurrence of common α-L-Rhap4NAc-(1→2)-α-L-Rhap4NAc and α-L-Rhap4NAc-(1→3)-α-L-Rhap4NAc disaccharides is sufficient for providing cross-reactivity between the A. hydrophila JCM The serological results (Western blotting and ELISA) allowed recognition of structural determinants that are most probably responsible for antibody binding (putative epitopes). The occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides is sufficient for providing cross-reactivity between the A. hydrophila JCM 3968 O6 antiserum and the LPS of A. veronii bv. sobria K557 and a similar reaction in an opposite system. On the other hand, an additional epitope (epitopes) most probably related to a 4-substituted α-d-GalpNAc residue or a →4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc disaccharide fragment, which has not been found in the O-antigen studied here, determines the specificity of the A. hydrophila JCM 3968 O6 serotype. This putative epitope (epitopes) seems to play an important role in the immunospecificity of the reference O6 antiserum (Figure 9).
3968 O6 antiserum and the LPS of A. veronii bv. sobria K557 and a similar reaction in an opposite system.
On the other hand, an additional epitope (epitopes) most probably related to a 4-substituted α-D-GalpNAc residue or a →4)-α-D-GalpNAc-(1→3)-α-L-Rhap4NAc disaccharide fragment, which has not been found in the O-antigen studied here, determines the specificity of the A. hydrophila JCM 3968 O6 serotype. This putative epitope (epitopes) seems to play an important role in the immunospecificity of the reference O6 antiserum (Figure 9).

Discussion
Almost every year, health disorders in freshwater fish are recorded on many farms in Poland. Although the development of a particular fish disease depends largely on climate conditions prevailing in a given zone and region, infections caused by Aeromonas spp. are the most common among bacterial fish diseases. As demonstrated recently by data collected during the last several years in Poland by Pękala-Safińska, health disorders caused by Aeromonas species were mostly observed in carp (Cyprinus carpio L.) and were usually manifested by skin lesions (MAI) in the form of ulceration as well as fish mortalities [48].
Previous studies performed on 558 isolates of mesophilic Aeromonas collected during 5 years in Polish culture facilities, among which 427 isolates were obtained from common carp and 121 from rainbow trout, revealed predominant occurrence of A. veronii bv. sobria, A. sobria and A. salmonicida species. In turn, A. veroni bv. sobria A. bestiarum and A. salmonicida were most frequently identified in both carp and trout samples; A. veroni bv. sobria was one of the dominant species only among carp isolates, and which is worth emphasizing, almost 80% of these isolates were classified as virulent [28,36]. Serological typing of all collected isolates using 44 antisera of the NIH scheme extended by 20 provisional serogroups for selected Polish isolates showed that O6 was one of the five most commonly identified serogroups, especially among carp isolates, and the other ones were O11, PGO1, O16, and O18. The report mentioned above also showed that, with the use of the antisera for serogroups from O45 to O96, there was little possibility for Aeromonas typing, since these groups occur rather rarely among fish isolates, especially in Polish aquacultures. In turn, when the 44 antisera of the NIH scheme were used, about 53% isolates were positively classified to appropriate somatic serotypes and this increased to about 88% when the NIH collection was extended by the sera for provisional serogroups of Polish origin [28,36]. Therefore, to obtain the most positive serotyping

