Introduction

Aquaculture is the primary operational solution for food scarcity, capable of filling the gap in food consumption (Jennings et al. 2016). The aquaculture sector is not limited to affluent countries; it has spread to all developing countries, including Egypt. Fish diseases are regarded as one of the most important restrictions in aquaculture, causing annual fish losses, economic downturns, and food poverty (Assefa and Abunna 2018). Fish, like all other aquatic organisms, are susceptible to a variety of diseases, particularly those caused by bacteria.

Vibrio strains are the causative agents of vibriosis, a dangerous bacterial disease that can cause high mortality and significant economic losses in aquaculture sectors (Sanches-Fernandes et al. 2022). Among strains, Vibrio campbellii is a Gram-negative bacterium that is common in marine habitats and serves as a global disease affecting fish and shellfish (Petersen et al. 2021). The bacteria are often associated with luminous vibriosis affecting early stages of penaeid shrimp cultivation and resulting in lower survival rates (Nuidate et al. 2021; Srisangthong et al. 2023). V. campbellii possesses a pVA1-type plasmid and is recently considered one of the major pathogenic strains causing acute hepatopancreatic necrosis disease (AHPND) in penaeid shrimp (Dong et al. 2019).

The quality of water parameters as well as the total microbial load of aquaculture systems, play a critical role in determining the overall success or failure of aquaculture operations. Microorganisms present in these systems have significant impacts on various factors, including productivity, nutrient cycling, disease control, and environmental consequences, leading to economic losses (Moriarty 1997). In the aquatic environment, the spread of virulence factors can occur horizontally, raising concerns that any Vibrio cell has the potential to transform into a toxic strain. The precise mechanisms underlying the emergence of novel toxigenic strains are still unknown. However, it has been claimed that genetic changes and natural selection, driven by environmental conditions, may play a role in this process (León Robles et al. 2013).

Vibriosis serves as a microbiological barometer of temperature warming or climate change, and its incidence is strongly associated with environmental conditions (Hassan et al. 2021). Several environmental variables like dissolved oxygen (DO), nitrogen, pH, phosphate, and turbidity also affect total Vibrio numbers, but they have inconsistent effects (Takemura et al. 2014). Several studies have summarized the prevalence of V. campbellii among various marine shrimps. However, little was known about their pathogenicity and the stressors that could confront the fish host during infection. As far as we know, this is the first study that investigates the molecular typing, prevalence, pathogenicity, and environmental risk factors of V. campbellii, a newly emerging pathogen affecting cultured seabream (Sparus aurata).

Materials and methods

Sampling procedure

Fish and water samples were gathered monthly from private fish-producing sectors located in the Suez Canal area from September 2021 to August 2022.

Fish sampling

About 160 gilthead seabream, Sparus aurata (40 fish/season) with average body weight (230 ± 15 g) and length (20 ± 1.5 cm) were collected randomly and seasonally for clinical investigation and bacteriological assay. Specimens of moribund live and freshly dead fish were transferred in sterile aerated plastic bags and/or insulated iceboxes to the laboratory of Aquaculture Diseases and Control at Fish Farming and Technology Institute, Suez Canal University, for further examination. Fish handling and all experiments were conducted following the guidelines of the Animal Ethics Review Committee of Suez Canal University (AERC-SCU), Egypt, with approved No. 2023017).

Water sampling and analysis

Forty-eight water samples (n = 12/season) were collected from different fishponds meanwhile the survey with a sampling of four samples/month. The water samples were collected from pre-determined distances and depths from the edge of the ponds. We selected carefully sampling points that were representative of the pond environment ensuring that the collected samples were indicative of the water quality in the entire pond. To minimize potential variations due to diurnal changes, we collected water samples at similar times of the day across all sampling locations. By doing so, we aimed to control temporal differences that could affect the water quality; the water samples were cooled during transportation. Maintaining appropriate temperatures during transportation is crucial to preserve the integrity and accuracy of the water quality data. We used insulated containers with ice packs to ensure that the samples remained at the required temperature during transportation. The duration of transportation varied depending on the specific location of each sampling point. On average, the transportation time ranged from < 30 min to approximately 60 min. We made every effort to minimize transportation time to ensure the samples’ quality and integrity for accurate laboratory examination. Water temperature (°C), pH, and DO (mg L−1) were measured onsite using water thermometer, electrometric pH meter, and dissolved oxygen meter (DO meter; Crison OXI 45 P, EU), respectively. Water samples (300 mL) were collected in sterile beakers with stoppers and analyzed on arrival to measure some physico-chemical parameters, including salinity (g L−1) by a conductivity meter (Anderson and Cummings 1999), O. M (mg L−1) by a modified WB–Meibus method (Mebius 1960), and toxic ammonia (NH3) (mg L−1) by a phenate UV screening spectrophotometric method (Koroleff 1976).

