Introduction

Aquaculture is currently the most important, cheapest, and eco-friendly animal protein source for the expanding human population, which reached 8 billion in November 2020 (UN 2022). The importance of aquaculture has increased, particularly with the complicated economic situation and high global living expenses accompanied by food shortages (GRFC 2022).

Meagre (Argyrosomus regius) is the most promising fish species for aquaculture in the Mediterranean region as it has a fast growth rate, efficient food conversion ratio, and high marketability value with a short production cycle (Roo et al. 2010; Duncan et al. 2013). Egypt ranked as the first world producer of cultured meager with about 70% of the global production (Ali et al. 2022).

Enterococcus faecalis is an emerging pathogen affecting many cultured fish species worldwide (Akter et al. 2021); E. faecalis is facultative anaerobic Gram-positive cocci present as single, pairs or short chains and classified in the group D Streptococcus. Enterococci are members of the normal gastrointestinal microbiota (Hashem et al. 2021). E. faecalis is responsible for one of the most severe systemic bacterial infections affecting many fish species; infection has been reported in many cultured fish species such as Nile tilapia (Oreochromis niloticus), silver barb (Barbonymus gonionotus), Crucian carp (Carassius auratus), and Barbonymus gonionotus (Zahran et al. 2019; Ehsan et al. 2021; Xu et al. 2022; Akter et al. 2023).

The role of the fish immune system is to destroy or inhibit the growth of pathogenic bacteria by the immune cells and the effector proteins (Smith et al. 2019). Humoral immunity components such as the complement system, antibodies, and lysozymes have valuable functions in destroying bacterial infection (Dalmo and Bogwald et al. 2022; Mokhtar et al. 2023).

The complement system is one of the most critical immune defences; complement has a regulatory role for acquired immunity (Li and Zhang 2022). Complement 3 (C3) is the most important and abundant protein in the complement system; C3 acts as a point that gathers activation pathways and initiates the amplification of the complement response (Ricklin et al. 2016). C3 participates in several defence mechanisms, including the opsonization process (Abdollahi et al. 2016). Also, C3 directly covers and destroys pathogens (Baharloei et al. 2021).

Lysozyme (Lyz) is an effective defence molecule of fish’s innate immune system; lysozyme exerts its role through the lysis of microbial cell walls by hydrolyzing the glycosidic bond in the peptidoglycan layer to prevent bacterial invasion (Saurabh and Sahoo 2008). Lysozymes exert their effect against gram-positive bacteria, while gram-negative are generally resistant (Kim et al. 2023a).

Immunoglobulins (antibodies) play a vital role in adaptive immune responses by destroying invading pathogens (Zhang et al. 2022). Teleost fish immunoglobulin is structurally like mammalian Immunoglobulin-M; fish IgM has a tetrameric configuration and is the most predominant immunoglobulin in fish serum (Choudhury and Prasad 2011; Mokhtar et al. 2023).

Cyclooxygenase 2 (Cox2) is the enzyme responsible for the biosynthesis of prostaglandins from arachidonic acid. Cox2 is expressed at low levels in healthy tissues (Wang et al. 2016), but the expression level increases in response to inflammation or infection to maintain homeostasis (Norambuena et al. 2012).

The present work aims to determine the cause of mortality affecting cultured A. regius and study the innate immunity response to E. faecalis infection via detecting the expression pattern of complement 3, cyclooxygenase, and lysozyme gene and estimating complement 3, immunoglobulin M, and lysozyme concentration in infected fish serum.

Materials and methods

Diseased fish samples

Eighteen clinically diseased meager (Argyrosomus regius) were collected from an earthen pond located at Barbahary, Kafrelsheikh province, Egypt, 31°35′14.8″N and 31°00′40.6″E. The affected farm suffered from a high mortality rate; the onset of mortality started 1 week before sampling during the summer season of 2023. Samples ranged between 925 and 1360 g in body weight and 34.5 to 42.1 cm in total length. Each sample was kept separately in a polyethylene bag after cleaning with pre-sterilized tape water. Samples were preserved during transportation to the laboratory as mentioned by Aboyadak et al. (2023).

Clinical examination

The clinical examination was performed during sample collection, while the necropsy findings were reported in the laboratory during the bacteriological examination following the method described by El-Bahar et al. (2019).

