Identification and Phylogenetic Analysis of Flavobacterium spp. Associated with Aquaculture Fish Diseased from Brazil

Abstract

Due to the recent taxonomic reclassification of the species Flavobacterium columnare into four new species—Flavobacterium columnare, Flavobacterium davisii, Flavobacterium covae, and Flavobacterium oreochromis—it is necessary to re-evaluate isolates of previous outbreaks to better understand the epidemiology related to this bacterial group. Therefore, the aim of this study was to determine the taxonomic profile of Brazilian isolates of Flavobacterium spp. associated with columnaris disease using available diagnostic methods. Fifty isolates from different outbreaks (17 clinical cases) occurring in five different Brazilian states previously identified as F. columnare were selected and identified by multiplex PCR and MALDI-ToF methods. In addition, at least one isolate from each clinical case was analyzed by 16S rRNA gene sequencing. After inclusion of the MSPs (main spectra profiles), the isolates were identifiable, and when compared with the multiplex PCR results, they showed almost perfect agreement (94.2% Kappa = 0.85). Only F. davisii, F. covae, and F. oreochromis were found among the Brazilian isolates, with these species causing disease in neotropical fish hosts not previously reported (e.g., Siluriformes, Serrasalmidae, and Bryconidae), while F. columnare was not detected. This study provides evidence of Flavobacterium species associated with columnaris disease circulating in various aquaculture facilities across different regions of Brazil. This information is crucial for developing control programs and advancing epidemiologic studies on columnaris disease in Brazilian aquaculture.

1. Introduction

The Flavobacterium genus includes 330 species validly published under the International Code of Nomenclature of Prokaryotes [1]. Several of these Flavobacterium species are host-associated in fish [2], including Flavobacterium columnare, which was appointed as the causative agent of columnaris disease in cold, temperate, and tropical fish from both wild and aquaculture freshwater environments [3].

Columnaris disease was described first in 1922 and manifests as lesions on the body surface and gills, appearing as dirty-white or yellowish areas on diseased fish. As the disease progresses, it can lead to high mortality rates [3,4]. This disease has a significant economic impact on various aquaculture production systems for farmed fish species worldwide [3,5,6,7].

Since the first successful isolation of the bacterial agent of columnaris disease in 1944 [8], its definitive taxonomy was believed to have been established in 1996 with the designation of Flavobacterium columnare [9]. However, a recent study has demonstrated that Flavobacterium columnare can be distinguished into four species: Flavobacterium columnare, Flavobacterium davisii, Flavobacterium covae, and Flavobacterium oreochromis [10]. Species identification of these new species of Flavobacterium bacteria associated with columnaris disease was originally conducted using phylogenetic and phylogenomic analyses [10]. Additionally, multiplex PCR targeting Flavobacterium columnare genotypes and MALDI-TOF MS have been proposed as methods for distinguishing Flavobacterium species associated with columnaris disease [10,11], and they have been applied in recent reports [12,13,14,15,16,17,18].

Columnaris disease was suspected to occur in Brazilian aquaculture as early as the late 20th century [19], but it was only isolated and characterized around 2005 from an outbreak in Nile tilapia (Oreochromis niloticus) [20,21,22]. Since then, reports of columnaris disease associated with F. columnare isolation and DNA sequencing and phylogenetic identification in other fish species have been registered in the country [5,13]. Nowadays, columnaris disease is an important disease for Brazilian freshwater aquaculture, affecting O. niloticus and some neotropical fish species, especially during the fingerling phase or after fish are exposed to environmental stress (such as water temperature reduction) or management stress (such as fish transport) [13,17,23].

Pathogenicity capabilities of F. columnare Brazilian isolates for O. niloticus and neotropical fish have already been proven [13,17,21,24,25]. However, with the new division of F. columnare into four taxonomic groups, a new epidemiologic approach for columnaris disease can be applied according to the specific Flavobacterium species involved. For instance, some authors have suggested that the incidence of F. oreochromis and F. covae is more closely associated with Nile tilapia and channel catfish, respectively [12,26].

Given the recent classification of F. columnare into four species associated with columnaris disease, it is essential to update the classification of isolates previously identified as F. columnare. This study aimed to determine the taxonomic profiles of Brazilian columnaris-causing bacteria through phylogenetic analyses and MALDI-ToF MS.

2. Materials and Methods

2.1. Bacterial Isolates

Isolates previously identified as F. columnare using species-specific PCR [27] were selected from the culture collection of the Laboratory of Aquatic Animal Diseases (AQUAVET) (n = 46) and the Laboratory of Applied Microbiology of Aquatic Organisms (LAMAO) (n = 4), as well as the reference strains ATCC23463 and ATCC49512. The selected isolates were isolated between 2004 and 2022 from columnaris disease outbreaks in five different states (Amazonas, Minas Gerais, Mato Grosso, Rondônia, and São Paulo) and from six different host species (Colossoma macropomum Lophiosilurus alexandri, Oreochromis niloticus, and Brycon amazonicus and the hybrids Colossoma macropomum × Piaractus brachypomus and Leiarius marmoratus × Pseudoplatystoma fasciatum) (Table 1). The isolates and reference strains that were preserved in Hsu–Shotts broth (MHS) with 15% glycerol and maintained at −70 °C were thawed, streaked, and incubated in Hsu–Shotts agar (MHSA) at 25 °C for 5 days.

2.2. Molecular Biology Analysis

2.2.1. DNA Extraction

Colonies of all isolates and strains of Flavobacterium spp. were collected from agar plates after incubation using sterile 10 uL plastic loops and subjected to DNA extraction using the Maxwell^® 16 Tissue DNA Kit on the Maxwell 16 Research Instrument (Promega, Madison, WI, USA), following the manufacturer’s instructions. The total DNA underwent quality and quantity verification with the aid of the NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); those samples with purity values of 1.8 ± 0.5 were stored at −70 °C until use.

2.2.2. 16S rRNA Gene Sequencing

To determine the species of Flavobacterium involved in different columnaris disease outbreaks, the DNA of 29 isolates from those extracted was selected, along with that of two reference strains, ATCC23463 and ATCC49512. In order to represent the different affected farms, at least one isolate from each clinical case was selected randomly (Table 1). The 16S rRNA gene was sequenced following the polymerase chain reaction (PCR) using the universal primers B37 (5′-TACGGYTACCTTGTTACGA-3′) and C70 (5′-AGAGTTGATYMTGGC-3′), exactly as reported by Fox et al. [30].

The obtained amplicons were purified using Agencourt AMPure XP (Beckman Coulter, Pasadena, CA, USA), as recommended by the manufacturer, and sequenced with the BigDye™ Terminator v.3.1 Cycle sequencing kit (Applied Biosystems, UK) and an ABI 3500 genetic analyzer (Life Technologies, USA). Subsequently, quality control, sequence trimming, and contig generation were performed using BioEdit, version 7.2 [31], as follows: visual inspection of chromatograms, followed by the removal of nucleotide bases with suboptimal or noisy peaks in the ABI trace file for each forward and reverse read [32], and, finally, contig assembly using the CAP application (minimum base overlap = 20, minimum percent match = 85). All assembled sequences were evaluated using NCBI BLAST version 2.15.0 (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 February 2025) on the nt/nr database with the default parameters of the megablast algorithm. After the BLAST procedure, identity (greater than 97%) and coverage (greater than 99%) were determined based on the lowest significant E-values.

