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Oxidative stress, gene expression and histopathology of cultured gilthead sea bream (Sparus aurata) naturally co-infected with Ergasilus sieboldi and Vibrio alginolyticus

BMC Veterinary Research volume 19, Article number: 277 (2023) Cite this article

Abstract

Background

Parasitic and bacterial co-infections have been associated with increasing fish mortalities and severe economic losses in aquaculture through the past three decades. The aim of this study was to evaluate the oxidative stress, histopathology, and immune gene expression profile of gilthead sea bream (Sparus aurata) co-infected with Ergasilus sieboldi and Vibrio alginolyticus.

Results

Vibrio alginolyticus and Ergasilus sieboldi were identified using 16 S rRNA and 28 S rRNA sequencing, respectively. The collagenase virulence gene was found in all Vibrio alginolyticus isolates, and the multiple antimicrobial resistance index ranged from 0.286 to 0.857. Oxidant-antioxidant parameters in the gills, skin, and muscles of naturally infected fish revealed increased lipid peroxidation levels and a decrease in catalase and glutathione antioxidant activities. Moreover, naturally co-infected gilthead sea bream exhibited substantial up-regulation of il-1β, tnf-α, and cyp1a1. Ergasilus sieboldi encircled gill lamellae with its second antennae, exhibited severe gill architectural deformation with extensive eosinophilic granular cell infiltration. Vibrio alginolyticus infection caused skin and muscle necrosis in gilthead sea bream.

Conclusion

This study described some details about the gill, skin and muscle tissue defense mechanisms of gilthead sea bream against Ergasilus sieboldi and Vibrio alginolyticus co-infections. The prevalence of co-infections was 100%, and no resistant fish were detected. These co-infections imbalance the health status of the fish by hampering the oxidant-antioxidant mechanisms and proinflammatory/inflammatory immune genes to a more detrimental side. Our results suggest that simultaneous screening for bacterial and parasitic pathogens should be considered.

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Background

The aquaculture sector has an essential role in supplying more than 50% of fish and fish products to the world population [1]. Several bacterial, viral, and parasitic illnesses have plagued gilthead sea bream (Sparus aurata) during the last few decades, posing major challenges to fish production and profitability [2].

Copepods are among the most prevalent destructive ectoparasites of farmed marine fish [3, 4], whereas vibriosis is the most dangerous bacterial infection [5, 6]. Ergasilus sieboldi is a common ectoparasitic copepod that infects primarily the gill filaments of fresh, brackish, and seawater fish, while additional microhabitats include the base of the fins, the external surface of the operculum, and the urinary bladder [7, 8]. Ergasilus sieboldi-mediated infections are seasonal, with parasite populations peaking during late summer and autumn. They induce respiratory distress, slow growth, sluggish behavior, and increased vulnerability to secondary illnesses such as bacterial infections [9,10,11].

Vibrio alginolyticus is pathogenic to gilthead sea bream through a series of events that begin with adhesion to sea bream mucus, then proliferation, colonization of the underlying epithelium, and finally secretion of hydrolytic enzymes, which may be responsible for the development of ulcers and extensive tissue damage [12, 13]. Various virulence factors encoded by virulence genes have been proposed as key contributors to the pathogenicity of V. alginolyticus [14].

Co-infections are infections of a host by two or more genetically distinct pathogens. Each pathogen is responsible for its pathogenic effects and contributes to the overall damage to the host when combined with other pathogens [15, 16]. Parasitic infections increase the risk of secondary bacterial diseases and can act as a vehicle to transmit bacterial pathogens [17]. This synergistic effect has been explained as a result of the stress caused by parasites reducing the resistance of fish to other secondary bacterial infections [18]. Co-infections significantly impact fish health and can potentially change several fish diseases’ progression and severity [19, 20]. Several studies reported parasitic and bacterial co-infections in Nile tilapia (Oreochromis niloticus) [21] goldfish (Carassius auratus) [22], Atlantic salmon (Salmo salar) [23] and flathead grey mullet (Mugil cephalus) [24].

The histopathological alteration, oxidative stress response, and immune-related gene expression may contribute to understanding the host response to pathogenic invasion [25, 26]. The innate immune system is the first to respond to infection and disease and does not retain memory of previous responses [27]. It is a crucial factor in resistance to disease [28], comprising the epithelial/mucosal barrier, humoral parameters, and immune cells [27, 29]. Mucus is the first line of defense and consists of mucins, which can be categorized as structural, associated with the plasmalemma, and secreted, which form the outer mucus gel layer [30]. As well as being a physical barrier, glycoprotein components of mucus are active in combating pathogens and parasites [30, 31]. Hyperplasia and hypertrophy of mucous cells have been described in fish with crustacean ectoparasite infections [32].

Parasitic copepods induce damage to the host through feeding or attachment to the skin or other surfaces by means of clawed limbs. On fish body surfaces, attachment can provoke epidermal erosion and necrosis and host responses including fibroblast proliferation, recruitment of immune cells, and increased collagen fibers at site of attachment [33]. Attachment to fish gill induces fusion of secondary lamellae, consequently reducing the respiratory surface of the organ [34]. The means by which E. sieboldi attaches and feeds cause considerable gill pathology. The insertion of the copepod antennae deep into gill tissue causes disruption of the gill filaments [32, 33, 35]. Heavy infections by E. sieboldi result in severe respiratory problems in fish, especially during warms months [10, 33].

Oxidative burst and the reactive oxygen species (ROS) production is an important defense mechanism in the immune response of aquatic organisms to infections [36]. Assessment of oxidant and antioxidant defense pathways is considered an excellent diagnostic and prognostic tool in aquatic organisms [37]. Glutathione is a non-enzymatic co-factor for glutathione-S-transferase, prevents the deleterious effects of ROS with the production of glutathione disulfide (GSSG), which play a vital role in counteracting oxidative stress and protecting cells from lipid peroxidation, cellular injury, and cellular apoptosis [35, 38]. Moreover, catalase (CAT) is a common antioxidant enzyme that utilizes oxygen and acts as a critical immune-related gene in the teleost, playing an essential role in the immune defense against pathogenic invasion [39, 40].

Immune gene expression profiles are crucial for understanding the molecular pathogenesis and disease development process associated with infections [41,42,43]. Cytochrome P450 family 1 subfamily A member 1 (cyp1a1) is considered the most active xenobiotic-metabolizing enzyme of cytochrome P450 [44], whereas interleukin-1β (il-1β) and tumor necrosis factor alpha (tnf-α) are classical proinflammatory cytokines [45]. Il-1β plays an essential role in coordinating fish responses to infections. It increases the expression of inflammation-related molecules and induces the release of other cytokines [45]. The expression dynamics of immune gene profile, prooxidant-antioxidant biomarker, and pathological changes can help to reveal fish defensive mechanisms against infections [25, 26]. Therefore, this study unlocks the enigmatic causes behind the summer mortalities in cultured gilthead sea bream reared in semi-intensive earthen pond-based marine fish farming and analyses the oxidative stress, histopathology, and immune gene expression profile of gilthead sea bream in response to parasitic and bacterial co-infections.