Clinical and postmortem examination

The collected fish samples were examined for the presence of external abnormalities and characteristic internal lesions after dissection using three-line incision methods, according to Austin and Austin (2007).

Bacteriological assay

Fresh specimens were examined upon arrival under complete aseptic conditions. For initial recovery of Vibrio isolates, loopful samples from the lesions, liver, kidneys, and spleen were inoculated directly onto tryptic soy agar (TSA) supplied with different concentrations of NaCl (0–8%) and incubated at 28 °C for 24 h. Plates were observed to determine the presence of dense virtually pure culture growth. From these plates, colonies (3 colonies/plate) were subcultured for purity onto TCBS medium (Hi-media, Mumbai) with incubation at 28 °C for 24 h. Pure colonies on TCBS were identified phenotypically by the Gram staining reaction and biochemical tests according to Bergey’s Manual of Determinative Bacteriology (Baumann et al. 1994). The API 20E rapid identification system (BioMérieux) was used following the manufacturer’s instructions.

Molecular approach and sequence analysis

The genomic DNA of purified representative isolates (n = 30) was extracted using a DNA zole kit (Invitrogen, USA) according to manufacturer’s instructions. A high-quality input (hybridized DNA) is required for unbiased and reproducible results; thus, the extracted templates were tested for concentration and integrity using a running gel assay and a Nanodrop machine (Nanodrop 1000, Thermo Scientific, UK). DNA templates at 100 ng μL−1 were kept frozen at −80 °C until further analysis. PCR was performed using set of primers targeting 16SrRNA-conserved gene of Vibrio sp. following to the standard protocol of Tarr et al. (2007). Reaction volume (25 μL) was amplified in T Gradient Thermocycler (Biometra) using a 2X DreamTaq Green Master Mix kit. Amplified products were detected by horizontal 1% (w/v) agarose gel electrophoresis for 45 min at 100 V in TAE 1 × (0.04 M Tris, 0.0001 M EDTA, pH 8.0) electrophoresis buffer. After gel separation, the amplified products were visualized using 15 μL of DNA gel stain (Invitrogen) under UV transilluminator (Fotodyne), photographed using a polaroid MP-4 camera and computer digitized (Gel Doc 100, Bio-Rad). A 100 bp ladder (NZYDNA Ladder V) was used as a molecular mass marker.

Since Vibrio isolates have similar molecular patterns, the PCR product of one selected culture was sent for direct sequencing after the purification by the QIAquick PCR Product extraction kit (Qiagen Inc. Valencia CA). A purified PCR product was sequenced in the forward and/or reverse directions on an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA) using a ready reaction BigDye Terminator V3.1 Cycle Sequencing Kit. (Perkin-Elmer/Applied Biosystems, Foster City, CA), with Cat. No. 4336817. A BLAST® analysis (Basic Local Alignment Search Tool) (Altschul et al. 1990) was initially performed to establish sequence identity to GenBank accessions. A comparative analysis of sequences was performed using the CLUSTAL W multiple sequence alignment program, version 1.83 of MegAlign module of Lasergene DNAStar software Pairwise (Thompson et al. 1994) and phylogenetic tree was drawn using neighbor-joining in MEGA6 (Tamura et al. 2013). The obtained sequence was deposited in the GenBank with accession number OR125508.

Histopathological examination

Fresh specimens of the liver, kidney, and spleen of naturally infected fish were collected and immediately preserved in 10% natural buffered formalin for histopathological study (Suvarna et al. 2018). After being cleaned in two changes of xylene and dehydrated in ethyl alcohol at ascending concentrations, the fixed tissues were embedded and blocked in paraffin wax. Using the rotary microtome (Chin, YD-315), the tissues were sectioned into 5-μm thickness. The dewaxed tissue sections were stained with standard hematoxylin and eosin (H&E) stains. The specimen was examined and photographed by a light microscope (Carl Zeiss, Primo Star, Germany) equipped with a digital camera (AxioCam, ERc 5s, Germany).