Initial bacterial isolation and purification

A loopful from heart-blood, liver, and posterior kidney was added to a sterile brain heart infusion broth. Broth tubes were incubated in the dry bath at 33 °C for 24 h. Brain heart infusion agar plates were streaked from the broth tubes, and then plates were incubated at 33 °C for 24–48 h (Ali et al. 2021a). The recovered colonies were sub-cultured on KF streptococcal agar media, Hardy Diagnostics, USA, for selective isolation of E. faecalis (Rahman et al. 2017).

A single bacterial colony was taken from each agar plate and then inoculated into brain heart infusion broth, preserved at –80 °C in 25% glycerol for further studies.

Biochemical identification of bacterial isolates

Biochemical identification was performed using the Vitek 2 system, bioMerieux, France, following the method described by Ali et al. (2021b) for confirming the preliminary E. faecalis identification on the selective media (KF streptococcal agar media). One bacterial colony from a fresh bacterial culture was suspended in 5 ml of 0.5% sodium chloride by vortex mixer; after that, the solution was adjusted to 0.6 McFarland standards. Identification cards were inoculated with bacterial suspensions in the Vitek 2 system, and the biochemical profile was monitored.

Experimental fish

One hundred and twenty-five healthy meager fingerlings ranging between 22 and 30 g in body weight were collected from a private A. regius nursery pond. Experimental fish were transported to the fish diseases laboratory, Baltim station, National Institute of Oceanography and Fisheries, as previously described by Ali et al. (2022).

Virulence test

The virulence of E. faecalis for A. regius was determined as described by Saleh et al. (2021) for one random isolate. Sixty-four fingerlings were randomly divided into four groups (16 fishes per group in two replicates); fish in groups 1, 2, and 3 were intraperitoneally inoculated with 0.2 ml of bacterial suspension containing 105, 106, or 107 CFU fish−1, respectively, and group 4 was inoculated with sterile phosphate buffer saline. Groups were observed for 7 days to monitor the mortality rate. LD50 was calculated according to Aboyadak et al. (2024).

Histopathological examination

Hepatopancreas, posterior kidney, and elementary tract were collected from the experimentally infected fish and cut into small cubes. Tissues were fixed in 10% neutral buffered formalin, then dehydrated, cleared, and impeded in soft paraffin for 2-h at 60 °C and then in hard paraffin wax as described by Suvarna et al. (2018). Five micrometer-thickness tissue sections were mounted over a glass slide and stained with Hematoxylin and Eosin.

The innate immune response of A. regius to E. faecalis infection

Experimental design

To estimate the effect of E. faecalis infection on the innate immune response of A. regius, 45 fish were intraperitoneally inoculated with 4.6 × 106 CFU fish−1 and then randomly subdivided into three replicates.

Serum and tissue samples

Three fish were sampled before the bacterial infection at 0 h to estimate the baseline value of complement 3, lysozyme, and cyclooxygenase. Samples were collected from three fishes at 24, 48, 72, and 96-h post-infection to monitor the changes in fish serum. After anaesthesia, 2 ml of blood was collected from each fish via heart puncture. Blood samples were kept for 1 h, centrifuged at 2800 RCF, and then serum was separated and preserved at −85 °C. Just after blood collection, the anterior kidney tissue was carefully dissected and then immediately frozen in liquid nitrogen and stored at −85 °C for gene expression analysis (Wang et al. 2016).

Gene expression study

RNA extraction and cDNA synthesis

Total RNA was extracted from the head kidney tissue using RNeasy Mini Kit, Qiagen, according to the manufacturer’s instructions. Briefly, 10 mg from anterior kidney tissue was taken, homogenised, and lysed, followed by ethanol addition. The lysate was loaded on the spin column and then eluted in 10 µl of molecular-grade water. The purified RNA was checked using NanoDrop, Thermo Scientific, USA. An absorbance value over 1.8 at 260/280 nm was acceptable. Samples were reverse transcribed using iScript™ cDNA synthesis kit, Bio-Rad; 1 µg of RNA was added to 4 µl of 5X iScript reaction mixture. After that, 1 µl of iScript reverse transcriptase was added and completed to 20 µl with nuclease-free water. In the SensoQuest thermal cycler, Germany, the cycle started with priming at 25 °C for 5 min, reverse transcription at 46 °C for 20 min, and final RT inactivation at 95 °C for 1 min.