Results

Case history, clinical and parasitological examinations

Based on clinical, parasitological and bacteriological examination, the uninfected control fish were free from macroscopic and microscopic parasites as well as pathogenic bacteria.

Naturally infected gilthead sea bream showed abnormal swimming behavior, respiratory distress with apparent signs of asphyxia. A clinical examination indicated skin darkening, detached scales, skin hemorrhage and skin abrasion (Fig. 1a) that progressed to skin ulceration (Fig. 1b) and muscle ulceration (Fig. 1c). On the other hand, the infected fish had pale gills (Fig. 1d). Furthermore, the abdominal cavity was filled with serosanguinous fluids, hemorrhagic muscle, and pale liver with hemorrhagic borders (Fig. 1e).

Fig. 1
figure 1

Clinical signs, and postmortem examination of naturally infected gilthead sea bream (Sparus aurata), (a) detached scales, skin hemorrhage and abrasion (black arrow), (b) detached scales, skin hemorrhage, skin and muscle ulceration (black arrow), (c) severe skin and muscle ulceration (black arrow), (d) Pale gills (dotted yellow circles), (e) hemorrhagic muscle (black arrow), pale liver with congested borders (green arrow) and serosanguinous fluids in abdominal cavity (red arrow)

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Microscopic examination of gill filaments revealed microscopic parasite. This parasite had a total body mean length of 1.2 ± 0.05 mm (range: 1.1–1.4 mm), pyriform cephalothorax with lateral constriction toward the posterior section and thoracic segments that narrow posteriorly. The parasite had two elongated egg sacs at the end of the adult female body. Moreover, it had two well-developed antennae. Each antenna consisted of coxobasis and three endopodite segments with a large, strong bent claw at the end, responsible for grasping the gills. Based on the previous morphometric characteristics, we confirm that these parasites belong to parasitic copepopds, genus Ergasilus Nordmann, 1832 and identified as Ergasilus spp. Further, Ergasilus spp. were confirmed as E. sieboldi by molecular characterization based on 28 S rRNA gene sequencing.

All forty-five diseased gilthead sea bream were infected with E. sieboldi. Therefore, the prevalence of E. sieboldi infestation was high (100%) in the gills, no parasites were found in any other organs (fin, skin, operculum, and other microhabitats) of the forty-five-gilthead sea bream. The mean infection intensity of E. sieboldi adult parasitic female in the gill filaments of gilthead sea bream was 32.4 ± 1.29 per fish gills. Infection intensity of copepod in the four gill arches of gilthead sea bream revealed that the highest infection intensity in the third gill arch (GA III) followed by the second gill arch (GA II), while the fourth gill arch (GA IV) showed the lowest infection intensity with E. sieboldi (Supplementary Table 1).

Water quality parameters

The values of physicochemical parameters of water in the culture pond were temperature (30 ± 3 °C), pH (8.2 ± 0.2), dissolved oxygen (5 ± 0.5 mg/L), salinity (38 ± 2 PSU), ammonium-nitrogen (2.5 ± 0.3 mg/L), un-ionized toxic ammonia (0.37 ± 0.1 mg/L), nitrate (6 ± 3 mg/L), nitrite (0.09 ± 0.02 mg/L) and iron (0.40 ± 0.1 mg/L).

Morpho-biochemical bacterial characterization

All isolates were gram-negative, curved rod shaped, and motile. They formed large yellow colonies on TCBS agar (sucrose fermenter isolates) and were positive for cytochrome oxidase, indole, and catalase tests. They showed swarming growth on blood agar and TSA. All the isolates were tolerant to high NaCl concentrations (up to 10%) and showed a similar carbohydrate fermentation profile. All isolates were confirmed as V. alginolyticus (99.5–99.9%) by API® (Table 1). Pathogenic V. alginolyticus were isolated from skin ulcer, muscle, and kidney. By contrast, no pathogenic V. alginolyticus were isolated from gill tissues.

Table 1 Biochemical characteristics of V. alginolyticus isolates retrieved from gilthead sea bream
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Molecular identification of parasite specimen and bacterial isolate

All forty-five diseased gilthead sea bream were co-infected with E. sieboldi and V. alginolyticus. Ergasilus sieboldi and V. alginolyticus were confirmed by molecular characterization based on 28 S rRNA and 16 S rRNA gene sequencing analysis, respectively. Ergasilus sieboldi and V. alginolyticus isolates co-infecting gilthead sea bream were sequenced, and the nucleotide sequences of the 28 S rRNA produced a total length of 688 bp, and 16 S rRNA produced a total length of 981 bp. Both nucleotides’ sequences were deposited in the GenBank database under accession numbers ON706996 (E. sieboldi) and ON041091 (V. alginolyticus). ON706996 was homologous with E. Sieboldi (MW810242 and OM812074), and the range of identity was 98–99% with an E value of 0.00. While ON041091 shared more than 99% identity (E value of 0.00.) with the accession numbers of V. alginolyticus (MN733128, MT368033, MH169304, OM654367, HQ827779 and KC884627).

Virulence genes of Vibrio alginolyticus isolates

Multiplex PCR amplification was employed to detect collagenase, VptoxR, and tdh virulence genes in V. alginolyticus isolates. A 738 bp amplicon fragment matching collagenase was obtained in all V. alginolyticus strains whereas no bands were observed for VptoxR (296 bp) and tdh (270 bp) virulence genes (Fig. 2).

Fig. 2
figure 2

Vibrio alginolyticus virulence genes (collagenase, VptoxR, tdh) using Multiplex PCR. Agarose gel electrophoresis (1.5% agarose) of the collagenase (lanes 1–4) amplification products (738 bp) of V. alginolyticus strains. Lane: M, molecular weight marker. Neither amplification products (296 bp) for VptoxR, nor amplification products (270 bp) for tdh. Lanes 1, 2, 3, and 4 refer to VAK4, VASk3, VAM6, and VASp1 isolates, respectively

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Antimicrobial susceptibility test and detection of florfenicol resistance gene in V. alginolyticus isolates

Antimicrobial susceptibility tests revealed that all V. alginolyticus isolates were resistant to ampicillin (AMP 10) and erythromycin (E 15). Apart from ampicillin and erythromycin, 50% of isolates were resistant for trimethoprim/sulfamethoxazole (SXT 25) and novobiocin (NV 30). Regarding tetracyclines group, a considerable proportion of antimicrobial resistance to oxytetracycline (OT 30) and doxycycline (DO 30) was detected in this study. In the present study, only two isolates were susceptible to tetracyclines, while all isolates were sensitive to florfenicol (FFC 30). The multiple antibiotic resistance (MAR) index of V. alginolyticus ranged from 0.286 to 0.857 (Supplementary Table 2). The florfenicol resistant gene (florR) was not detected in all V. alginolyticus isolates.