2.2.3. Multiplex PCR and Amplicon Sequencing

All extracted DNA was also subjected to multiplex PCR to determine the species, using the only the available protocol for species differentiation among columnaris-causing bacteria, as described by LaFrentz et al. [11], with some modifications. Briefly, multiplex PCR assays were performed with the HotStart GoTaq DNA polymerase kit (Promega, Madison, WI, USA), with 50 ng of bacterial DNA per reaction. To carry out the multiplex PCR, there was an initial denaturation step at 95 °C for 5 min, followed by 20 cycles of denaturation at 95 °C for 30 s, an annealing step at 54 °C for 20 s, and an extension phase at 72 °C for 60 s. The final extension step was performed at 72 °C for 5 min. Thermal cycling was performed in a 96-well Veriti^® thermal cycler (Life Technologies). The resulting amplicons were assessed for size using a QIAxcell Advanced and a QIAxcell DNA Screening Kit (Qiagen, Valencia, CA, USA). The primer sequences for multiplex PCR used in this study, as well as the amplicon sizes, are displayed in Supplementary Table S2.

For multiplex PCR sequencing, two random amplicons of clinical samples were chosen for only three available product sizes (320, 659, and 894 bp). These six samples were then subjected to sequencing with the primers of interest for further evaluation, as described in Section 2.2.2.

2.2.4. Phylogenetic Analysis

Phylogenetic analysis was performed on the sequences of the 16S rRNA gene obtained from the Brazilian isolates (n = 29) and other Flavobacterium spp. obtained from the NCBI database (n = 26). For this purpose, sequence alignment was performed on MAFFT v7.471 (settings: —adjustdirection—auto—reorder) [33], followed by phylogenetic tree building in MEGA7 [34] by fitting the General Time-Reversible model [GTR + G + I] based on the maximum likelihood (ML) method. All positions containing gaps and missing data were eliminated, leaving a total of 1255 positions in the final dataset submitted to MEGA7 (bootstrap = 1000, Discrete Gamma categories = 5, ML Heuristic Method = NNI, Initial Tree for ML = default NJ/BioNJ, Branch Swap Filter = none). The final phylogenetic tree was edited in FigTree v.3.4.4 software [35] and rooted using Capnocytophaga ochracea (strain ATCC 27872T; Accession Number: U41350) as an external group.

2.3. Mass Spectrometry by MALDI-ToF Analysis

2.3.1. Custom Main Spectra Profile (MSP) Creation

In order to identify the four species of Flavobacterium by the MALDI-ToF MS method, using the Bruker MALDI Biotyper database (v7), the first step was to insert a main spectra profile (MSP) of each one. For this, after detection of the species by multiplex PCR and confirmation by Sanger sequencing (Supplementary Table S1), representative isolates were determined (FC05—F. covae, FC14—F. davisii, FC27—F. oreochormis, and ATCC49512—F. columnare) for inclusion in the device database as standard MSPs. The strains that had been previously cultivated in MHSA (as mentioned in Section 2.1) were collected to perform protein extraction, which was performed as described in the previous works of the AQUAVET research team [36,37].

For spectra acquisition and evaluation, 1 μL of the supernatant obtained from protein extraction was added to 8 different spots on the MSP 96 target polished steel plate. After drying, 1 μL of the α-cyano-4-hydroxycinnamic acid (HCCA) matrix was added. Triplicate readings were performed at each point to obtain 24 spectra, via the FlexControl MicroFlex LT software (version 3.4, Bruker Daltonics), which were then analyzed using the FlexAnalysis software (Bruker Daltonics). The 3 worst-quality spectra were discarded, while the selected spectra were forwarded to the MALDI Biotyper 3 software (Bruker Daltonics) to create the MSPs. Those MSPs that, when evaluated by the software quality control, presented a minimum score of 2.700 and a peak frequency of 75% were considered approved. The protocols were performed according to the manufacturer’s recommendations.

2.3.2. Bacterial Identification by MALDI-ToF MS

The Flavobacterium isolates underwent the identification process after the creation and inclusion of the MSPs. For this, the 50 isolates already cultivated in MHSA had between 1 and 3 colony forming units (CFUs) collected with the aid of a sterile disposable wooden toothpick and placed in an identification spot on the MSP 96 target polished steel plate. Then, 1 µL of formic acid (70%) was added, and after the solution dried, 1 µL of HCCA matrix was applied and allowed to dry naturally. All spectra were acquired using the FlexControl MicroFlex LT software (Bruker Daltonics) with the same parameters described in Assis et al. [22]. The standard bacterial test (BTS) (E. coli DH5 alpha; Bruker Daltonics) was used to calibrate the mass detection. The scoring criteria for confirming the microorganisms were those recommended by the manufacturer, where scores ≥ 2000 indicate identification of the species, scores ≥1700 and <2000 indicate identification relative to the genus, while scores < 1700 are considered as an absence of reliable identification.

2.4. Statistical Analysis

Comparisons between the multiplex PCR and MALDI-TOF MS identification methods were performed using Jamovi software version 1.6 [38,39,40], with agreement rates determined by the Kappa coefficient (ρ < 0.05).

3. Results

3.1. Species Identification by 16S rRNA Gene Sequencing

The 16S rRNA gene of 29 Brazilian isolates and 2 F. columnare ATCC references were successfully sequenced and identified as Flavobacterium species after the BLAST procedure (Table 1). Identity evaluation showed that F. oreochromis (13/17 clinical cases, 76.5%) and F. covae (3/17 clinical cases, 17.6%) were present in columnaris disease cases of Brazilian aquaculture fish. Additionally, there were two isolates (FC14 and FC50) that displayed high identity (> 98.9%) with strain CUVET1215, which was registered as F. columnare in the NCBI database. A further phylogenetic evaluation (Figure 1) showed that both these isolates, FC14 and FC50, and the strain CUVET1215 were all clustered inside the branch of F. davisii (2/17 clinical cases, 11.8%), as highlighted in a previous study [41]. Interestingly, although most clinical cases evaluated (n = 17) displayed isolates belonging to just one Flavobacterium species, there was one clinical case (Clinical Case 6) involving two species simultaneously (F. davisii and F. covae) in an outbreak in O. niloticus (Table 2).

Figure 1 displays a phylogenetic tree constructed with 29 Brazilian isolates, representing all 17 clinical cases of Flavobacterium spp. reported in this study. The tree also includes 16S rRNA gene sequences from 20 Flavobacterium sp. fish strains used by LaFrentz et al. (2022) [3] and 5 additional Flavobacteriaceae strains available in the NCBI database: F. columnare (strain CUVET1215; Accession Number: KF274040.1), Capnocytophaga ochracea (strain ATCC 27872-T; Accession Number: U41350), F. lacunae (strain AHQ-46; Accession Number: LN717318.1), F. inkyongense (strain IMCC27201; Accession Number: KX025140.1), and F. verecundum (strain TTM-46; Accession Number: LN849950.1). Overall, all Brazilian isolates clustered within the phylogenetic branches of F. oreochromis (n = 21), F. covae (n = 6), and F. davisii (n = 2), with high bootstrap support (≥0.7).