Quantitative real-time PCR assay

cDNA was used as a template to determine the relative expression of the following immune-related genes: complement 3 (c3), cyclooxygenase 2 (cox2), and lysozyme c (lyzc) using the previously designed primer by Campoverde et al. (2019) for A. regius genes. The primer sequences are presented in Table 1.

Table 1 The primers sequence used in the real-time PCR assay
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The innate immune gene expression was performed in Quant Studio R-T PCR system, Applied Biosystems, according to the method described by Campoverde et al. (2019). The final reaction volume (10 µl) consisted of 5 µl iTaq Universal SYBR Green Supermix (Bio-Rad), 4 µl of cDNA, and 10 µM of each forward and reverse primer (Intron, Korea) in 1 µl PCR grade water (Milli Q). RT-qPCR cycle started with initial denaturation for 10 min at 95 °C, then 40 cycles of (denaturation at 95 °C for 25 s, annealing step at 57 °C for 25 s, and extension at 72 °C for 15 s), each cycle ended with a final dissociation step at 95 °C for 10 s. β-actin gene was used as an internal control to calculate fold-change in targeted genes.

Immune parameters

Complement 3, lysozyme activity, and IgM were assayed in V-750 UV-visible spectrophotometer, Jasco, Japan.

Lysozyme activity assay

Lysozyme activity was assayed according to the method described by Ali et al. (2018) with modifications. Tested samples were heat-inactivated in a moist bath for 30 min at 56 °C to avoid the other lytic factors except lysozymes (Biller et al. 2021). Lyophilized Micrococcus lysodeikticus (Sigma Aldrich) was prepared as 0.25 mg/ml in 0.1 mM sodium phosphate buffer solution at 7.1 (pH). Fifty microliters of the inactivated serum sample was added to 4.95 ml of M. lysodeikticus suspension followed by vortex mixing at 1000 rpm for 5 s; lysozyme activity was expressed as unit ml−1 and determined by the reduction in absorption at 450 nm after 5 min in which one unit of activity results in the reduction of absorption by 0.001/min.

Complement 3 and IgM assay

Quantitative determination of complement (C3) and IgM was assayed in fish serum using complement C3 and IgM MonlabTest® turbidimetry test kit, Monlab, Spain. The wavelength was adjusted at 340 nm, and then 10 µl of serum sample was added and well mixed to 800 µl of diluent reagent in quartz cuvette; the first absorbance was recorded after that, 200 µl of the antibody reagent was added and well mixed followed by recording the second absorbance.

Statistical analysis

Serum Complement 3, lysozyme, IgM values, and gene expression results were checked for normality by the Shapiro-Wilk test using the online version designed by Simon (2009). The baseline mean value at different sampling time points (24–96) h were compared with the baseline mean value at 0 h using paired samples Student T-test as described by Snedecor and Cochran (1989) using IBM SPSS Statistics for Windows, version 22 (2013), Armonk, NY: IBM Corp. Data were considered statistically significant at P ≤ 0.05.

Results

Clinical examination

Naturally and experimentally infected A. regius fingerlings showed similar signs as skin haemorrhages. Internally, the elementary tract (stomach, pyloric caeca, and intestine) was very congested and haemorrhagic. The hepatopancreas, posterior kidney, abdominal wall, and gonads were inflamed with petechial haemorrhages, as shown in supplementary file Figs. (1–7).

Identification of bacterial isolates

Seventeen out of the thirty-one bacterial isolates were grown as pinkish-red colonies with dark centres characteristic for E. faecalis. Isolates were further biochemically confirmed as E. faecalis with 99% probability using the Vitek 2 system; biochemical characteristics for the recovered isolates are represented in Table 2.

Table 2 The biochemical profile of E. faecalis
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Virulence test

E. faecalis was highly virulent for A. regius; the experimental infection induced similar clinical signs of natural infection, calculated was LD50 estimated by 4.6 × 106 CFU fish−1. Daily and cumulative mortality are shown in Fig. 1.

Fig. 1
figure 1

Mortality rate following experimental infection of A. regius fingerlings with E. faecalis

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Histopathological investigation

The posterior kidney of experimentally infected fish showed severe inflammation represented by diffused mononuclear cell infiltration and interstitial haemorrhage, degenerated glomeruli, hypertrophied as well as atrophied, and degenerated renal tubules (Fig. 2a, b).