Oxidant/ antioxidant biomarkers in infected gilthead sea bream

The activity of antioxidant enzymes (CAT and GSH) in the gills, skin and muscles of gilthead sea bream are shown in Fig. 3. Catalase and reduced glutathione antioxidant enzymes were decreased in the gills, skin, and muscles of gilthead sea bream co-infected with E. sieboldi and V. alginolyticus compared with uninfected control fish. The gills of diseased gilthead sea bream had the lowest levels of catalase and reduced glutathione antioxidant enzymes, followed by the skin and muscle. On the other hand, gilthead sea bream co-infected with E. sieboldi and V. alginolyticus showed a significant increase in Malondialdehyde (MDA) as a lipid peroxidation level (LPO) biomarker in the muscle, gills and skin compared with uninfected control fish. The muscle of diseased gilthead sea bream had the highest lipid peroxidation level followed by gills and skin (Fig. 3).

Fig. 3
figure 3

Malondialdehyde (MDA), catalase (CAT) and reduced glutathione (GSH) levels in gilthead sea bream naturally co-infected with E. sieboldi and V. alginolyticus analyzed in the gills, skin, and muscle tissues. White bar represents uninfected control fish, while black bar represents infected fish. The bars represent the mean ± standard error of the mean. Values are statistically significant at p value < 0.05 (Independent sample T-test, R 4.1.2). * (p value < 0.05), ** (p value < 0.01) and *** (p value < 0.001)

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Gene expression analysis of cyp1a1, il-1β and tnf-α in infected gilthead sea bream

Significant up-regulation of tnf-α, cyp1a1, and il-1β expressions were detected in the gills of gilthead sea bream infected with E. sieboldi compared with uninfected control fish. On the other hand, il-1β, cyp1a1, and tnf-α expressions were up-regulated in the skin and muscle of gilthead sea bream infected with V. alginolyticus compared with uninfected control fish (Fig. 4).

Fig. 4
figure 4

Fold changes comparing infected vs. uninfected control gilthead sea bream are shown. Relative expression of cytochrome P450 family 1 subfamily A member 1 (cyp1a1), interleukin-1β (il-1β) and tumor necrosis factor alpha (tnf-α) in gilthead sea bream naturally co-infected with E. sieboldi and V. alginolyticus analyzed in the gills, skin, and muscle tissues. The bars represent the mean ± standard error of the mean. Values are statistically significant at p value < 0.05 (Independent sample T-test, R 4.1.2). * (p value < 0.05), ** (p value < 0.01) and *** (p value < 0.001)

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Histopathology

The gills of uninfected control fish showed normal lamellar epithelium, with no evidence of proliferative or inflammatory reactions (Fig. 5a). By contrast, the gill filaments of infected gilthead sea bream revealed various histopathological observations represented by severe congestion of the venous sinuses and blood vessels associated with marked expansion of the primary gill lamellae with edematous fluid and inflammatory mononuclear cell infiltration (Fig. 5b). Acute inflammatory reaction was a frequent lesion in the gill filament, characterized by congestion of the blood vessels with intense infiltration of the primary and secondary gill lamellae with mononuclear cells in addition to the accumulation of faint bluish mucous exudate (Fig. 5c). Focal epithelial proliferation with focal epithelial fusion was demonstrated in some gill lamellae (Fig. 5d). Mucous cell hyperplasia was also demonstrated (Fig. 5e). Hyperplasia of respiratory epithelium with complete fusion of secondary gill lamellae. The proliferating cells appeared large with round vesicular basophilic nuclei (Fig. 5f).

Fig. 5
figure 5

Photomicrograph representing histological sections from the gills of, (a) normal gills of uninfected control fish showing normal lamellar epithelium, with no evidence of proliferative or inflammatory reaction, (b to f) infested gilthead sea beam showing congestion of the venous sinuses and blood vessels associated with marked expansion of the primary gill lamellae with edematous fluid and inflammatory mononuclear cell infiltration (b), congestion of the blood vessels with intense infiltration of the primary and secondary gill lamellae with mononuclear cells in addition to accumulation faint bluish of mucous exudate (c), focal epithelial proliferation with focal epithelial fusion (d), Mucous cell hyperplasia (e), and hyperplasia of respiratory epithelium with complete fusion of secondary gill lamellae. The proliferating cells appeared large with round vesicular basophilic nuclei (f). (Stain: H and E; Scale bar: 100 μm)

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Variable histopathological observations in the gill arch of infected gilthead sea bream were demonstrated in Fig. 6. The gill arch showed severe congestion of the blood vessels (Fig. 6a) associated with extensive edema and infiltration of inflammatory cells, mainly eosinophilic granular cells (EGCs) and lymphocytes (Fig. 6b), in addition to massive hemorrhage (Fig. 6c). The base of the gill filament revealed congestion of the blood vessels associated with edema and multi-focal infiltration of their epithelial lining with mononuclear cells (Fig. 6d) and melanomacrophages (Fig. 6e). Hyperplastic proliferation of the epithelial cells lining the base of gill filaments concurrently with intense infiltration of the hyperplastic epithelium with mononuclear cells was frequently demonstrated (Fig. 6f). The hyperplastic epithelial cells appeared hypertrophied with large round vesicular basophilic nuclei (Fig. 6f).

Fig. 6
figure 6

Photomicrograph representing histological sections from the gill arch and base of gill filaments of infected gilthead sea beam showing (a) severe congestion of the blood vessels (asterisk) associated with EGCs (arrows) and mononuclear cell infiltration, (b) extensive edema (asterisk) and infiltration of eosinophilic granular cells (EGCs) and lymphocytes (arrow), (c) massive hemorrhage (star), (d) focal infiltration of the epithelial lining the base of the gill filament with mononuclear cells (arrow), (e) focal infiltration of melanomacrophages (arrows), and (f) severe epithelial hyperplasia of the base of the gill filament (asterisk) concurrently with intense mononuclear cell infiltration (arrows). (Stain: H and E; Scale bar: 100 μm)

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No pronounced severe lesions were demonstrated in the skin of gilthead sea bream except for the focal erosive area with focal desquamation of the superficial epidermal cell layer, leaving an intact basement membrane (Fig. 7a). The underlying muscle appeared normal in most examined sections except for focal necrosis of individual myocytes, which is infiltrated by mononuclear cells (Fig. 7b).

Fig. 7
figure 7

Photomicrograph representing histological sections from skin and muscle of infected gilthead sea beam showing (a) skin with focal erosive lesion with intact basement membrane (arrows), and (b) muscle with focal necrosis of individual myocyte which was infiltrated by mononuclear cells (arrow) (Stain: H and E; Scale bar: 100 μm)

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Discussion

Fish co-infected with parasitic and bacterial infections induce synergistic interaction resulting in severe illness and significantly higher mortalities [46]. The current study aimed to evaluate the oxidative stress, histopathology and gene expression of cultured gilthead sea bream co-infected with V. alginolyticus and E. sieboldi during the onset of summer mortalities.