3.2. Species Identification by Multiplex PCR and MALDI-TOF MS

Of the 52 isolates identified by multiplex PCR, 51 had their species determined by the technique. The results showed that the species F. oreochromis, F. covae, F. davisii, and F. columnare were identified in 76.92% (n = 40), 11.54% (n = 6), 5.77% (n = 3), and 3.85% (n = 2) of the isolates, respectively. Only isolate PD02, 1.92%, did not present any detectable amplicon, its identification not being allowed by the method (Table 1). Additionally, multiplex PCR amplicon identities of six selected isolates were confirmed by BLAST analysis (Supplementary Table S1). As expected, two isolates each of F. davisii, F. covae, and F. oreochromis were identified, in agreement with the amplicon size and Flavobacterium species described by LaFrentz et al. [10,11].

In order to evaluate the applicability of the MALDI Biotyper in performing the identification of Flavobacterium isolates, an MSP was created for each of the four species analyzed in this study using the isolates FC05—F. covae, FC14—F. davisii, FC27—F. oreochormis, and the reference strain ATCC49512—F. columnare. The MSPs presented a range of m/z peaks from 3086.50 to 13922.38, X to Y, with reproducibility between 76.2% and 100%, and were considered suitable for identification of the strains.

After the inclusion of MSPs, 98.08% (n = 51) of the isolates were identified at the species level, with mean, maximum, and minimum scores of 2.226, 2.022, and 2.601, respectively (Table 1). Isolate FC32 was not identified by MALDI at the species level; only the genus was reliably identified (score = 1.917) (Table 1). The proportions in relative frequencies were preserved in relation to the results obtained by multiplex PCR. The identification methods by multiplex PCR and MALDI-TOF MS were compared using the Kappa agreement method; it was found that the two methods tested obtained 94.2% agreement (Kappa = 0.852 and ρ < 0.001); this value indicates an almost perfect agreement.

3.3. Epidemiological Dispersion of Flavobacterium spp.

The observed frequencies of identified Flavobacterium species by origin and host are shown in Table 2. F. oreochromis was the only species present in all five Brazilian states considered in this study. In addition, this species was mainly isolated from diseased O. niloticus (6/17 clinical cases) and the neotropical fish C. macropomum (5/17 clinical cases). Other neotropical fish species, such as B. amazonicus (1/17 clinical cases) and Colossoma macropomum × Piaractus brachypomus (1/17 clinical cases), were also associated with fish disease by F. oreochromis. A similar host diversity infection was observed with F. covae, which was isolated from O. niloticus and two siluriform species (L. alexandri and L. marmoratus × P. fasciatum), but with a lower observed frequency (all with 1/17 clinical cases) and restricted to only one Brazilian state. Finally, F. davisii was only isolated from diseased O. niloticus (2/17 clinical cases) and from one Brazilian state.

4. Discussion

The etiology of columnaris disease in fish was recently associated with four different Flavobacterium species [3]. This study showed that Brazilian Flavobacterium isolates associated with columnaris diseases in aquacultured fish belong to at least three new species (F. covae, F. davisii, and F. oreochromis) described by LaFrentz et al. [3]. Interestingly, none of the Brazilian isolates reported in this study were identified as F. columnare by biomolecular diagnostic tools (16S rRNA gene phylogeny and multiplex PCR) or by proteomic tools (MALDI-ToF MS). Similar results were observed in other studies, where all Flavobacterium isolates recovered from tilapia did not belong to F. columnare [10,11,26]. Additionally, to our knowledge, F. columnare has not been reported in tilapia according to the new taxonomic classification in the specialized literature. However, this does not imply that this bacterial species is absent in farmed tilapia worldwide or in Brazil, as columnaris disease continues to be reported in various aquaculture fish species in the country [22,42]. On the other hand, it is likely that F. columnare is adapted to cause disease in fish species from temperate or cold-water environments, as highlighted in another study [26]. Following the taxonomic reclassification of Flavobacterium species, a new diagnostic approach is needed to improve the epidemiological data in Brazil.

Despite this study reporting Flavobacterium sp. isolates obtained from six different fish hosts, 47% (8/17) of all clinical cases were related to farmed O. niloticus. This may also explain the higher frequency of F. oreochromis (35.2%, 6/17) observed in this fish species compared to the others, as well as the high frequency of this pathogen when the observed frequencies are confined to the O. niloticus species itself (75%, 6/8). According to LaFrentz et al. [26], F. oreochromis would be more frequently associated with columnaris disease in fish species of the Cichlidae family, such as tilapia (Oreochromis sp.) and Nile tilapia (O. niloticus), farmed in countries in Asia (Thailand, Malaysia, Vietnam, China, and Indonesia) and the Americas (Brazil, Ecuador, Honduras, and Costa Rica). Although F. oreochromis was more frequently associated with clinical cases in O. niloticus, the observed frequency in diseased C. macropomum, a species of fish in the Serrasalmidae family, was also quite significant (29.4%, 5/17). Furthermore, this study presents the first report identifying F. oreochromis as causing columnaris disease in native fish, in the species Brycon amazonicus, of the Bryconidae family, and in the hybrid Colossoma macropomum × Piaractus brachypomus, of the Serrasalmidae family. Considering the higher observed frequency of F. oreochromis isolation (76.4%, 13 out of 17) in various diseased fish species from different Brazilian aquaculture locations in this present report and its proven virulence in O. niloticus, C. macropomum, Lates calcarifer, and Ictalurus punctatus [12,13,21,43,44], the epidemiological significance of this pathogen in aquaculture fish is noteworthy.

Regarding F. davisii and F. covae, both bacteria were also isolated from diseased O. niloticus, although at low observed frequencies of 11.8 and 5.9%, respectively. The association of columnaris disease with these bacteria has been previously reported [10,11]. However, in this study, these two bacterial species were detected in two different fish from the same clinical case. To our knowledge, this is the first Brazilian report of concurrent infection of F. davisii and F. covae in a single clinical case of columnaris disease in cultured O. niloticus. Similarly, LaFrentz et al. [12] reported the simultaneous detection of F. davisii, F. columnare, and F. covae in diseased channel catfish in aquaculture facilities from the USA, but without isolating these bacteria species. On the other hand, those authors also observed a probable coinfection of F. columnare and F. covae in an individual diseased I. punctatus through multiplex PCR. They suggested a possible role for F. columnare and F. davisii as secondary pathogens following primary infection with F. covae in channel catfish. In Nile tilapia, the pathogenicity of F. oreochromis and F. davisii has already been demonstrated [14,21], but not for F. covae. Further studies are needed to elucidate the pathogenic capabilities of F. covae and its role in coinfections with other Flavobacterium species.