Challenge test

Healthy gilthead seabream (n = 120), weighing 30 ± 6g and measuring 12 ± 1 cm in length, were procured from a private fish farm located in the Suez Canal region. Prior to the experimental phase, the fish were acclimatized for 2-week period in fiberglass tanks filled with aerated sand-filtered seawater. Subsequently, the fish were divided into two equal groups (n = 60), each contributing three replicates of 20 fish. These fish were placed in flat-bottomed fiberglass aquaria with a volume of 100 L. The first group served as a control and was injected intraperitoneally with 0.1 ml of sterile saline; the second group was injected with 0.1 ml of V. campbellii bacterial suspension at a final concentration of 1 × 106 CFU (according to Balebona et al. 1998).

All groups were closely monitored for 10 days after challenge, and mortalities and disease signs were recorded. Throughout the experiment, strict adherence to optimal environmental conditions was maintained. Prior to their inclusion in the challenge trial, the fish were closely observed to ensure their overall health. To prepare the inoculum, V. campbellii, previously isolated from gilthead seabream, identified phenotypically and molecularly was used. A bacterial culture was prepared in nutrient broth containing 2% (w/v) NaCl and suspensions prepared by washing and re-washing in sterilize 9.9% (w/v) saline. The number of bacterial cells was determined using the McFarland Turbidity Standard, and finally adjusted to 1 × 106 CFU/mL (according to Balebona et al. 1998).

Statistical analyses

The analysis utilized the XLSTAT 2018.1.49386 software, specifically employing the ROC Curves module. The event data consisted of a single variable V. campbellii, while the test data included six variables (temperature, dissolved oxygen, salinity, toxic ammonia, organic matter, and pH), all having the same number of observations. The area under the curve (AUC) was calculated using the Bamber variance estimator. The challenge test data and logistic regression analysis were analyzed using the SPSS 27.0 (SPSS Inc., USA). Mean values ± S.E.M. were employed to represent the data, and statistical tests such as one-way and two-way ANOVA, as well as chi-square, were used to determine significant differences among groups. To establish group differences, Duncan’s Multiple Range test (Duncan 1955) was applied. The data were considered statistically significant if the p-value was less than 0.05.

Results

Clinical and postmortem examination

The majority of naturally infected seabream, Sparus aurata, showed slight abdominal distension and corneal opacity (Fig. 1a), skin darkness, caudal fin erosions, and big ulcerations with detached scales (Fig. 1b). The necropsy findings revealed petechial hemorrhages on the internal organs including swimming bladder and intestine. Congested gills, pale liver, engorged spleen, and distended intestine with profuse serosanguinous fluid and decaying odor were the most predominant signs (Fig. 2).

Fig. 1
figure 1

Naturally infected gilthead seabream, Sparus aurata, showing (a) slight abdominal distension and corneal opacity, b skin darkness, caudal fin redness and erosions, and ulcerations with detached scales

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Fig. 2
figure 2

Naturally infected gilthead seabream, Sparus aurata, showing congested gills, pale liver, engorged spleen, and distended intestine with profuse serosanguinous fluid and decaying odor

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Bacteriological assay

All isolates (n = 90) comprised motile, Gram-negative, straight, or curved short motile. The predominant colonies on TCBS media exhibited characteristic round green morphology, approximately 2–3 mm in diameter, with swarming activity. The colonies were able to grow on nutrient agar with 2– 4% (w/v) NaCl but showed no growth at higher salt concentrations, i.e., 6–10 %. On blood agar, the bacteria exhibited beta-hemolytic activity. Biochemically, the isolated strains revealed were oxidase and catalase positive, sensitive to the vibriostatic agent O/129–150 μg and novobiocin. These results were matched the characteristics of V. campbellii in Bergey’s Manual. The presumptive identification of selected isolates using the API 20E system revealed 96% probability of V. campbellii (code 4045105).