In this study, the obtained behavioral and clinical signs of cultured gilthead sea bream were abnormal swimming behavior, evidence of asphyxia, and skin ulceration with fish mortalities. These clinical findings were described in previous studies [3, 4]. Further microscopic examination of gills revealed adult female Ergasilus species with elongated egg sacs embedded in the mucous exudates of the gill filament. Similar findings were recently described in gills of infected gilthead sea bream [47]. The insertion of the copepod antennae deep into gill tissue causes disruption of the gill filaments, results in severe respiratory problems in fish, especially during warms months [32, 33, 35]. Adult E. sieboldi were confirmed by 28 S rRNA sequencing, which produced a total length of 688 bp amplicon. It was found that the morphometric characteristics of E. sieboldi were similar to those described in previous studies [7, 47,48,49,50]. Ergasilus sieboldi is one of the most widespread ergasilid species that has been reported from fifteen fish families in Europe, Asia, and Africa [47, 48, 51]. The molecular data available for E. sieboldi includes species of African, Asian, and European origin [47, 48, 52, 53]. The analysis of the 28 S rRNA gene sequence of E. sieboldi (Accession number: ON706996) revealed identity (99%) with E. sieboldi of European origin (Accession number: MW810242) [48] and African origin (Accession number: OM812074) [47].

Typical signs of V. alginolyticus infection in gilthead sea bream were described in earlier studies [5, 13, 54]. Further, V. alginolyticus strains were confirmed by 16 S rRNA sequencing and revealed more than 99% identity with the V. alginolyticus strains deposited previously in NCBI.

In this study, forty-five cultured gilthead sea bream fish were co-infected with E. sieboldi and V. alginolyticus. No parasites were detected in the fish skin, fin, operculum, mouth, and other microhabitats. Co-infection with bacteria and parasites is a common occurrence in aquaculture. Infections with parasites not only increase the risk of secondary bacterial diseases but also have the potential to function as a vehicle for the transmission of disease-causing bacteria [17]. The majority of ergasilids infest the gills of their hosts are rarely found on the skin or other tissues [55]. The prevalence and infection intensity of E. sieboldi copepods in the gills of naturally infected gilthead sea bream was high (100%) and (32.4 ± 1.29) per fish gills, respectively. The copepods Ergasilus spp. in the gills of Scatophagus argus showed the highest prevalence (78.6%) and intensity [17.8 (1–233)] [56].

Physiological stress and physical injury are the primary contributing factors of fish disease and mortality in aquaculture [57, 58]. In this study, high water temperatures (30 ± 3 °C), salinity (38 ± 2 PSU), non-ionized toxic ammonia (0.37 ± 0.1 mg/L) and iron (0.40 ± 0.1 mg/L) were found to constitute likely stress factors resulting in increased fish susceptibility to infections [5, 25]. Poor water quality in fishponds was linked with mortalities of cultured fish due to parasitic and bacterial co-infections [59]. Temperature can also influence the severity of co-infection in fish by affecting the activity of the innate immune system. An increase in temperature accelerates co-infection intensity and adversely affects immunological and physiological parameters [60]. Parasites readily respond to changes in temperature. Commonly, increases in temperature can boost parasite reproduction, accelerate and shorten their life cycles and extend their transmission periods [61]. The high intensity of E. sieboldi in gills of cultured gilthead sea bream could be attributed to high water temperature in late summer, considering that rates of oviposition and egg hatching of Ergasilus spp. are greater at higher temperature [62]. At water temperature above 23 °C, the rates of oviposition and egg hatching of Ergasilus spp. are increased, thus affecting availability of copepod’s infective stages [63, 64]. Salinity is considered one of the main factors influencing the infection with ectoparasitic copepods. Ergasilid copepods are euryhaline metazoan ectoparasites which can tolerate a wider range of (19.7–31.2 PSU) [56]. Ergasilus labracis are an estuarine or freshwater parasite that are indeed tolerant of a wide range of salinities (10.2–30.2 PSU) [65]. In contrast, E. labracis was found to be the most prevalent parasitic copepod in fish reared in low to moderate salinities ranging from 13.56 to 21.11 PSU, and rarely on fish reared in high-salinity zone (more than 29 PSU). Moreover, parasite intensities were highest in the low-salinity zone and decreased significantly in high salinity zone [66].

Vibrio species are ubiquitous inhabitants of aquatic environments including estuaries, marine coastal waters, sediments and aquaculture facilities [67]. Currently, genus Vibrio comprises more than 130 species grouped in fourteen clades [68]. PCR-based approaches of Vibrio species targeting species-specific virulence genes have proven useful in discerning between closely related Vibrio species [69]. Several V. alginolyticus and V. parahemolyticus molecular protocols have been established based on the detection of collagenase and hemolysin encoding genes [70, 71]. PCR targeting species-specific virulence genes is an efficient approach to accurately determine the presence of opportunistic/pathogenic bacteria in complex microbial communities inhabiting aquaculture facilities [69]. Vibrio alginolyticus screening for the presence of virulence genes (collagenase, VptoxR, and tdh) revealed that all isolates produce only a 738-bp amplicon fragment matching collagenase gene, while VptoxR and tdh genes were negative in all tested strains. Vibrio alginolyticus strains had a MAR index ranging from 0.286 to 0.857. It was higher than findings from earlier studies in Egyptian aquaculture [5] or in different areas [72, 73]. A considerable proportion of antimicrobial resistance to oxytetracycline and doxycycline might be attributed to the frequent application of tetracyclines to control vibriosis in Egyptian marine fish farms during the past decade [5].

In the present study, a significant decrease in the activity of the antioxidant enzymes (CAT and GSH) as well as a significant increase in the activity of lipid peroxidation level were reported in the gills, skin, and muscle of gilthead sea bream infected with E. sieboldi and V. alginolyticus compared with uninfected control fish. The cellular damage of gills due to E. sieboldi leads to significant decrease in the activity of antioxidant enzymes in gills. Additionally, the cellular damage of skin and muscle tissues due to V. alginolyticus also leads to significant decrease in the activity of antioxidant enzymes in skin and muscle. Malondialdehyde is a byproduct of the peroxidative breakdown of polyenic fatty acids in the lipid peroxidation process, and its accumulation in tissues indicates the degree of free radical formation, lipid peroxidation, and oxidative stress [74]. Lipid peroxidation is essentially a toxic response to oxidative damage to cellular and tissue components [75]. The elevated levels of LPO affect the cell membrane permeability and mitochondrial oxidative phosphorylation, which could damage cellular membrane and stimulate cell apoptosis [76]. Oxidative stress is defined as the imbalance between oxidants and antioxidant defenses [77]. The infection leads to the imbalance between oxidant and antioxidant defense mechanisms through inhibiting the antioxidant activity as well as the inability to neutralize the impact of ROS released [25, 78, 79].