In this study, the only two clinical cases of columnaris disease in siluriformes farmed in Brazil were associated with F. covae. In addition, a previous report demonstrated the pathogenic capability of F. covae in two species of siluriformes fish from South America [5]. Similarly, reports from the USA have shown a moderate frequency of F. covae in diseased I. punctatus, an important siluriforme species for aquaculture purposes in that country [12,15]. Although this information suggests the epidemiological relevance of F. covae in siluriformes, it is important to note that this bacterium has recently been proven pathogenic and associated with outbreaks in perciform fish, such as L. calcarifer [45]. Furthermore, experimental challenges with various F. covae strains in channel catfish have shown variable pathogenic effects, with mortalities ranging from 0 to 100% [12,15,16]. In contrast, the susceptibility of South American siluriformes of aquaculture importance to F. davisii and F. columnare has not yet been evaluated, unlike the variable susceptibility observed in channel catfish to these three bacteria [12,21] and the experimental susceptibility of F. oreochromis to Rhamdia quelen [25], indicating that this area deserves further investigation.

Of the 50 isolates evaluated in this study, only one isolate, PD02, could not be identified as a Flavobacterium species using multiplex PCR. However, species identification of this isolate as F. oreochromis could be achieved through phylogenetic analysis of the 16S rRNA gene and MALDI-ToF analysis. The multiplex PCR for Flavobacterium species identification is based on the amplification of a region that includes a partial hypothetical protein (targeted by the forward degenerated primer) and an intergenic space (targeted by the reverse degenerated primer) [11]. The failure of multiplex PCR identification could be attributed to a possible lack of DNA integrity. However, this seems unlikely, as the 16S rRNA gene (~1500 bp) was successfully amplified in two DNA extraction attempts of this isolate, while the expected multiplex PCR amplicon (~659 bp) was not. Another possible explanation for the absence of amplicon generation could be nucleotide variability in the target region for this F. oreochromis isolate, which may have prevented primer annealing. Further genomic analysis of this isolate could provide more information about this potential divergent variant.

MALDI-ToF MS has been widely used in the clinical diagnosis of several microorganisms in recent years [46], including emerging etiological agents of infections in fish [36,37,47]. In this study, after the inclusion of MSPs, the Flavobacterium sp. isolates were identified with 98.08% accuracy, which was not possible before, since the scores presented were below 1700. The isolate FC32 was the only one that presented a score of 1.913, below the 2.000 required for a reliable species result by MALDI-ToF MS, despite being identified as F. orechromis by multiplex PCR. Additionally, other isolates recovered from the same outbreak, clinical case 9, were consistently identified as F. oreochromis by both methods. Furthermore, four of these isolates were subsequently analyzed phylogenetically, grouping them within the F. oreochromis branch. Although MALDI-ToF MS proved to be a highly accurate tool for identifying the analyzed Flavobacterium isolates, species identification was not always possible. This limitation may have arisen from minor variations during growth and sample preparation, which can slightly alter ribosomal protein expression and, consequently, the intensity of m/z peaks used for identification [48]. In our study, the isolates were identified using the standard method (whole bacterial cells), achieving scores close to 2.000. To optimize identification, protein extracts could be used, as they increase the likelihood of detecting specific m/z peaks [49].

In order to verify the results obtained via MALDI-ToF MS and multiplex PCR, all isolates were identified by both methods, with the exception of isolate FC32, identified only by multiplex PCR as F. oreochromis, and PD02, identified only by MALDI-ToF MS as F. oreochromis, a result that corroborates the species identified by sequencing the 16S rRNA gene of this isolate, as previously described. The almost perfect agreement is explained by the Kappa value presented in this study, suggesting that both methods can be used to differentiate between Flavobacterium species. These data are in line with what was described by LaFrentz et al. [10], where the analyzed strains were identified by MALDI-ToF MS and multiplex PCR. Although LaFrentz et al. achieved a better result using the algorithm model the Supervised Neural Network (SNN) [3], in this study an almost perfect congruence was obtained using the standard Maldi Biotyper software, which even allowed the assignment of isolates to the currently recognized Flavobacterium species, allowing the validation of previous reports made with these isolates [7,10,12,36,37]. To make a full validation for species-specific identification of the use of MALDI-ToF MS in Flavobacterium in future studies, a more representative number of strains of each species would be necessary.

Overall, the information obtained in this study is valuable for designing control and prevention programs for columnaris-causing bacteria and for encouraging further epidemiologic studies to better understand the impact of columnaris disease in Brazilian aquaculture.

5. Conclusions

This study provides evidence of F. covae, F. davisii, and F. oreochromis circulating in various aquaculture facilities across different regions of Brazil by 16S rRNA gene sequencing, multiplex PCR, and MALDI-ToF MS methods. Additionally, this is the first report of F. covae associated with clinical cases in two South American siluriformes species (Leiarius marmoratus × Pseudoplatystoma fasciatum and Lophiosilurus alexandri) and F. oreochromis associated with diseased fish from two neotropical families, Serrasalmidae (Colossoma macropomum × Piaractus brachypomus) and Bryconidae (Brycon amazonicus).

Author Contributions

Conceptualization, P.C.J.-S., H.L.C., G.C.T. and H.C.P.F.; methodology, P.C.J.-S., H.L.C., J.C.C.R., G.C.T. and H.C.P.F.; validation, J.C.C.R., G.C.T. and H.C.P.F.; formal analysis, P.C.J.-S. and H.L.C.; investigation, P.C.J.-S., H.L.C. and H.C.P.F.; resources, G.C.T. and H.C.P.F.; data curation, P.C.J.-S. and H.L.C.; writing—original draft preparation, P.C.J.-S. and H.L.C.; writing—review and editing, P.C.J.-S., H.L.C., J.C.C.R., G.C.T. and H.C.P.F.; visualization, P.C.J.-S. and H.L.C.; supervision, J.C.C.R., G.C.T. and H.C.P.F.; project administration, P.C.J.-S., H.L.C. and H.C.P.F.; funding acquisition, H.C.P.F. All authors have read and agreed to the published version of the manuscript.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequences used in this study are available on NCBI under the following accession numbers: F. oreochromis (PQ578965, PQ578966, PQ578968, PQ578969, PQ578970, PQ578971, PQ578973, PQ578974, PQ578975, PQ578976, PQ578977, PQ578978, PQ578983, PQ578984, PQ578985, PQ578986, PQ578987, PQ578988, PQ578989, PQ578990, PQ578991, PQ578992, and PQ578993), F. davisii (PQ578967 and PQ578972), and F. covae (PQ578979, PQ578980, PQ578981, and PQ578982).

Acknowledgments

The authors thank Silvia Umeda Gallani for sharing and allowing the use of four isolates from the Laboratory of Applied Microbiology of Aquatic Organisms (LAMAO).

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figure and Tables

Figure 1. Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic positions of Brazilian isolates (in black) among the four Flavobacterium species reported by LaFrentz et al. [10] (F. oreochromis in red, F. columnare in blue, F. covae in green, and F. davisii in yellow) and other Flavobacteriaceae isolates (in purple). Relatedness was inferred using the maximum likelihood method based upon the General Time-Reversible model [GTR + G + I; 50] and rooted with Capnocytophaga ochracea ATCC 27872T. The percentage of trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) is shown next to the main branches. The analysis involved 58 nucleotide sequences, all positions containing gaps and missing data were eliminated, and there was a total of 1255 positions in the final dataset.

Table 1

Identification of strains used in the study; clinical cases; and origin, host, and species results determined by 16S rRNA gene sequencing, multiplex PCR, and MALDI-ToF MS (before and after inclusion of MSPs).