Molecular approach and sequence analysis

At the molecular level, all isolates (n = 30 ) were positive for the 16S rRNA conserved gene and gave an expected amplicons of 663 bp. Unequivocally, the represented isolate was identified as V. campbellii based on 99.85% nucleotide identity to the partial sequence of the 16S rRNA conserved gene. The obtained sequence was deposited in GenBank under accession number OR125508. A phylogenetic tree was drawn following blasting the 16S rRNA gene sequence of the isolated strain with other homologous sequences available in GenBank using a neighbor joining method (Fig. 3).

Fig. 3
figure 3

It demonstrates the phylogenetic analysis of V. campbellii 16S rRNA gene sequencing. The tree explains the genetic relationship of the tested V. campbellii strain and other homologues deposited in the GenBank database. The isolated strain in this study is marked with a solid green square

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Prevalence of V. campbellii among cultured gilthead seabream (Sparus aurata)

The total number of cases with V. campbellii was 24, representing 15% of the total sample. Although the chi-square value of 1.9608 was not statistically significant (p = 0.5806), the individual analyses for each season demonstrated clear associations. These findings suggest that different seasons may be associated with variations in the occurrence of V. campbellii. Specifically, summer exhibited a higher proportion of cases with V. campbellii, while spring, autumn, and winter had lower proportions. These results provide valuable insights into the relationship between seasonal variation and V. campbellii, highlighting the importance of considering seasonal factors when studying or addressing this outcome (Table. 1).

Table 1 The logistic regression analysis to examine the association between each season and the outcome variable V. campbellii
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The calculation was based on the number of gilthead seabream (Sparus aurata) per season (n = 40 season)

Pond water characteristics and the frequency of V. campbellii

ROC (Receiver Operating Characteristic) curve analysis was performed on different water quality parameters to evaluate its diagnostic performance. The analysis involved calculating sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the combination of sensitivity and specificity for various threshold values of different water quality parameters concentration. The analysis conducted on the different variables suggests varying levels of predictive performance in relation to the outcome variable V. campbellii. Firstly, when examining NH3 as a predictor, the analysis indicates that there is no significant deviation of the area under the curve (AUC) from 0.5. This suggests that the classifier’s performance using NH3 as a predictor is not significantly better than a random classifier. Therefore, NH3 may not be a reliable predictor for the outcome represented by V. campbellii (Fig. 4). Similarly, when evaluating the variable DO, the AUC for the ROC curve is not significantly different from 0.5. This finding suggests that the predictive performance of DO may not be significantly better than a random classifier, indicating limited utility in predicting the V. campbellii variable (Fig. 5).

Fig. 4
figure 4

ROC curve was performed on toxic ammonia (NH3), p-value (two-tailed) = 0.71, and the test is positive if the NH3 concentration is greater than or equal to the threshold value (≥ 0.03)

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Fig. 5
figure 5

ROC curve was performed on dissolved oxygen (DO), p-value (two-tailed) = 0.149, and the test is positive if DO concentration is greater than or equal to the threshold value (≥ 5.7)

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Furthermore, the analysis reveals that the temperature variable has a limited ability to predict the “V. campbellii” variable. Although the AUC is slightly above 0.5, the difference is not statistically significant. Consequently, temperature alone may not be a strong predictor for the V. campbellii variable (Fig. 6). Moreover, based on the analysis, there is insufficient evidence to suggest that the AUC for the relationship between Temp and compb significantly deviates from 0.5. This implies that there is not enough statistical support to establish a meaningful relationship between temperature and the outcome represented by V. campbellii.

Fig. 6
figure 6

ROC curve was performed on Temperature (Temp), p-value (two-tailed) = 0.389, and the test is positive if the temperature concentration is greater than or equal to the threshold value (≥ 17)

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Conversely, the analysis provides evidence to support the significance of the AUC for the relationship between ammonia and V. campbellii. The AUC value is significantly different from 0.5, indicating a meaningful predictive power of the ammonia variable in determining the outcome represented by V. campbellii. This suggests that ammonia can be considered a meaningful predictor for the variable of interest (Fig. 7).

Fig. 7
figure 7

ROC curve was performed on pH, p-value (two-tailed) = 0.042, and the test is positive if the ammonia concentration is greater than or equal to the threshold value (≥ 8.1)

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Additionally, the variable organic matter exhibits a moderate AUC value of 0.533, indicating that its diagnostic performance is slightly better than random chance. However, the AUC is not significantly different from 0.5, suggesting that the variable may not be a strong predictor for the tested condition, despite performing slightly better than random chance (Fig. 8). Lastly, the variable salinity demonstrates some predictive ability in distinguishing between the outcomes, although the overall performance is not statistically significant. This implies that while salinity may have some predictive value, it is not consistently reliable in determining the outcome represented by V. campbellii (Fig. 9).