The innate immune system of fish is a vital defence mechanism against various pathogens. Co-infections in fish can elicit various alterations in the expression of immune genes in fish, which may include the up-regulation of genes associated with innate immunity. Up-regulation of tnf-α, cyp1a1, and il-1β expressions occurred in the three tissues of gilthead sea bream. Most inflammatory genes were up-regulated in the skin of Indian major carp, Labeo rohita in response to crustacean ectoparasite infection [80, 81]. Il-1ß is a proinflammatory cytokine with a significant role in the initial stage of inflammation by attracting fish leucocytes. Hence, up-regulation of il-1ß expression is a common response to microbial infections [45, 82, 83]. Tnf-α is an important mediator in response to parasitic, bacterial and viral infections [28, 41, 45]. Cytokines (il-1β and tnf-α) were up-regulated in kidney and brain of gilthead sea bream infected with nodavirus [84]. Significant up-regulation of cyp1a1 was detected in the liver of gilthead sea bream reared in mixed sediments. Cyp1a1 is activated by one specific heavy metal, or a consequence of exposure to all heavy metals existing in the sediments [85]. Up-regulation of cyp1a1 expression was observed in channel catfish infected with Edwardsiella ictaluri infection [86]. Up-regulated cyp1a1 expression is a common response to viral infection by reovirus in grass carp, Ctenopharyngodon idella [87]. This significant up-regulation in cyp1a1, il-1ß, and tnf-α expression might contribute to proinflammatory and inflammatory responses due to bacterial and parasitic infections [26, 44, 45, 88].

The skin and gills are important mucosal barriers that protect fish from the external environment. Fish mucosal surfaces harbor microbiome and mucosal immunity [89]. Mucosal surfaces can be colonized by commensals that provide benefits to the host by providing nutrients or protection from pathogens [90]. Homeostasis between the microbiome and mucosal immunity is crucial for the overall health of fish. Changes in the environment could affect the microbiome, thus altering mucosal immunity [91]. Dysbiosis in the commensal microbes can turn a commensal into a pathogen. Parasite-induced dysbiosis modulating host microbiota composition and host immune system [92]. For instance, this microbial dysbiosis can disrupt homeostasis on the mucosal surface, facilitating pathogen invasion and colonization by opportunist pathogenic bacteria (Vibrio spp.) from the surrounding environment [93]. Finally, parasites can directly carry new microbes that co-infect the host, and thus causing problems for fish [94].

Histopathological investigations of gilthead sea bream co-infected with E. sieboldi and V. alginolyticus revealed considerable damage and distortion of the normal tissue architecture. The extensive damage of gills were due to E. sieboldi copepods, while V. alginolyticus induces skin and muscle tissue reaction. Histopathological investigations of infected gills showed extensive tissue damage due to attachment and feeding of E. sieboldi copepods [3, 32]. Ergasilus sieboldi attaches to the outer surfaces of the gill filaments by powerful specialized antennae inserted deep into the gill tissues leading to severe mechanical injury, deformation of the gill filaments and puncturing of blood vessels [3]. Adhesions between gill filaments are shown as a response to copepods attachement. Consequently, fish respiration is impaired and reduced feeding, weight loss, and general deterioration of health can result [3, 4, 32]. The severity of damage caused by ergasilid infection of fish specimens is directly proportional to the number of copepods on the gills [86]. In the present study, the mean infection intensity of E. sieboldi copepods on the gills were low to moderate (32.4 ± 1.29) per fish gills. Accordingly, less than 20 E. sieboldi may have little effect, but, when their intensity exceeds 100, gill damage may be serious [95,96,97].

Histopathology of diseased fish with Vibrio spp. included sloughing of epithelial cells and severe necrotic muscle with massive infiltration of immune-related cells [98, 99]. Skin and muscle necrosis due to V. alginolyticus in gilthead sea bream was also reported [12].

Conclusion

We have identified E. sieboldi and V. alginolyticus co-infections in gilthead sea bream cultured in a land-based farm. The prevalence of co-infections was 100%, no resistant fish were detected. Ergasilus sieboldi copepods infest the gill filaments of gilthead sea bream and are rarely found on the skin or other microhabitates. High water temperature and poor water quality accelerate the intensity of co-infections which imbalance the health status of the fish by hampering the oxidant-antioxidant mechanisms and proinflammatory/inflammatory immune genes to more detrimental side. Our results suggest that simultaneous screening for bacterial and parasitic pathogens should be considered. Also, we recommend conducting a screening for potential viral pathogens. These microbial co-infections may occur in farmed fish. Our findings provide valuable information on the relationship between fish immune systems and co-infections. Thus, maintaining good water quality and accurate diagnosis of diseases will aid in establishing effective management and treatment to control pathogens in gilthead sea bream aquaculture.

Methods

Culture conditions, sampling and clinical signs

In late-Summer 2020, cultured gilthead sea bream was reared on a semi-intensive earthen pond-based marine fish farm, located in East of Al Tafrreah, Port Said, Egypt. The Fish seed (average: 5 g) were purchased from governmental hatchery. The stocking density of fish was 0.5–0.75 kg/m3. The fishpond was rectangular in shape (120 m x 60 m x 1.25 m) supplemented with two or three paddle wheel aerators. The fish were fed 4.0 mm pellets with 42% crude protein and 16% crude fat. The fish were fed about 1.8% of their total body weight. This feed ratio could be optimized depending on water temperature, water quality and fish size. At the onset of disease, the age of cultured fish ranged from 12 to 13 months. The first observation of behavioral signs was at the end of July. The fish were monitored for one month (August 2020), the fish exhibited signs of abnormal behavior, respiratory distress, and skin ulceration. The cumulative mortalities of diseased gilthead sea bream were moderate and ranged from 15 to 20%. The mortality pattern was irregular and ranged from a few dead fish to tens of dead fish daily.

To investigate the cause of respiratory distress and skin ulceration, forty-five naturally infected gilthead sea bream (average weight: 250 ± 50 g) were collected. Also, We sampled fifteen control un-infected gilthead sea bream (average weight: 250 ± 50 g) from uninfected pond of the same farm. These fish exhibited neither behavioral signs nor clinical signs. The clinically affected fish and uninfected control fish were immediately placed in separate plastic bags containing water, under conditions of artificial aeration, and transported alive to the Aquatic Animal Medicine and Management Department, Faculty of Veterinary Medicine, Cairo University, where they were measured, euthanized with overdose of benzocaine (0.2 g/L). The clinical, parasitological, and microbiological examination were conducted on the euthanized fish.

Water quality parameters

The physicochemical parameters of the water were measured two times daily (at 9 am and 4pm) using multi-parameter portable measuring instrument (HI-9829, Hanna Instruments Inc., Romania). Briefly, dissolved oxygen (DO, ppm) was measured using HI-7609829-2 probe, while water temperature (°C), salinity (PSU) and pH were measured using HI-7609829-1 probe. Moreover, ammonium-nitrogen (NH4-N, ppm) and nitrate-nitrogen (NO3-N, ppm) were measured weekly (at 9 am) using HI-7609829-10 and HI-7609829-12 probes, respectively. Nitrite (NO2−, ppm) and total iron (ppm) were measured monthly (at 9 am) using HI-3873 nitrite test kit and HI-3834 iron test kit according to manufacturer’s protocol (Hanna Instruments Inc., Romania).