\	Clinical Case	Origin	Host	Geographical Location	Reference	16S rRNA Sequencing			Multiplex PCR	MALDI-ToF MS
						Species	Identity (%)	Coverage (%)	Species	Before Custom MSP Inclusion †	Organism Best Match	After Custom MSP Inclusion
										Score Value †		Score Value
FC01	1	MG	O. niloticus	19°42′23.3″ S + 44°18′46.6″ W	[5]	F. oreochromis	98.54 a	100	F. oreochromis	1.151	F. oreochromis	2.357
FC02	2	MG	O. niloticus	21°16′52.4″ S + 45°03′16.7″ W	[5]	F. oreochromis	99.64 a	100	F. oreochromis	1.218	F. oreochromis	2.194
FC03	3	MG	L. marmoratus × P. fasciatum	19°54′09.4″ S + 43°59′09.8″ W	[5]	F. covae	97.95 b	99	F. covae	1.180	F. covae	2.212
FC04	3	MG	L. marmoratus × P. fasciatum	19°54′09.4″ S + 43°59′09.8″ W	[5]	F. covae	99.35 c	99	F. covae	1.313	F. covae	2.334
FC05	4	MG	L. alexandri	19°54′09.4″ S + 43°59′09.8″ W	[5]	F. covae	99.50 b	99	F. covae	1.324	F. covae	2.255
FC06	4	MG	L. alexandri	19°54′09.4″ S + 43°59′09.8″ W	[5]	F. covae	99.85 b	100	F. covae	1.340	F. covae	2.157
FC07	5	MG	O. niloticus	18°35′26.5″ S + 45°20′10.9″ W	[5]	F. oreochromis	99.71 a	100	F. oreochromis	1.268	F. oreochromis	2.107
FC08	5	MG	O. niloticus	18°35′26.5″ S + 45°20′10.9″ W	[5]	F. oreochromis	99.43 a	99	F. oreochromis	1.332	F. oreochromis	2.098
FC09	5	MG	O. niloticus	18°35′26.5″ S + 45°20′10.9″ W	[5]	F. oreochromis	99.71 a	99	F. oreochromis	1.323	F. oreochromis	2.022
FC10	5	MG	O. niloticus	18°35′26.5″ S + 45°20′10.9″ W	[5]	F. oreochromis	98.69 a	100	F. oreochromis	1.253	F. oreochromis	2.194
FC11	5	MG	O. niloticus	18°35′26.5″ S + 45°20′10.9″ W	[5]	F. oreochromis	99.78 a	99	F. oreochromis	1.222	F. oreochromis	2.231
FC12	6	MG	O. niloticus	21°18′09.1″ S + 45°53′47.2″ W	[5]	F. covae	99.14 c	99	F. covae	1.581	F. covae	2.214
FC13	6	MG	O. niloticus	21°18′09.1″ S + 45°53′47.2″ W	[5]	F. covae	99.28 c	99	F. covae	1.365	F. covae	2.262
FC14	6	MG	O. niloticus	21°18′09.1″ S + 45°53′47.2″ W	This study	F. columnare	99.21 d	99	F. davisii	1.358	F. davisii	2.109
FC21	7	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	F. oreochromis	99.07 a	99	F. oreochromis	1.302	F. oreochromis	2.088
FC22	8	MT	C. macropomum × P. brachypomus	13°48′59.6″ S + 56°05′32.7″ W	This study	-	-	-	F. oreochromis	1.662	F. oreochromis	2.087
FC23	8	MT	C. macropomum × P. brachypomus	13°48′59.6″ S + 56°05′32.7″ W	This study	F. oreochromis	99.85 a	100	F. oreochromis	1.196	F. oreochromis	2.241
FC24	8	MT	C. macropomum × P. brachypomus	13°48′59.6″ S + 56°05′32.7″ W	This study	F. oreochromis	100 a	100	F. oreochromis	1.307	F. oreochromis	2.290
FC25	8	MT	C. macropomum × P. brachypomus	13°48′59.6″ S + 56°05′32.7″ W	This study	-	-	-	F. oreochromis	1.411	F. oreochromis	2.373
FC26	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.655	F. oreochromis	2.091
FC27	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	F. oreochromis	98.72 e	99	F. oreochromis	1.264	F. oreochromis	2.099
FC28	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.578	F. oreochromis	2.200
FC29	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	F. oreochromis	98.47 a	100	F. oreochromis	1.255	F. oreochromis	2.328
FC30	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.611	F. oreochromis	2.121
FC31	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.308	F. oreochromis	2.338
FC32	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.613	F. oreochromis	1.917
FC33	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.696	F. oreochromis	2.421
FC34	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.591	F. oreochromis	2.231
FC35	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.613	F. oreochromis	2.056
FC36	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	F. oreochromis	99.64 a	99	F. oreochromis	1.300	F. oreochromis	2.304
FC37	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.640	F. oreochromis	2.413
FC38	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.628	F. oreochromis	2.097
FC39	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.295	F. oreochromis	2.290
FC40	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	F. oreochromis	99.28 a	99	F. oreochromis	1.244	F. oreochromis	2.319
FC41	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.549	F. oreochromis	2.036
FC42	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.642	F. oreochromis	2.348
FC43	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.251	F. oreochromis	2.406
FC44	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.659	F. oreochromis	2.179
FC46	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.347	F. oreochromis	2.281
FC47	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.525	F. oreochromis	2.026
FC48	9	RO	C. macropomum	8°41′29.1″ S + 63°54′33.2″ W	This study	-	-	-	F. oreochromis	1.608	F. oreochromis	2.220
FC49	10	MG	O. niloticus	19°54′09.4″ S + 43°59′09.8″ W	This study	-	-	-	F. davisii	1.580	F. davisii	2.082
FC50	10	MG	O. niloticus	19°54′09.4″ S + 43°59′09.8″ W	This study	F. columnare	98.91 d	100	F. davisii	1.348	F. davisii	2.252
PD01	11	SP	O. niloticus	23°39′8.212″ S + 47°13′17.9″ W	This study	F. oreochromis	99.85 a	99	F. oreochromis	1.312	F. oreochromis	2.361
PD02	12	SP	O. niloticus	23°39′8.212″ S + 47°13′17.9″ W	This study	F. oreochromis	99.49 a	99	NA*	1.255	F. oreochromis	2.328
PD03	13	SP	O. niloticus	23°39′8.212″ S + 47°13′17.9″ W	This study	F. oreochromis	99.78 a	100	F. oreochromis	1.282	F. oreochromis	2.325
AM-FC08	14	AM	C. macropomum	2°30′32.695″ S + 59°36′1.8″ W	[25]	F. oreochromis	98.97 a	99	F. oreochromis	1.192	F. oreochromis	2.354
AMFC30	15	AM	C. macropomum	1°13′14.862″ S + 60°1′22.8″ W	[17]	F. oreochromis	99.85 a	99	F. oreochromis	1.232	F. oreochromis	2.196
AM-FC42	16	SP	C. macropomum	20°47′1.05″ S + 49°21′34.2″ W	This study	F. oreochromis	99.42 a	99	F. oreochromis	1.548	F. oreochromis	2.060
AM-FC56	17	AM	B. amazonicus	3°6′26.669″ S + 59°58′36.545″ W	This study	F. oreochromis	99.93 a	99	F. oreochromis	1.373	F. oreochromis	2.113
ATCC23463	-	USA	Oncorynchus tshawytscha		[28]	F. columnare	99.93 f	100	F. columnare	1.441	F. columnare	2.057
ATCC49512	-	France	Salmo truta		[29]	F. columnare	99.57 g	100	F. columnare	1.562	F. columnare	2.601