Fig. 2
figure 2

Photomicrography of A. regius experimentally infected with E. faecalis. a Posterior kidney showed inflammation and diffused mononuclear cell aggregation (black Asterix), degenerated hypertrophied glomeruli (red arrow), degenerated shrunken glomeruli (blue arrow), X = 100. b Posterior kidney with shrunken glomeruli (blue arrow), degenerated renal tubules (D) necrosis (N), and interstitial haemorrhage (grey arrow), X = 400. c Hepatopancreas showed congestion and engorgement of blood vessels (C) with the presence of leucocytic infiltration (white arrow), X = 100. d Hepatopancreas with diffuse haemorrhage (H), leucocytic infiltration (white arrow), and vacuolar degeneration of hepatocytes (black arrow) X = 400

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The hepatopancreas was also severely affected by bacterial infection, congestion with engorgement of blood vessels, and the presence of leucocytic infiltration, vacuolar degeneration of hepatocytes was also observed, as in Fig. 2c and d.

Regarding the pathological lesions in the elementary tract, the stomach showed inflammation and diffused mononuclear cell infiltration, necrosis, and gastric blood vessels were congested and engorged with blood. Detachment of gastric glands, degeneration, and necrosis were remarkable in the lamina propria of fish intestines. Leucocytic infiltration, detachment of intestinal villi, and massive inflammation represented in diffused inflammatory cell aggregation were clear in the studied tissue sections (Fig. 3a–d).

Fig. 3
figure 3

Tissue sections of A. regius experimentally infected with E. faecalis. Stomach showed inflammation, focal mononuclear cell aggregation (a) and diffused mononuclear cell infiltration (b) (blue arrows), necrosis (N), congested engorged blood vessels (black arrows), detachment of gastric glands (Asterix), X = 100 (a) and X = 400 (b). Intestine with degeneration and necrosis of lamina propria (N) with leucocytic infiltration (blue arrows), detachment of intestinal villi (D) with massive inflammation and diffused inflammatory cell aggregation (blue arrows), X = 400 (c) and X = 100 (d)

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Effect of E. faecalis infection on immune gene expression in A. regius

Results of gene expression study

Figure 4 illustrates gene expression patterns of the studied immune-related genes. The expression of the complement 3 gene was characterised by an initial significant increase that peaked at 24 h; subsequently, it decreased to half before re-increasing at 72 h, followed by a final decrease at 96-h post-infection.

Fig. 4
figure 4

Change in gene expression of c3, cox2, and lyzc genes to B-actin housekeeping genes in head kidney tissue of infected A. regius at 24, 48, 72, and 96-h post-infection. The significant change is calculated from baseline value at 0 h. a Complement 3 gene expression, (*) significantly different at P < 0.005. b Cox2 geneexpression, (*) significantly different at P < 0.01, (#) significantly different at P < 0.04. c Lysozymes gene expression, (*) significantly different at P < 0.004, (#) significantly different at P < 0.02, (&) significantly different at P < 0.01

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The cyclooxygenase gene expressed a 17-fold initial increase at 24 h followed by a gradual decrease until it reached near the baseline at 72-h post-infection.

Lysozyme gene expression started with a sharp increase at 24 h, followed by a gradual decrease until it reached near the baseline at 96-h post-infection.

Immune parameters values

The complement 3 value has significantly increased throughout the sampling time compared to the baseline value, Fig. 5a. Regarding immunoglobulin M, the IgM level has gradually increased from 48 h and reached the peak 96-h post-infection, as in Fig. 5b. Lysozyme concentration increased significantly throughout the sampling time, peaked at 48 h, then gradually decreased but was still higher than the baseline value (Fig. 5c).

Fig. 5
figure 5

Change in gene expression of c3, cox2, and lyzc genes to B-actin housekeeping genes in head kidney tissue of infected A. regius at 24, 48, 72, and 96-h post-infection. The significant change is calculated from baseline value at 0 h. a Complement 3 gene expression, (*) significantly different at P < 0.005. b Cox2 gene expression, (*) significantly different at P < 0.01, (#) significantly different at P < 0.04. c Lysozymes gene expression, (*) significantly different at P < 0.004, (#) significantly different at P < 0.02, (&) significantly different at P < 0.01

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Discussion

The present study was carried out to understand the influence of bacterial infection on the expression of some immune genes in Argyrosomus regius. The concentration of some innate immune parameters in fish serum was evaluated to determine the interaction between the immune system and pathogenic bacteria. This work is the first documentation of Enterococcus faecalis infection in cultured Meagre and the first gene expression study in this fish species under the influence of bacterial infections.