Parasitological examination

Skin, gills and muscles of naturally infected fish and uninfected control fish were carefully examined by naked eye and handheld lens for macroscopic parasites and then dissected under a dissecting microscope. Parasitic copepods were collected, cleaned and then preserved in 70% ethanol. Permanent slide preparations were made using the phenol-balsam method [100]. Different structures of the collected copepods were carefully observed to be identified [7]. Total body length of the collected copepods was measured. Body measurements were demonstrated by millimeters (mm) and provided as a mean value followed by the minimum and maximum values. The total number of copepods collected from gills of each fish were counted; the parasitic prevalence and mean intensity were calculated [101].

Bacterial isolation and characterization

Under complete aseptic conditions, Loopfuls from skin, muscles, gills and kidney of naturally infected fish and uninfected control fish were streaked onto Tryptic soy agar supplemented with 2% NaCl (TSA, Lab M, UK), blood agar (Oxoid) supplemented with (5% sheep blood, 2% NaCl), motility agar (TSB + 0.3% agar + 2% NaCl) and TCBS agar (Oxoid). Inoculated plates were incubated for 48–72 h at 28 °C and 37 °C. Pure colonies were re-streaked onto TSA + 2% NaCl for phenotypic and biochemical identification. Presumptive identification was accomplished using different phenotypic and biochemical tests (APIID Test Strips®, APIWEB™, Biomérieux, USA) following the manufacture instructions. Presumptively identified Pure cultures were stored at − 20 °C in tryptic soy broth (TSB) (Lab M) supplemented with 2% NaCl (Lab M) and 16% glycerol (Sigma-Aldrich) for further characterization.

DNA extraction, amplification, and sequencing

The genomic DNA of parasite specimen and bacterial isolates was extracted using a QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany) following the manufacturer’s instructions. The 28 S rRNA and 16 S rRNA fragment was amplified using the 28 S and 16 S primers (Supplementary Table 3). PCRs were performed in a final volume of twenty-five µL, containing 2 µL of DNA (50 ng/ µL) template,12.5 µL of 2x DreamTaq® Green Master Mix (Thermo Fisher Scientific) and 0.5 µL (10 nmol L− 1) of each primer. The PCR amplifications were performed using a MyCycler™ thermal cycler (Bio-Rad, USA) with the following cycling conditions according to [53] (Supplementary Table 4). The amplified products were analyzed by electrophoresis (100 V, 400 mA, and 60 min) on a 1.5% (W/V) agarose gel.

The PCR products were purified with a QIA quick PCR purification column (Qiagen, GmbH, Hilden, Germany) following the manufacturer’s instructions and then sequenced with an ABI 3730XL DNA sequencer (Biosystems™, USA). The sequences obtained were analysed using Sequencer software and newly generated sequences were searched against the NCBI using blastn (BLAST®) and deposited in GenBank under the following accession numbers: ON041091 for 16 S rRNA and ON706996 for 28 S rRNA.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing of V. alginolyticus isolates was performed using the standard disc diffusion method [102], using the fellowing antimicrobial discs : ampicillin (AMP 10), erythromycin (E 15), novobiocin (NV 30), florfenicol (FFC 30), oxytetracycline (OT 30), doxycycline (DO 30), and trimethoprim/sulfamethoxazole (SXT 25). The diameters of inhibition zones (mm) were measured and then interpreted according to previously published guidelines [103].

Detection of the virulence genes

The detection of V. alginolyticus virulence genes; collagenase, VptoxR, tdh gene (Supplementary Table 3) were performed using a Multiplex PCR. PCRs were performed in a final volume of twenty-five µL, containing 2 µL of DNA (50 ng/ µL) template,12.5 µL of 2x DreamTaq® Green Master Mix (ThermoFisher Scientific) and 0.5 µL (10 nmol L− 1) of each primer. The PCR amplifications were performed using a MyCycler™ thermal cycler (Bio-Rad, USA) with the following cycling conditions according to [71] (Supplementary Table 4). The template-free reactions were included in the PCR setup as negative controls. The amplified products were analyzed by electrophoresis on a 1.5% (W/V) agarose gel.

Detection of florfenicol resistance gene

The detection of V. alginolyticus florfenicol resistant gene; florR gene [104] (Supplementary Table 3) was performed using PCR in a final volume of 25 µL, containing 1 µL of DNA (50 ng/ µL) template,12.5 µL of 2x DreamTaq® Green Master Mix (Thermo Fisher Scientific) and 0.5 µL (10 nmol L− 1) of each primer. The PCR amplifications were performed using a MyCycler™ thermal cycler (Bio-Rad, USA) with the following cycling conditions according to [104] (Supplementary Table 4). The amplified products were analyzed by electrophoresis on a 1.5% (W/V) agarose gel.

Oxidant/ antioxidant biomarkers

Gills, skin and muscle tissues from naturally infected fish and uninfected control fish were homogenized in ice-cold 0.1 M phosphate buffer saline (pH 7.4) using a Teflon tissue homogenizer. The crude tissue homogenate was centrifuged at 4000 rpm for 15 min at 4°C, then the supernatants were stored at − 80 °C until use. Catalase activity (CAT) was measured using Catalase assay colorimetric method (Bio-diagnostic Co., Egypt) [105]. Malondialdehyde (MDA) as a lipid peroxidation level (LPO) biomarker was measured spectroscopically at 532 nm using the thiobarbituric acid method (Bio-diagnostic Co., Egypt) [106]. Reduced glutathione content (GSH) was measured using Reduced glutathione colorimetric method (Bio-diagnostic Co., Egypt) along with the manufacturer’s guides.

Quantitative real-time PCR analysis of cyp1a1 and of immune response genes (il-1β and tnf-α)

Sampling and RNA extraction

Gills, skin and muscle tissues from naturally infected fish and uninfected control fish were sampled and kept in RNA latter at 4 °C for 24 h and then stored at – 80 °C. Approximately, 30 mg of gill, skin, and muscle tissues was used for RNA extraction using total RNA Extraction Kit (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. The RNA concentration (ng/µL) and quality (A260/A280 ratios were 1.8–2.0) was assessed using Nanodrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, USA).

Primer design and validation

Primer pairs, GenBank accession number, amplicon size and primer efficiencies for the target genes (cyp1a1, il-1β and tnf-α) [85, 107] and reference gene (gapdh) [108] and were summarized (Supplementary Table 5). These primers were selected due to their higher efficiency and their sequences best matches the sequences of the reference and target genes.