Lowercase letters indicate strains used for NCBI identities and their accession numbers: ^a TI2056 (KX711900.1); ^b AL-02-36 (MW353013.1); ^c C#2 (CP015107.1); ^d CUVET1215 (KF274040.1); ^e TI1690 (KX711899.1); ^f B185R (CP013012.1); ^g 2023717G (OR946449.1). AM, Amazonas State; MG, Minas Gerais State; MT, Mato Grosso State; SP, São Paulo State; RO, Rondônia State. NA*, No amplicon detected. †, Indicates that the scores were less than 1.700 and that the strains were considered as “not reliably identified”.

Table 2

Frequency of identified Flavobacterium species by origin and host. Percentage values are relative to the total clinical cases (N = 17).

		Flavobacterium Species (Number of Clinical Cases; Percentage)
		F. oreochromis	F. covae	F. davisii	Concurrent Infection(F. covae and F. davisii)	Total
Origin	AM	n = 3; 17.6%	-	-	-	n = 3; 17.6%
	MG	n = 3; 17.6%	n = 2; 11.8%	n = 1; 5.9%	n = 1; 5.9%	n = 7; 41.2%
	MT	n = 1; 5.9%	-	-	-	n = 1; 5.9%
	RO	n = 2; 11.8%	-	-	-	n = 2; 11.8%
	SP	n = 4; 23.5%	-	-	-	n = 4; 23.5%
Host	O. niloticus	n = 6; 35.2%	-	n = 1; 5.9%	n = 1; 5.9%	n = 8; 47.0%
	C. macropomum	n = 5; 29.4%	-	-	-	n = 5; 29.4%
	B. amazonicus	n = 1; 5.9%	-	-	-	n = 1; 5.9%
	L. alexandri	-	n = 1; 5.9%	-	-	n = 1; 5.9%
	C. macropomum × P. brachypomus	n = 1; 5.9%	-	-	-	n = 1; 5.9%
	L. marmoratus × P. fasciatum	-	n = 1; 5.9%	-	-	n = 1; 5.9%
	Total	n = 13; 76.4%	n = 2; 11.8%	n = 1; 5.9%	n = 1; 5.9%	n = 17; 100%

AM, Amazonas State; MG, Minas Gerais State; MT, Mato Grosso State; SP, São Paulo State; RO, Rondônia State.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/pathogens14030219/s1: Table S1: BLAST identities of Brazilian F. columnare isolates based on sequencing of multiplex amplicons. Table S2: Sequences of primers used for the multiplex PCR for Flavobacterium species, including amplicon sizes.

References

1. Parte, A.C.; Sardà, C.J.; Meier-Kolthoff, J.P.; Reimer, L.C.; Göker, M. List of Prokaryotic Names with Standing in Nomenclature (LPSN) Moves to the DSMZ. Int. J. Syst. Evol. Microbiol.; 2020; 70, pp. 5607-5612. [DOI: https://dx.doi.org/10.1099/ijsem.0.004332] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32701423]

2. Kim, M.; Cha, I.-T.; Lee, K.-E.; Li, M.; Park, S.-J. Pangenome Analysis Provides Insights into the Genetic Diversity, Metabolic Versatility, and Evolution of the Genus Flavobacterium. Microbiol. Spectr.; 2023; 11, e01003-23. [DOI: https://dx.doi.org/10.1128/spectrum.01003-23] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37594286]

3. Declercq, A.M.; Haesebrouck, F.; Van den Broeck, W.; Bossier, P.; Decostere, A. Columnaris Disease in Fish: A Review with Emphasis on Bacterium-Host Interactions. Vet. Res.; 2013; 44, 27. [DOI: https://dx.doi.org/10.1186/1297-9716-44-27]

4. Davis, H.S. A New Bacterial Disease of Fresh-Water Fishes. Bulletin of Bureau of Fisheries-USA; Government Printing Office: Washington, DC, USA, 1922; Volume 38.

5. Barony, G.M.; Tavares, G.C.; Assis, G.B.N.; Luz, R.K.; Figueiredo, H.C.P.; Leal, C.A.G. New Hosts and Genetic Diversity of Flavobacterium columnare Isolated from Brazilian Native Species and Nile Tilapia. Dis. Aquat. Organ.; 2015; 117, pp. 1-11. [DOI: https://dx.doi.org/10.3354/dao02931] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26575151]

6. Çağatay, İ.T. Use of Proteomic-Based MALDI-TOF Mass Spectra for Identification of Bacterial Pathogens in Aquaculture: A Review. Aquacult Int.; 2024; 32, pp. 7835-7871. [DOI: https://dx.doi.org/10.1007/s10499-024-01544-x]

7. Malecki, J.K.; Roy, L.A.; Arias, C.R.; Nhat Truong, T.; Hanson, T.R.; Lange, M.D.; Shoemaker, C.A.; Beck, B.H. Bioeconomic Analysis of Flavobacterium columnare Vaccine Pond Trials with Channel Catfish. N. Am. J. Aquac.; 2021; 83, pp. 207-217. [DOI: https://dx.doi.org/10.1002/naaq.10191]

8. Ordal, E.J.; Rucker, R.R. Pathogenic Myxobacteria. Proc. Soc. Exp. Biol. Med.; 1944; 56, pp. 15-18. [DOI: https://dx.doi.org/10.3181/00379727-56-14572]

9. Bernardet, J.-F.; Segers, P.; Vancanneyt, M.; Berthe, F.; Kersters, K.; Vandamme, P. Cutting a Gordian Knot: Emended Classification and Description of the Genus Flavobacterium, Emended Description of the Family Flavobacteriaceae, and Proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978). Int. J. Syst. Evol. Microbiol.; 1996; 46, pp. 128-148. [DOI: https://dx.doi.org/10.1099/00207713-46-1-128]

10. LaFrentz, B.R.; Králová, S.; Burbick, C.R.; Alexander, T.L.; Phillips, C.W.; Griffin, M.J.; Waldbieser, G.C.; García, J.C.; de Alexandre Sebastião, F.; Soto, E. et al. The Fish Pathogen Flavobacterium columnare Represents Four Distinct Species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and Emended Description of Flavobacterium columnare. Syst. Appl. Microbiol.; 2022; 45, 126293. [DOI: https://dx.doi.org/10.1016/j.syapm.2021.126293]

11. LaFrentz, B.R.; García, J.C.; Shelley, J.P. Multiplex PCR for Genotyping Flavobacterium columnare. J. Fish Dis.; 2019; 42, pp. 1531-1542. [DOI: https://dx.doi.org/10.1111/jfd.13068]