Vitek 2 is the most reputable biochemical system for identifying pathogenic bacteria. In the present study, Vitek 2 efficiently identified all seventeen E. faecalis isolates with 99% probability. Accordingly, Fang et al. (2012) reported the high accuracy of the Vitek 2 system in identifying E. faecalis as compared to molecular identification (Multiplex PCR assay); Vitek 2 correctly identified 32 isolates out of 32 (100%). Tan et al. (2014) and Kim et al. (2023b) also reported the same result; they identified 983 E. faecalis isolates using the Vitek-2 compact system with 100% accuracy when compared with the conventional phenotypic tests, phylogenetic analysis, and with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), Bruker, Germany.

In the present work, the LD50 of E. faecalis for A. regius fingerlings was 4.6 × 106 CFU fish−1. Experimentally infected fish showed the same clinical signs reported for natural infection to satisfy Koch’s postulates. Ehsan et al. (2021) reported similar results; they found the LD50 of E. faecalis was 5.54 × 106 in silver barb (Barbonymus gonionotus). While Rizkiantino et al. (2021) found the LD50 in red tilapia equals 0.79 × 108 CFU fish−1, this difference in the LD50 could be due to the difference in the pathogenicity of challenging isolate or different fish species.

Skin haemorrhage was the most prominent external sign that appeared on infected fish. Internally, the hepatopancreas, posterior kidney, and elementary tract were congested and haemorrhagic. The previously mentioned clinical picture was also reported by Rahman et al. (2017); Rizkiantino et al. (2021); Hassan et al. (2022); and Akter et al.(2023) in diseased Clarias batrachus, red tilapia, Mugil cephalus, and Barbonymus gonionotus fish. Nieto et al. (1995) observed extensive haemorrhages, ulceration on the skin, desquamative enteritis, and fatty changes in the liver of turbot (Scophthalmus maximus) experimentally infected with Enterococcus.

Regarding the histopathological examination, the hepatopancreas and posterior kidney were severely affected by E. faecalis infection; severe inflammation, diffused leucocytic infiltration, haemorrhage, and vacuolar degeneration were dominant. These findings were nearly identical to those described by Zahran et al. (2019); they found caseous necrosis, degenerated hepatocytes with congestion of hepatic tissue, and focal areas of fatty degeneration in the liver of O. niloticus infected with E. faecalis. Abu-Elala et al. (2020) also reported the presence of mild vacuolar degeneration of hepatocytes with individual cell necrosis with severe haemorrhage and the proliferation of hemopoietic element in kidney. Similarly, Elgohary et al. (2021) recorded the abundance of hemorrhage, congestion, inflammation, hyper-cellularity, and degeneration in the hepatic and renal tissue of naturally diseased Nile tilapia and experimentally infected with E. faecalis.

The gastrointestinal tract also showed remarkable inflammation, necrosis, congestion, detachment of gastric glands, and intestinal villi. Nieto et al. (1995) mentioned that desquamative enteritis was the most prominent pathological lesion affecting cultured turbot infected with Enterococcus.

Clinical picture and pathological tissue changes concerning the experimentally infected fish can be attributed to the high pathogenicity of challenging E. faecalis isolate. E. faecalis has two major virulence factors: cell surface factors and secreted virulence factors. The cell surface factors include biofilm formation that protects against the host immune system and adhesins (Geraldes et al. 2022). Adhesins are surface proteins that facilitate bacterial adherence to host cells. E. faecalis has two types of adhesins: Agg (aggregation substance), which mediates the adherence to host epithelial cells, and Ace is responsible for the adherence to collagen (Hashem et al. 2021). The secreted virulence factors include cytolysin, gelatinase, and protease. Cytolysin has a cytolytic activity for the host blood cells, gelatinase hydrolyzes gelatin and collagen, and protease can catalyze the proteolysis of host tissue (Marques et al. 2023).