Reverse transcription quantitative polymerase chain reaction

Complementary DNA (cDNA) was synthesized from 1 µg of RNA using M-MuLV Reverse Transcriptase (New England Biolabs® Inc., MA, USA) according to the manufacturer’s protocol. The expression of the nominated genes (Supplementary Table 5) was analyzed by real-time qPCR with Bio-Rad iCycler thermal cycler and MyiQrealtime PCR detection system (BIO-RAD, USA). The qPCR reaction mixtures were performed in a total volume of 10 µL, containing 5 µL of Maxima SYBR Green/ROX qPCR Master Mix (2X) (Cat. No. K0221, Thermo Fisher Scientific, USA), 0.5 µL of primers (10 mM each), 1.5 µL of cDNA template and 2.5 µL nuclease free water. The real-time qPCR conditions were performed according to [85, 107, 108] as follows: 95 °C for 5 min (initial denaturation) and then 40 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s. A melting curve analysis was performed after the amplification to confirm the specificity of the PCR products, through one cycle of 95 °C for 15 s, 55 °C for 15 s, and 95 °C for 15 s. In all cases, each PCR was performed with triplicate samples. The specificity of the reactions was analyzed using samples without cDNA as negative controls (NTC). For each mRNA, target gene expression was normalized with reference gene in each sample. The gene expression was analyzed using the 2−ΔΔCT method [109].

Histopathological investigations

Pieces of gills, skin and muscle tissues were excised from naturally infected fish and uninfected control fish, then rinsed in physiological saline and fixed in aqueous fixative (10% formal saline) for 24 h to preserve the structure and chemical constituents of tissues and cells. The tissues were dehydrated in ethyl alcohol series of ascending concentrations, then embedded in molten paraffin wax and cooled to harden the wax. Tissue blocks were cut into 5 μm thick sections using a rotatory microtome and then mounted onto glass microscope slides. After clearing and rehydration, the tissue sections can be stained using Hematoxylin and Eosin (H and E) according to the methods of [110]. Ten sections of each tissue from each fish were examined by a light microscope for histopathological evaluation.

Statistical analyses

Statistical analyses were performed using R program [111] and GraphPad Prism (8.2.0, 2019). The normality of residuals and heteroscedasticity of variances were assumed using Shapiro-Wilk test and Levene’s test. Infection intensity of copepod was expressed as mean ± SEM (One way ANOVA, Tukey post hoc.). Oxidant/antioxidant biomarkers were expressed as mean ± SEM, while genes expression was presented as mean of copies of each gene (fold change) ± SEM (Independent sample T-test). The significance level was set at a probability value of less than 0.05 (p ˂0.05).

Accession numbers

Ergasilus sieboldi (GenBank accession number ON706996) and Vibrio alginolyticus (GenBank accession number ON041091).

Data Availability

The datasets analysed during the current study are available in the GenBank database under the accession numbers: ON706996 and ON041091.

References

  1. Bartley DM. World Aquaculture 2020 – A brief overview. FAO Fisheries and Aquaculture Circular No. 1233. Rome, FAO; 2022. https://doi.org/10.4060/cb7669en.

  2. Rigos G, Katharios P. Pathological obstacles of newly-introduced fish species in Mediterranean mariculture: a review. Rev Fish Biol Fisheries. 2010;20:47–70. https://doi.org/10.1007/s11160-009-9120-7.

    Article  Google Scholar 

  3. Dezfuli BS, Squerzanti S, Fabbri S, Castaldelli G, Giari L. Cellular response in semi-intensively cultured sea bream gills to Ergasilus sieboldi (Copepoda) with emphasis on the distribution, histochemistry and fine structure of mucous cells. Vet Parasitol. 2010;174(3–4):359–65. https://doi.org/10.1016/j.vetpar.2010.08.024.

    Article  PubMed  CAS  Google Scholar 

  4. Dezfuli BS, Giari L, Lui A, Lorenzoni M, Noga EJ. Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream. Fish Shellfish Immunol. 2011;30(4–5):1087–94. https://doi.org/10.1016/j.fsi.2011.02.005.

    Article  PubMed  CAS  Google Scholar 

  5. Abdel-Aziz M, Eissa AE, Hanna M, Abou Okada M. Identifying some pathogenic Vibrio/Photobacterium species during mass mortalities of cultured Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) from some Egyptian coastal provinces. Int J Veterinary Sci Med. 2013;1(2):87–95. https://doi.org/10.1016/j.ijvsm.2013.10.004.

    Article  Google Scholar 

  6. Abou-Okada M, El-Gendy NM, Elhelw R. Effect of booster vaccination on immunoprotection in European seabass vaccinated against vibriosis. Aquaculture Res. 2021;52:736–48. https://doi.org/10.1111/are.14930.

    Article  CAS  Google Scholar 

  7. Boxshall GA, Halsey SH. An introduction to copepod diversity. Ray Society; 2004.

  8. Rosim DF, Boxshall GA, Ceccarelli PS. A novel microhabitat for parasitic copepods: a new genus of Ergasilidae (Copepoda: Cyclopoida) from the urinary bladder of a freshwater fish. Parasitol Int. 2013;62(4):347–54. https://doi.org/10.1016/j.parint.2013.03.003.

    Article  PubMed  Google Scholar 

  9. Boxshall GA. Host specificity in copepod parasites of deep-sea fishes. J Mar Syst. 1998;15:215–23. https://doi.org/10.1016/S0924-7963(97)00058-4.

    Article  Google Scholar 

  10. Falkenberg JM, Lacerda ACF, Vieira GHC. Co-occurrence and niche overlap among gill parasites of the white mullet (Mugil curema Valenciennes, 1836) (Osteichthyes: Mugilidae) from the western Atlantic, Brazil. Parasitol Res. 2021;120(3):849–59. https://doi.org/10.1007/s00436-020-07015-5.

    Article  PubMed  Google Scholar 

  11. Tavares-Dias M, Dias-Júnior MBF, Florentino AC, Silva LMA, Cunha ACD. Distribution pattern of crustacean ectoparasites of freshwater fish from Brazil. Revista Brasileira De Parasitologia Veterinária. 2015;24:136–47. https://doi.org/10.1590/S1984-29612015036.

    Article  PubMed  Google Scholar 

  12. Balebona MC, Andreu MJ, Bordas MA, Zorrilla I, Moriñigo MA, Borrego JJ. 1998. Pathogenicity of Vibrio alginolyticus for cultured gilt-head sea bream (Sparus aurata L.). Applied and environmental microbiology 1998; 64(11), 4269–4275. https://doi.org/10.1128/AEM.64.11.4269-4275.1998.

  13. Balebona MC, Zorrilla I, Moriñigo MA, Borrego JJ. Survey of bacterial pathologies affecting farmed gilt-head sea bream (Sparus aurata L.) in southwestern Spain from 1990 to 1996. Aquaculture. 1998;166(1–2):19–35. https://doi.org/10.1016/S0044-8486(98)00282-8.

    Article  Google Scholar 

  14. Jun LI, Woo NY. Pathogenicity of vibrios in fish: an overview. J Ocean Univ Qingdao. 2003;2(2):117–28. https://doi.org/10.1007/s11802-003-0039-7.

    Article  Google Scholar 

  15. Cox FEG. Concomitant Infections, parasites and immune responses. Parasitology. 2001;122(S1); S23-S38. https://doi.org/10.1017/S003118200001698X.

  16. Bakaletz LO. Developing animal models for polymicrobial Diseases. Nat Rev Microbiol. 2004;2(7):552–68. https://doi.org/10.1038/nrmicro928.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Holzer AS, Sommerville C, Wootten R. Molecular studies on the seasonal occurrence and development of five myxozoans in farmed Salmo trutta L. Parasitology. 2006;132(2):193–205. https://doi.org/10.1017/S0031182005008917.