Discussion
Almost every year, health disorders in freshwater fish are recorded on many farms in Poland. Although the development of a particular fish disease depends largely on climate conditions prevailing in a given zone and region, infections caused by Aeromonas spp. are the most common among bacterial fish diseases. As demonstrated recently by data collected during the last several years in Poland by Pękala-Safińska, health disorders caused by Aeromonas species were mostly observed in carp (Cyprinus carpio L.) and were usually manifested by skin lesions (MAI) in the form of ulceration as well as fish mortalities [48].
Previous studies performed on 558 isolates of mesophilic Aeromonas collected during 5 years in Polish culture facilities, among which 427 isolates were obtained from common carp and 121 from rainbow trout, revealed predominant occurrence of A. veronii bv. sobria, A. sobria and A. salmonicida species. In turn, A. veroni bv. sobria A. bestiarum and A. salmonicida were most frequently identified in both carp and trout samples; A. veroni bv. sobria was one of the dominant species only among carp isolates, and which is worth emphasizing, almost 80% of these isolates were classified as virulent [28,36]. Serological typing of all collected isolates using 44 antisera of the NIH scheme extended by 20 provisional serogroups for selected Polish isolates showed that O6 was one of the five most commonly identified serogroups, especially among carp isolates, and the other ones were O11, PGO1, O16, and O18. The report mentioned above also showed that, with the use of the antisera for serogroups from O45 to O96, there was little possibility for Aeromonas typing, since these groups occur rather rarely among fish isolates, especially in Polish aquacultures. In turn, when the 44 antisera of the NIH scheme were used, about 53% isolates were positively classified to appropriate somatic serotypes and this increased to about 88% when the NIH collection was extended by the sera for provisional serogroups of Polish origin [28,36]. Therefore, to obtain the most positive serotyping results, the postulate to include new provisional antisera against strains occurring in a given area and thus to extend the collection of antisera seems reasonable.
The inland aquaculture sector in Poland is mainly based on the culture of two species of freshwater fish -carp (49% of total production in 2015) and rainbow trout (38% of total production in 2015) [49]; thus, the lack of a commercially available vaccine dedicated to carp seems to be alarming. The most effective prevention of fish disease should involve immunoprophylaxis based on auto-vaccines chosen according to the needs of culture facilities and prepared from bacterial strains isolated from the fish or even the entire region. Auto-vaccines are confirmed to be highly effective against conditionally pathogenic microorganisms like Aeromonas sp., Yersinia ruckeri, and Pseudomonas sp. [50][51][52][53]. According to recently published studies, a vaccine containing whole cells and LPS of Aeromonas sp. seems to protect fish against MAS disease [54][55][56]. However, to avoid a failure of implemented prophylactic programs based on auto-vaccination, the serological and structural similarities and differences in O-chain polysaccharides in various serogroups and strains, which contribute to the immunospecificity of Aeromonas, should be carried out [57].
Here The chemical and mass spectrometry analyses of the phenol-soluble LPS of A. veronii bv. sobria strain K557 demonstrated the LPS glycoforms had hexaacylated lipid A species with a conserved architecture and a backbone composed of 1,4 -bisphosphorylated-β-(1→6)-linked-d-GlcN disaccharide. The residues of 3-hydroxytetradecanoate were predominant among fatty acids, similarly as previously reported for A. hydrophila [37]. However, some differences were found in the acylation profile of lipid A species, in comparison to those of A. bestiarum [59]. Amongst the ester-linked saturated fatty acids, not only dodecanoic (12:0) but also tetradecanoic (14:0) residues were found. The latter fatty acids were also detected in the lipid A of A. veronii strain Bs19, O16 [60].
The compositional analysis of the core oligosaccharide revealed two isomers of heptose (d,d-Hep and l,d-Hep), and MALDI-TOF MS confirmed that the core decasaccharide, with the following composition: Hep 6 Hex 2 HexN 1 Kdo 1 P 1 , has a structure shared by the LPS core regions of the A. hydrophila and A. bestiarum species [37,59]. Interestingly, the mass spectrum of the core OS of A. hydrophila JCM 3968, which was isolated from R-LPS molecules after mild acid hydrolysis and separation by gel-permeation-chromatography, showed an ion at m/z 1856.59, which corresponded to the dephosphorylated core variant with the composition Hep 6 Hex 2 HexN 1 Kdo anh . On the other hand, the structure of the core OS in the SR-LPS molecules of A. hydrophila JCM 3968 was slightly different: Hep 6 Hex 1 HexN 1 HexNAc 1 Kdo 1 P 1 [37]. In these species, the O-antigen was linked to the GalNAc residue, whereas in the rough R-LPS glycoforms, the galactose appeared to be a terminal outer core sugar, similarly as has been established for the core OS of the rough mutant strain of A. hydrophila AH-3, O34 [39].
In conclusion, the immunochemical studies of LPS, which is a glycolipid characterized by high heterogeneity amongst Aeromonas sp. bacteria, may facilitate selection of vaccine strains suitable for immunoprophylaxis of MAI/MAS diseases. The O-antigen of A. veronii bv. sobria strain K557, serotype O6, studied here is composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). The consideration of the serological results in view of the known O-antigen structures enabled recognition of domains that could be responsible for antibody binding (putative epitopes). The occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides is sufficient for providing cross-reactivity between A. hydrophila JCM 3968 O6 antiserum and the LPS of A. veronii bv. sobria 557, and vice versa. On the other hand, the additional epitope related to a 4-substituted α-d-GalpNAc residue, which has not been found in the O-antigen studied here, determines the specificity of the A. hydrophila JCM 3968 O6-serotype. This putative epitope seems to play an important role in the immunospecificity of the reference O6 antiserum.

Bacterial Strain, Cultivation Conditions, and Isolation of LPS
Aeromonas veronii strain K557 was isolated from a common carp during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The isolate was identified to the species level by restriction analysis of 16S rRNA gene amplified by polymerase chain reaction [28] and classified as Aeromonas veronii bv. sobria because of the positive reaction for arginine dihydrolase, and negative reactions for ornithine decarboxylase and aesculin hydrolysis [30,31]. Based on virulence-associated markers (hemolytic, gelatinolytic, and caseinolytic activities), strain K557 was classified as virulent for fish. For the LPS analysis, A. veronii bv. sobria K557 bacterium was cultivated with shaking (120 rpm) on tryptic soy broth (TSB) for 72 h at 28 • C. The cells were harvested by low-speed centrifugation (8000× g, 20 min). The recovered bacterial cell pellet was washed twice with 0.85% saline and once more with distilled water.
The bacterial cells (5 g dry mass) were digested with lysozyme, RNAse, and DNAse (24 h, 1 mg/g) and then with Proteinase K (36 h, 1 mg/g) in 50 mM phosphate buffer (pH 7.0) containing 5 mM MgCl 2 . The suspension was dialyzed against distilled water and freeze-dried. The digested cells were extracted three times with aqueous 45% phenol at 68 • C, [38] and the separated layers were dialyzed against tap and distilled water. LPS species recovered from the phenol phase were purified by ultracentrifugation at 105,000× g and freeze-dried to give a yield of 3.9% of dry bacterial cell mass.