12. LaFrentz, B.R.; Khoo, L.H.; Lawrence, M.L.; Petrie-Hanson, L.; Hanson, L.A.; Baumgartner, W.A.; Hemstreet, W.G.; Kelly, A.M.; García, J.C.; Shelley, J.P. et al. Flavobacterium covae Is the Predominant Species of Columnaris-Causing Bacteria Impacting the Channel Catfish Industry in the Southeastern United States. J. Aquat. Anim. Health; 2024; 36, pp. 3-15. [DOI: https://dx.doi.org/10.1002/aah.10207] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37859458]

13. de Abreu Reis Ferreira, D.; Assane, I.M.; Vaneci-Silva, D.; do Vale Oliveira, A.; Tamashiro, G.D.; Hashimoto, D.T.; Pilarski, F. Morpho-Molecular Identification, Pathogenicity for Piaractus mesopotamicus, and Antimicrobial Susceptibility of a Virulent Flavobacterium columnare Isolated from Nile Tilapia Cultured in Brazil. Aquaculture; 2022; 560, 738486. [DOI: https://dx.doi.org/10.1016/j.aquaculture.2022.738486]

14. Guan, B.; Cai, Y.; Zhou, Y.; Zhao, Z.; Wang, Y.; Zhang, D.; Wang, S. Pathogen Identification, Risk Factor and Preventive Measure of a Columnaris Disease Outbreak in Tilapia (Oreochromis niloticus) Eggs and Larvae from a Tilapia Hatchery. Aquaculture; 2022; 561, 738718. [DOI: https://dx.doi.org/10.1016/j.aquaculture.2022.738718]

15. Wise, A.L.; LaFrentz, B.R.; Kelly, A.M.; Liles, M.R.; Griffin, M.J.; Beck, B.H.; Bruce, T.J. The Infection Dynamics of Experimental Edwardsiella Ictaluri and Flavobacterium covae Coinfection in Channel Catfish (Ictalurus punctatus). Pathogens; 2023; 12, 462. [DOI: https://dx.doi.org/10.3390/pathogens12030462]

16. Wise, A.L.; LaFrentz, B.R.; Kelly, A.M.; Liles, M.R.; Griffin, M.J.; Beck, B.H.; Bruce, T.J. Coinfection of Channel Catfish (Ictalurus punctatus) with Virulent Aeromonas hydrophila and Flavobacterium covae Exacerbates Mortality. J. Fish Dis.; 2024; [DOI: https://dx.doi.org/10.1111/jfd.13912] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38214100]

17. Mielke, T.D.; Francisco, C.J.; Dorella, F.A.; Figueiredo, H.C.P.; Tavares, G.C.; Gallani, S.U. The Strategic Use of Water Additives for Tambaqui Colossoma macropomum Transport: New Insights of Bacteriosis and Productivity Approach. Aquaculture; 2022; 558, 738406. [DOI: https://dx.doi.org/10.1016/j.aquaculture.2022.738406]

18. Han, R.; Hong, Y.; Xu, R.; Guo, W.; Zhang, M.; Lu, Z.; Han, Q.; Mo, Z.; Dan, X.; Li, Y. Genomic Evidence of Genetic Diversity and Functional Evolution in Flavobacterium columnare. Front. Microbiol.; 2023; 14, 1240471. [DOI: https://dx.doi.org/10.3389/fmicb.2023.1240471]

19. Shama, S.; Brandão, D.A.; de Vargas, A.C.; da Costa, M.M.; Pedrozo, A.F. Bactérias com potencial patogênico nos rins e lesões externas de jundiás (Rhamdia quelen) cultivados em sistema semi-intensivo. Ciência Rural.; 2000; 30, pp. 293-298. [DOI: https://dx.doi.org/10.1590/S0103-84782000000200016]

20. Arias, C.R.; Welker, T.L.; Shoemaker, C.A.; Abernathy, J.W.; Klesius, P.H. Genetic Fingerprinting of Flavobacterium columnare Isolates from Cultured Fish. J. Appl. Microbiol.; 2004; 97, pp. 421-428. [DOI: https://dx.doi.org/10.1111/j.1365-2672.2004.02314.x]

21. Figueiredo, H.C.P.; Klesius, P.H.; Arias, C.R.; Evans, J.; Shoemaker, C.A.; Pereira Jr, D.J.; Peixoto, M.T.D. Isolation and Characterization of Strains of Flavobacterium columnare from Brazil. J. Fish Dis.; 2005; 28, pp. 199-204. [DOI: https://dx.doi.org/10.1111/j.1365-2761.2005.00616.x]

22. Pilarski, F.; Rossini, A.J.; Ceccarelli, P.S. Isolation and Characterization of Flavobacterium columnare (Bernardet et al. 2002) from Four Tropical Fish Species in Brazil. Braz. J. Biol.; 2008; 68, pp. 409-414. [DOI: https://dx.doi.org/10.1590/S1519-69842008000200025]

23. Figueiredo, H.C.P.; Leal, C.A.G. Tecnologias aplicadas em sanidade de peixes. R. Bras. Zootec.; 2008; 37, pp. 8-14. [DOI: https://dx.doi.org/10.1590/S1516-35982008001300002]

24. Leal, C.A.G.; Carvalho-Castro, G.A.; Sacchetin, P.S.C.; Lopes, C.O.; Moraes, A.M.; Figueiredo, H.C.P. Oral and Parenteral Vaccines against Flavobacterium columnare: Evaluation of Humoral Immune Response by ELISA and in Vivo Efficiency in Nile Tilapia (Oreochromis niloticus). Aquacult. Int.; 2010; 18, pp. 657-666. [DOI: https://dx.doi.org/10.1007/s10499-009-9287-x]

25. Baldissera, M.D.; Souza, C.F.; Tavares, G.C.; Valladão, G.M.R.; Da Silva, A.S.; Antoniazzi, A.; Cunha, M.A.; Baldisserotto, B. Purinergic Signaling and Gene Expression of Purinoceptors in the Head Kidney of the Silver Catfish Rhamdia quelen Experimentally Infected by Flavobacterium columnare. Microb. Pathog.; 2020; 142, 104070. [DOI: https://dx.doi.org/10.1016/j.micpath.2020.104070] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32081613]

26. LaFrentz, B.R.; García, J.C.; Waldbieser, G.C.; Evenhuis, J.P.; Loch, T.P.; Liles, M.R.; Wong, F.S.; Chang, S.F. Identification of Four Distinct Phylogenetic Groups in Flavobacterium columnare With Fish Host Associations. Front. Microbiol.; 2018; 9, 452. [DOI: https://dx.doi.org/10.3389/fmicb.2018.00452]

27. Darwish, A.M.; Ismaiel, A.A.; Newton, J.C.; Tang, J. Identification of Flavobacterium columnare by a Species-Specific Polymerase Chain Reaction and Renaming of ATCC43622 Strain to Flavobacterium johnsoniae. Mol. Cell. Probes; 2004; 18, pp. 421-427. [DOI: https://dx.doi.org/10.1016/j.mcp.2004.07.002]

28. Stine, C.B.; Li, C.; Crosby, T.C.; Hasbrouck, N.R.; Lam, C.; Tadesse, D.A. Draft Whole-Genome Sequences of 18 Flavobacterium spp. Genome Announc.; 2017; 5, e00865-17. [DOI: https://dx.doi.org/10.1128/genomeA.00865-17]