Fig. 8
figure 8

ROC curve was performed on organic matters, p-value (two-tailed) = 0.628, and the test is positive if the organic matter concentration is greater than or equal to the threshold value (≥ 12.4)

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Fig. 9
figure 9

ROC curve was performed on salinity, p-value (two-tailed) = 0.611, and the test is positive if the salinity concentration is greater than or equal to the threshold value (≥ 33)

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Histopathological examination

The histopathological alteration caused by infection with Vibrio pathogens among cultured gilthead seabream is illustrated (Fig. 10). The hepato-pancreatic tissue showed significant congestion in the hepatic and pancreatic vessels as well as widespread vacuolar degeneration and coagulative necrosis in the hepatocytes and pancreatic acinar cells with influx of the inflammatory cells to the degenerative tissue (Fig. 10a, b). Likewise, the majority of the renal tubules and glomeruli showed tubular nephrosis in the form of vacuolar degeneration and coagulative necrosis with mononuclear cell infiltration in the interstitial tissue of the renal parenchyma. Furthermore, clear indications of melanomacrophage centers (MMCs) activity and localized hyperplasia within the hematopoietic tissue were observed (Fig. 10c, d). The splenic tissue exhibited noticeable aggregation of melanomacrophages associated with congested splenic blood vessels, alternative depilation in the lymphoid tissue, and focally active MMCs (Fig. 10e, f).

Fig. 10
figure 10

Histopathological alterations in hepatic, renal and splenic tissues of gilthead seabream infected with vibriosis (H&E stain). a and b Liver micrographs showing vacuolar degeneration (VD) in the hepatocytes and pancreatic acinar cells (PAC) with marked congestion (C) in hepatic and pancreatic vessels with inflammatory cell infiltration (black arrow). c and d Renal micrographs displaying marked activation of melanomacrophage centers (MMCs) with tubular nephrosis (RT, renal tubules) of the renal epithelium in the form of vacuolar degeneration (D) and coagulative necrosis (N) in the majority of renal epithelium and renal glomeruli accompanied with influx of inflammatory cells. e and f Splenic micrographs presenting aggregation of melanomacrophages with activated splenic melanomacrophage centers (MMCs) besides alternative depilation of splenic lymphoid follicles (SLF) with congestion in the spleen

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Challenge with V. campbellii

In the control, no mortalities were observed, and all individuals survived without any evidence of disease. However, in the challenged group, a mortality number of 6.00 ± 0.58 was recorded, with a survival rate of 70.00 ± 2.89%. The experimentally infected fish displayed pathognomonic clinical findings and signs of hemorrhagic septicemia similar to those observed during the natural episode.

Discussion

The present study investigated for the first time the clinical signs and bacteriological, molecular, and histopathological aspects of V. campbellii infection in gilthead seabream (Sparus aurata). Herein, the observed clinical signs are consistent with the previous reports on vibriosis in various fish species. For instance, a review by Manchanayake et al. (2023) indicated that naturally infected fish exhibited similar lesions, including abdominal distension and skin lesions. Likewise, the study performed by Eissa et al. (2017) revealed corneal opacity and skin lesions as common pathognomonic lesions in fish infected with Vibrio species. The manifested signs indicate the systemic nature of the infection and the involvement of multiple organs. The necropsy findings in this study support the clinical findings and point to systemic infection and tissue destruction caused by V. campbellii. Comparable lesions have also been documented in the naturally infected gilthead seabream with Vibrio (Abdel-Aziz et al. 2013; Sanches-Fernandes et al. 2022). Additionally, Eissa and Lester (2017) reported similar lesions in moribund common pandora fish.