Degradation of LPS and Isolation of O-polysaccharide
The phenol-soluble S-LPS (110 mg) was hydrolyzed with aqueous 2.5% acetic acid at 100 • C for 3 h, and lipid A precipitate was removed by centrifugation. The supernatant was concentrated and then fractionated by GPC on a column (1.8 × 80 cm) of Sephadex G-50 fine (Pharmacia, Sweden) using 1% acetic acid as the eluent and monitoring with a differential refractometer (Knauer, Berlin, Germany). The yield of the O-PS fraction was 33% of the LPS mass subjected to hydrolysis.

Chemical Analyses
For neutral and amino sugar analysis, the LPS and O-PS samples were hydrolyzed with 2 M CF 3 CO 2 H (100 • C, 4 h) or 10 M HCl for 30 min at 80 • C, respectively, and reduced with NaBD 4 ; this was followed by acetylation with a 1:1 (v/v) mixture of acetic anhydride and pyridine (85 • C, 0.5 h).
To release acidic sugar, LPS was dephosphorylated with 48% aqueous hydrofluoric acid, HF (4 • C, 18 h) and dried under vacuum over sodium hydroxide [40]. Methanolysis was performed with 1 M MeOH/HCl (85 • C, 1 h), and the sample was extracted twice with hexane. The methanol layer was concentrated and the residue was dried and acetylated. The monosaccharides were identified as alditol and aminoalditol acetates [61] as well as acetylated methyl glycosides by GLC-MS.
Methylation of the O-PS (1.0 mg) was carried out with methyl iodide in dimethyl sulfoxide in the presence of powdered sodium hydroxide [62]. The products were recovered by extraction with chloroform/water (1:1, v/v), hydrolyzed with 10 M HCl for 30 min at 80 • C, N-acetylated, and reduced with NaBD 4 . The partially methylated alditol acetates derivatives were analyzed by GLC-MS.
For fatty acid analysis, a sample of the lipid A (1 mg) was subjected to methanolysis in 2 M methanolic HCl (85 • C, 12 h). The resulting fatty acid methyl esters were extracted with hexane and converted to their O-trimethylsilyl (O-TMS) derivatives, as described [63,64]. The methanol layer containing methyl glycosides was dried and acetylated with a pyridine-acetic anhydride mixture. The fatty acid derivatives and acetylated methyl glycosides were analyzed by GLC-MS as above.
All the sample derivatives were analyzed on an Agilent Technologies 7890A gas chromatograph (Agilent Technologies, Wilmington, DE, USA) connected to a 5975C MSD detector (inert XL EI/CI, Agilent Technologies, Wilmington, DE, USA). The chromatograph was equipped with an HP-5MS capillary column (Agilent Technologies, 30 m × 0.25 mm, flow rate of 1 mL/min, He as carrier gas). The temperature program for all the derivatives was as follows: 150 • C for 5 min, then 150 to 310 • C at 5 • C/min, and the final temperature was maintained for 10 min.

NMR Spectroscopy
An O-PS sample was deuterium-exchanged by freeze-drying with D 2 O and then examined in 99.98% D 2 O using acetone as an internal standard (δ H 2.225, δ C 31.45). 1D and 2D NMR spectra were recorded at 32 • C on a 500 MHz NMR Varian Unity Inova instrument (Varian Associates, Palo Alto, CA, USA) using Varian software Vnmrj V. 4.2 rev. (Agilent Technologies, Santa Clara, CA, USA). The following homonuclear and heteronuclear shift-correlated two-dimensional experiments were conducted for signal assignments and determination of the sugar sequence: 1 H, 1 H DQF-COSY, 1 H, 1 H TOCSY, 1 H, 1 H NOESY, 1 H, 13 C HSQC, and 1 H, 13 C HMBC. The mixing time was set to 100 and 200 ms in the TOCSY and NOESY experiments, respectively. The 1 H, 13 C HSQC experiment with CRISIS based multiplicity editing was optimized for a coupling constant of 146 Hz. The heteronuclear multiple-bond correlation (HMBC) experiment was optimized for J H,C = 7 and 5 Hz, with 2-step low-pass filter 130 and 165 Hz to suppress one-bond correlations.