29. Tekedar, H.C.; Karsi, A.; Gillaspy, A.F.; Dyer, D.W.; Benton, N.R.; Zaitshik, J.; Vamenta, S.; Banes, M.M.; Gülsoy, N.; Aboko-Cole, M. et al. Genome Sequence of the Fish Pathogen Flavobacterium columnare ATCC 49512. J. Bacteriol.; 2012; 194, pp. 2763-2764. [DOI: https://dx.doi.org/10.1128/JB.00281-12] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22535941]

30. Fox, J.G.; Yan, L.L.; Dewhirst, F.E.; Paster, B.J.; Shames, B.; Murphy, J.C.; Hayward, A.; Belcher, J.C.; Mendes, E.N. Helicobacter bilis sp. nov., a Novel Helicobacter Species Isolated from Bile, Livers, and Intestines of Aged, Inbred Mice. J. Clin. Microbiol.; 1995; 33, pp. 445-454. [DOI: https://dx.doi.org/10.1128/jcm.33.2.445-454.1995] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/7536217]

31. Hall, T.A. BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symp. Ser.; 1999; 41, pp. 95-98.

32. Al-Shuhaib, M.B.S.; Hashim, H.O. Mastering DNA Chromatogram Analysis in Sanger Sequencing for Reliable Clinical Analysis. J. Genet. Eng. Biotechnol.; 2023; 21, 115. [DOI: https://dx.doi.org/10.1186/s43141-023-00587-6] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37955813]

33. Katoh, K.; Standley, D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol.; 2013; 30, pp. 772-780. [DOI: https://dx.doi.org/10.1093/molbev/mst010] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23329690]

34. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol.; 2016; 33, pp. 1870-1874. [DOI: https://dx.doi.org/10.1093/molbev/msw054] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27004904]

35. Rambaut, A. FigTree v1.3.1. Available online: http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 27 June 2024).

36. Assis, G.B.N.; Pereira, F.L.; Zegarra, A.U.; Tavares, G.C.; Leal, C.A.; Figueiredo, H.C.P. Use of MALDI-TOF Mass Spectrometry for the Fast Identification of Gram-Positive Fish Pathogens. Front. Microbiol.; 2017; 8, 1492. [DOI: https://dx.doi.org/10.3389/fmicb.2017.01492]

37. Rocha, V.P.; Gauer, D.; Costa, H.L.; Reis, F.Y.T.; Marcelino, S.A.C.; Nogueira, L.F.F.; Pierezan, F.; Figueiredo, H.C.P.; Tavares, G.C. Francisella Salimarina Infection in Farmed Cobia (Rachycentron Canadum) and Dusky Grouper (Epinephelus Marginatus) in Brazil: A Case Report. J. Fish Dis.; 2024; e13962.Available online: https://onlinelibrary.wiley.com/toc/13652761/0/0 (accessed on 1 February 2025).

38. Gamer, M.; Lemon, J.; Singh, I. Irr: Various Coefficients of Interrater Reliability and Agreement; 2019. Available online: https://CRAN.R-project.org/package=irr (accessed on 26 June 2024).

39. The Jamovi Project. Jamovi. 2021; Available online: https://www.jamovi.org (accessed on 26 June 2024).

40. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2020.

41. Dong, H.T.; LaFrentz, B.; Pirarat, N.; Rodkhum, C. Phenotypic Characterization and Genetic Diversity of Flavobacterium columnare Isolated from Red Tilapia, Oreochromis Sp., in Thailand. J. Fish Dis.; 2015; 38, pp. 901-913. [DOI: https://dx.doi.org/10.1111/jfd.12304]

42. Sebastião, F.D.A.; Pilarski, F.; Lemos, M. Isolation and Molecular Characterization of Flavobacterium columnare Strains from Fish in Brazil. J. Bacteriol. Res.; 2009; 2, pp. 22-29. Available online: https://academicjournals.org/journal/JBR/article-abstract/A41F7BB9085 (accessed on 26 June 2024).

43. Bunnoy, A.; Thangsunan, P.; Chokmangmeepisarn, P.; Yata, T.; Klongklaew, N.; Pirarat, N.; Kitiyodom, S.; Srisapoome, P.; Rodkhum, C. Mucoadhesive Cationic Lipid-Based Flavobacterium oreochromis Nanoencapsulation Enhanced the Efficacy of Mucoadhesive Immersion Vaccination against Columnaris Disease and Strengthened Immunity in Asian Sea Bass (Lates calcarifer). Fish Shellfish. Immunol.; 2022; 127, pp. 633-646. [DOI: https://dx.doi.org/10.1016/j.fsi.2022.06.059] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35779812]

44. Pereira, E.C.; Bontinelly, T.d.F.B.; Nogueira, L.F.F.N.; Tavares, G.C.; Kotzent, S.; Figueiredo, H.C.P.; Pirlaski, F.; Gallani, S.U. Eficácia de Protótipo Vacinal Para O Tambaqui Contra Flavobacterium Oreochromis. Anais do XVII Encontro Brasileiro de Patologistas de Organismos Aquáticos; ABRAPOA: Belo Horizonte, Brasil, 2023; ISBN 978-65-5465-040-3

45. Tumree, P.; Bunnoy, A.; Tang, X.; Srisapoome, P. Efficacy of Whole-Cell-Based Monovalent and Bivalent Vaccines against Streptococcus iniae and Flavobacterium covae in Fingerling Asian Seabass (Lates calcarifer). Fish. Shellfish. Immunol.; 2024; 144, 109269. [DOI: https://dx.doi.org/10.1016/j.fsi.2023.109269]

46. Elbehiry, A.; Aldubaib, M.; Abalkhail, A.; Marzouk, E.; ALbeloushi, A.; Moussa, I.; Ibrahem, M.; Albazie, H.; Alqarni, A.; Anagreyyah, S. et al. How MALDI-TOF Mass Spectrometry Technology Contributes to Microbial Infection Control in Healthcare Settings. Vaccines; 2022; 10, 1881. [DOI: https://dx.doi.org/10.3390/vaccines10111881] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36366389]

47. Segawa, S.; Nishimura, M.; Sogawa, K.; Tsuchida, S.; Murata, S.; Watanabe, M.; Matsushita, K.; Kamei, K.; Nomura, F. Identification of Nocardia Species Using Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry. Clin. Proteom.; 2015; 12, 6. [DOI: https://dx.doi.org/10.1186/s12014-015-9078-5] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25931991]

48. Josten, M.; Reif, M.; Szekat, C.; Al-Sabti, N.; Roemer, T.; Sparbier, K.; Kostrzewa, M.; Rohde, H.; Sahl, H.-G.; Bierbaum, G. Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus Aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages. J. Clin. Microbiol.; 2013; 51, 1809. [DOI: https://dx.doi.org/10.1128/JCM.00518-13] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23554199]

49. Sauget, M.; Valot, B.; Bertrand, X.; Hocquet, D. Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?. Trends Microbiol.; 2017; 25, pp. 447-455. [DOI: https://dx.doi.org/10.1016/j.tim.2016.12.006]