Complement 3 has a direct and potent antimicrobial effect against bacterial pathogens. In the present study, the expression of the C3 gene was upregulated at 24 h after the challenge. This upregulation could be due to the stimulating effect of infection and the need for more C3 production to exert its immunological role in fighting infection. C3 concentration in fish serum also peaked at 24-h post-infection under the influence of C3 gene overexpression in response to the activated C3 pathway. Accordingly, Maekawa et al. (2023) observed upregulation of C3 gene expression in the head kidney of Eleutheronema Eleutheronema after Streptococcus iniae infection. At 48-h post-infection, C3 gene expression decreased mainly to avoid excessive complement production to restrain the immunological reaction; overstimulation of the immune system can be prejudicial to the host. The subsequent decrease in C3 concentration in serum might be due to the rabid consumption of complement in the immunological reaction accompanied by the downregulation of C3 gene expression. Bai et al. (2022) reported the same result in Oreochromis niloticus experimentally infected with Streptococcus agalactiae. At 72 h and 96 h after the challenge, C3 expression and serum levels increased and then decreased; this fluctuation can be associated with activation and slowing down of the immune response and linked to the expression level of the C3 gene (Wu et al. 2022).

Lysozyme is an essential component of the humoral immune response, inducing lysis of the bacterial cell walls. In the present investigation, the expression pattern of the lysozyme gene initially peaked at 24 h PI and then decreased with time. The initial increase was in response to the activated immune system to withstand the bacterial infection behind expression decreased with time. An identical finding was documented by Guo et al. (2022) in Pinctada fucata martensii after stimulation with peptidoglycan. Meanwhile, Buonocore et al. (2014) stated that Lyz gene expression peaked in Dicentrarchus labrax at 72 h after infection with P. damselae. The serum lysozyme activity started to increase at 24 h after the challenge and reached the top activity at 48 h, then gradually declined. The initial increase in serum lysozyme activity was induced by the upregulation in Lyz gene expression that increased lysozyme synthesis. After that, the Lyz gene expression declined, which decreased the lysozyme synthesis. Decreased lysozyme activity is also associated with lysozyme exhaustion accompanied by pathogen neutralization. This result is consistent with Zahran et al. (2019), who reported declined serum lysozyme concentration in Oreochromis niloticus infected with E. faecalis. Kim et al. (2023a) recorded that the transcript level of Lyz was upregulated in the head kidney of yellowtail clownfish (Amphiprion clarkii) following the experimental infection using Vibrio harveyi.

In the present study, Cox2 displayed upregulation between 24 and 48 h PI, then decreased near the baseline at 96 h PI. The initial sharp upregulation might be due to the severe inflammation accompanied by the initial phases of primary infections. The piscine cox2 enzyme is mainly induced by inflammation, endotoxins, and bacterial infection (Olsen et al. 2012). Wang et al. (2016) results were also compatible with the present findings; they indicated the upregulation of the Cox2 gene in response to the induced inflammation in large yellow croaker (Larmichthys crocea). The Cox-2 mRNA expression was upregulated in many fish species during bacterial infections like Photobacterium damselae infection in sea bream and Streptococcus agalactiae infection in Nile tilapia (Kayansamruaj et al. 2014). Hwang et al. (2016) observed the induction of Cox-2 gene expression in zebrafish (Danio rerio) after LPS-induced inflammation.

Immunoglobulins are glycoproteins that recognize many antigen types (Yu et al. 2020). IgM is the prime and most abundant antibody in different fish species and is the systemic immunoglobulin; it is also responsible mainly for attacking pathogens. IgM concentration in fish serum is variable, depending on the immunological status and infection (Hordvik et al. 2015). In the present research, serum IgM concentration increased gradually and duplicated at 72 h, then peaked at 96 h PI. A significant increase in IgM was evident after 48 h PI; at this time, the immune cells were blustery to destroy the invading pathogen, and B cells produced the specific antibodies. With the persistence of infection, the IgM titer was further increased. Consistently with this research results, Yin et al. (2019) reported a significant increase in serum IgM levels of Nile tilapia (Oreochromis niloticus) after infection with S. agalactiae at 2 days then reached the maximum value at 5 days post-infection. Mu et al. (2022) also recorded a sharp elevation of serum IgM levels in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

Conclusion

E. faecalis is assumed pathogenic for cultured meager, experimental infection induced similar clinical signs of natural infection as skin haemorrhages. Internally, the elementary tract was very congested and hemorrhagic hepatopancreas, posterior kidney, abdominal wall, and gonads were inflamed. Pathologically, there were severe degenerative changes in the liver, kidney, and elementary tract as destruction, necrosis, and lymphocytic infiltration. The experimental infection stimulated immune systems by upregulating the expression of some immune-related genes. Serum complement 3 and lysozyme concentrations increased in the first stage of infection and then gradually decreased. On the other hand, the IgM level gradually increased with time and peaked at 96 h.