In the current study, the bacteriological assay confirmed the presence of V. campbellii in the infected gilthead seabream. The isolated strains exhibited characteristic morphological and biochemical properties that are consistent with the established phenotypic characteristics of V. campbellii (Lorenz et al. 2016; Nuidate et al. 2021). The molecular analysis based on 16S rRNA gene sequencing further confirmed the identity of the isolated strains as V. campbellii. The high nucleotide identity and phylogenetic analysis supported the accurate identification of the isolates. Molecular techniques, such as 16S rRNA gene sequencing, have been widely used for species identification and phylogenetic analysis of Vibrio species (Thompson et al. 2013). The 16S rRNA gene is a good candidate for bacterial identification due to its conserved nature across different species (Clarridge III 2004).

The obtained nucleotide sequences from the isolates showed a high similarity to known sequences of V. campbellii, with a nucleotide identity of 99.85%. To assess the relationship between the isolates and other related sequences, a phylogenetic tree was constructed using the neighbor-joining method. The phylogenetic analysis incorporated homologous sequences of V. campbellii available in the GenBank database. The tree demonstrated the clustering of the isolated strain with other homologous sequences of V. campbellii, providing additional evidence for the accurate identification of the isolates. The findings are in line with the previous study of Halet et al. (2007), who employed 16S rRNA gene sequencing to identify and characterize Vibrio species from marine samples, including V. campbellii. Their results demonstrated the effectiveness of the 16S rRNA gene in differentiating V. campbellii from other Vibrio species. The use of molecular analysis, specifically targeting the 16S rRNA gene, not only strengthens the identification of V. campbellii in this study but also provides a valuable tool for future research on the molecular epidemiology and phylogenetic relationships of this pathogen.

With regard to the prevalence analysis, the results revealed that V. campbellii was present in 15% of the sampled gilthead seabream. Although the chi-square test did not show statistical significance, the individual analyses for each season demonstrated clear associations. Summer and spring exhibited a higher proportion of V. campbellii cases, while autumn and winter had lower proportions. Similar findings have been reported in previous studies investigating the prevalence of Vibrio species in aquatic organisms. For example, a study by Zarei et al. (2012) found variations in the seasonal prevalence of Vibrio species in the collected shrimp samples. Specifically, Vibrios were detected in 18.6% of the samples collected during winter, 64% of the samples in spring, 70.6% of the samples in summer, and 41.3% of the samples in autumn. The results showed that the occurrence of Vibrio spp. was higher during warm months compared to cold ones. These results lend credence to the idea that seasonal fluctuations in environmental parameters could affect the occurrence and abundance of Vibrio species. The results were also highlighting the importance of considering seasonal factors in future studies related to this pathogen. Further studies are warranted to elucidate the underlying mechanisms behind these associations, such as the influence of temperature, water quality, and changes in host physiology. Additionally, understanding the potential implications of seasonal variations in V. campbellii prevalence is crucial for effective management and control strategies in aquaculture settings. Seasonal variation in the prevalence of Vibrio infections has been reported in various fish species. Hjelm et al. (2004) found that V. anguillarum infections in Atlantic cod (Gadus morhua) were more prevalent during warmer months. Similarly, Letchumanan et al. (2015) reported a higher prevalence of V. parahaemolyticus in fish during the summer season. These findings suggest that environmental factors such as temperature may influence the growth and abundance of Vibrio species in aquatic environments.

In addition to the seasonal variation, the relationship between V. campbellii infection and water quality parameters was assessed in this study. The ROC curve analysis also provided valuable insights into the predictive power of different water quality parameters. Among the variables investigated, NH3 (ammonia), DO (dissolved oxygen), temperature, and pH were found to have limited predictive ability for V. campbellii infection. These findings may suggest that while these parameters are essential for overall water quality assessment, they might not be the most reliable indicators of V. campbellii presence alone. Conversely, ammonia (NH3) demonstrated significant predictive ability for V. campbellii infection. High levels of ammonia in the water might create favorable conditions for the growth and survival of V. campbellii, concurrent with the previous findings of Alloul et al. (2021). Moreover, organic matter and salinity exhibited moderate predictive performance, although they did not reach statistical significance. The moderate relationship between organic matter and V. campbellii occurrence suggests that the availability of nutrients in the water might influence the proliferation of the bacterium. Salinity, being a key environmental parameter in aquatic systems, might also play a role in shaping the habitat and potential survival of V. campbellii.

It is essential to recognize that the presence and abundance of V. campbellii may not be solely determined by a single-water quality variable but rather by a combination of factors and their interactions. Therefore, comprehensive assessments that consider multiple water quality parameters and their interplay are crucial for a more accurate understanding of the dynamics of V. campbellii infection in aquatic ecosystems. Several studies have investigated the association between water quality parameters and Vibrio infections in fish. For instance, a study by Oberbeckmann et al. (2012) found a positive correlation between water temperature and Vibrio abundance in coastal waters. Moreover, Johnson et al. (2010) reported that higher levels of dissolved organic carbon were associated with increased V. parahaemolyticus abundance in estuarine waters. These findings indicate that water quality parameters can play a role in the ecology and epidemiology of Vibrio species.

The histopathological examination of infected gilthead seabream provided valuable insights into the tissue alterations associated with V. campbellii infection. Significant histopathological changes were observed in the hepatic, renal, and splenic tissues, indicating the impact of the infection on these organs. In the hepato-pancreatic tissue, congestion was evident in the hepatic and pancreatic vessels, suggesting compromised blood flow. Vacuolar degeneration and coagulative necrosis were observed in hepatocytes and pancreatic acinar cells, indicating cellular damage. Infiltration of inflammatory cells further indicated an immune response to the infection. These findings are consistent with previous studies, which have reported similar histopathological alterations in the liver and pancreas (Ringø et al. 2007; Yao et al. 2021). The renal tissue also exhibited significant histopathological changes. Renal tubules and glomeruli displayed tubular nephrosis characterized by vacuolar degeneration and coagulative necrosis. Mononuclear cell infiltration in the interstitial tissue indicated an inflammatory response. These findings align with previous studies that have reported renal tubular necrosis and inflammation as common pathological features in Vibrio infections (Mahmoud et al. 2018). In the splenic tissue, notable alterations were observed as well. Aggregation of melanomacrophages, congestion in splenic blood vessels, and alternative depilation in lymphoid tissue indicated immune responses and alterations in the splenic structure. Focal active melanomacrophage centers further indicated an active immune response to the infection. Similar histopathological findings have been reported in other fish species infected with Vibrio species (Zhang et al. 2020). Overall, the observed alterations in the different tissues indicate the systemic and septicemic impact of the infection on multiple organs. These findings contribute to our understanding of the pathogenesis of V. campbellii infection in gilthead seabream. Additionally, future research should focus on exploring potential therapeutic interventions and preventive measures to mitigate the detrimental effects of V. campbellii infection in aquaculture settings.

The results obtained from the challenge study provide valuable insights into the impact of V. campbellii on gilthead seabream, highlighting its potential role as a pathogen in aquaculture settings. This significant difference between the non-challenged and challenged groups strongly suggests that the presence of V. campbellii had a profound and adverse effect on the survival of gilthead seabream.

The findings from this study underscore the importance of comprehending the effects of pathogenic bacteria on aquatic species, particularly in the context of aquaculture. The implications of bacterial infections on fish health and survival can have substantial economic consequences for the aquaculture industry, including financial losses due to fish mortalities and reduced productivity. Therefore, it is crucial for researchers and aquaculture practitioners to be vigilant about the presence of pathogenic bacteria and their potential impact on fish populations. To address these challenges, further research efforts are warranted to delve deeper into the virulence mechanisms of V. campbellii and other similar pathogens. Understanding the pathogenesis and virulence factors of these bacteria can aid in the development of effective management strategies to mitigate the risks posed by such pathogens in aquaculture settings. Moreover, implementing proactive measures, such as regular health monitoring, early detection of infections, and appropriate biosecurity practices, can help safeguard the health and welfare of fish populations in aquaculture farms. Additionally, the use of probiotics and other alternative disease management approaches may provide promising avenues to reduce the impact of pathogenic bacteria and enhance the overall resilience of cultured fish species.

In conclusion, this study provides valuable insights into the clinical, bacteriological, molecular, and histopathological aspects of V. campbellii infection in gilthead seabream. The observed alterations are consistent with previous reports on vibriosis in fish. The identification of V. campbellii through bacteriological and molecular techniques further confirms its involvement in the infection. The study also highlights a potential association between seasonal variation and the occurrence of V. campbellii. Additionally, the relationship between V. campbellii infection and water quality parameters is complex and requires further investigation. Overall, understanding the clinical and epidemiological aspects of V. campbellii infection is crucial for the development of effective prevention and control strategies in aquaculture settings.