    Article  PubMed  CAS  Google Scholar 

  18. Bowers JM, Mustafa A, Speare DJ, Conboy GA, Brimacombe M, Sims DE, Burka JF. The physiological response of Atlantic salmon, Salmo salar L., to a single experimental challenge with sea lice, Lepeophtheirus salmonis. J Fish Dis 200; 23(3): 165–72. https://doi.org/10.1046/j.1365-2761.2000.00225.x.

  19. Kotob MH, Gorgoglione B, Kumar G, Abdelzaher M, Saleh M, El-Matbouli M. (2017). The impact of Tetracapsuloides bryosalmonae and Myxobolus cerebralis co-infections on pathology in rainbow trout. Parasites and vectors 2017a; 10: 1–14. https://doi.org/10.1186/s13071-017-2347-6.

  20. Kotob MH, Menanteau-Ledouble S, Kumar G, Abdelzaher M, El-Matbouliet M. The impact of co-infections on fish: a review. Vet Res. 2017b;47:98. https://doi.org/10.1186/s13567-016-0383-4.

    Article  Google Scholar 

  21. Xu DH, Shoemaker CA, Klesius PH. (2007). Evaluation of the link between gyrodactylosis and streptococcosis of Nile tilapia, Oreochromis niloticus (L.). Journal of fish diseases 2007; 30(4): 233–238. https://doi.org/10.1111/j.1365-2761.2007.00806.x.

  22. Zhang C, Li DL, Chi C, Ling F, Wang GX. (2015). Dactylogyrus intermedius parasitism enhances Flavobacterium columnare invasion and alters immune-related gene expression in Carassius auratus. Diseases of aquatic organisms 2015; 116(1): 11–21. https://doi.org/10.3354/dao02902.

  23. Lhorente JP, Gallardo JA, Villanueva B, Carabaño MJ, Neira R. Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi. PLoS ONE. 2014;9(4):e95397. https://doi.org/10.1371/journal.pone.0095397.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Mahmoud MA, Attia MM, Abdelsalam M, Abdel-Moneam DA, Zaki Ewiss MA. (2021). Ergasilus extensus and bacterial co‐infection in flathead grey mullet, Mugil cephalus (Linnaeus, 1758), are associated with pathological changes and immunological gene expression alterations. Aquaculture Research 2021; 52(12): 6143–6151. https://doi.org/10.1111/are.15476.

  25. Abou-Okada M, AbuBakr HO, Hassan A, Abdel-Radi S, Aljuaydi SH, Abdelsalam M, Taha E, Younis NA, Abdel-Moneam DA. Efficacy of Acriflavine for controlling parasitic Diseases in farmed Nile tilapia with emphasis on fish health, gene expression analysis, oxidative stress, and histopathological alterations. Aquaculture. 2021a;541:736791. https://doi.org/10.1016/j.aquaculture.2021.736791.

    Article  CAS  Google Scholar 

  26. Zhi T, Xu X, Chen J, Zheng Y, Zhang S, Peng J, Brown CL, Yang T. Expression of immune-related genes of Nile tilapia Oreochromis Niloticus after Gyrodactylus Cichlidarum and Cichlidogyrus sclerosus Infections demonstrating immunosupression in coinfection. Fish Shellfish Immunol. 2018;80:397–404. https://doi.org/10.1016/j.fsi.2018.05.060.

    Article  PubMed  CAS  Google Scholar 

  27. Smith NC, Rise ML, Christian SL. A comparison of the innate and adaptive immune systems in cartilaginous fish, ray-finned fish, and lobe-finned fish. Front Immunol. 2019;10:2292. https://doi.org/10.3389/fimmu.2019.02292.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Secombes CJ, Wang T. The innate and adaptive immune system of fish. In: Austin B, editor. Infectious Disease in aquaculture. Woodhead Publishing; 2012. pp. 3–68. https://doi.org/10.1533/9780857095732.1.3.

  29. Magnadottir B. Immunological control of Fish Diseases. Mar Biotechnol. 2010;12:361–79. https://doi.org/10.1007/s10126-010-9279-x.

    Article  CAS  Google Scholar 

  30. Hasnain SZ, Gallagher AL, Grencis RK, Thornton DJ. A new role for mucins in immunity: insights from gastrointestinal nematode Infection. Int J Biochem cell Biology. 2013;45(2):364–74. https://doi.org/10.1016/j.biocel.2012.10.011.

    Article  CAS  Google Scholar 

  31. Hosoya S, Kido S, Hirabayashi Y, Kai W, Kinami R, Yoshinaga T, et al. Genomic regions of pufferfishes responsible for host specificity of a monogenean parasite, Heterobothrium Okamotoi. Int J Parasitol. 2013;43(11):909–15. https://doi.org/10.1016/j.ijpara.2013.06.006.

    Article  PubMed  Google Scholar 

  32. Dezfuli BS, Giari L, Konecny R, Jaeger P, Manera M. Immunohistochemistry, ultrastructure and pathology of gills of Abramis brama from Lake Mondsee, Austria, infected with Ergasilus sieboldi (Copepoda). Diseases of aquatic organisms 2003; 53(3), 257–62. https://doi.org/10.1007/s00441-013-1627-5.

  33. Dezfuli BS, Giari L, Bosi G. Survival of metazoan parasites in fish: putting into context the protective immune responses of teleost fish. Adv Parasitol. 2021;112:77–132. https://doi.org/10.1016/bs.apar.2021.03.001.

    Article  Google Scholar 

  34. Boxshall GA, Copepoda K, editors. Marine Parasitology. Csiro Publishing; 2005. pp. 123–38.

  35. Tsotetsi AM, Avenant-Oldewage A, Mashego SN. Aspects of the pathology of Lamproglena clariae (Copepoda: Lernaeidae) on gills of Claria Gariepinus from the Vaal River system, South Africa. Afr Zool. 2005;40(2):169–78. https://hdl.handle.net/10520/EJC17977.

    Article  Google Scholar 

  36. Parrilla-Taylor DP, Zenteno-Savín T, Magallón-Barajas FJ. Antioxidant enzyme activity in pacific white leg shrimp (Litopenaeus vannamei) in response to Infection with white spot syndrome virus. Aquaculture. 2013;380:41–6. https://doi.org/10.1016/j.aquaculture.2012.11.031.

    Article  CAS  Google Scholar 

  37. Tkachenko H, Kurhaluk N, Grudniewska J. Oxidative stress biomarkers in different tissues of rainbow trout (Oncorhynchus mykiss) exposed to Disinfectant-CIP formulated with peracetic acid and hydrogen peroxide. Fisheries and Aquatic Life. 2014;22(3):207–19. https://doi.org/10.2478/aopf-2014-0021.

    Article  CAS  Google Scholar 

  38. Srikanth K, Pereira E, Duarte AC, Ahmad I. Glutathione and its dependent enzymes’ modulatory responses to toxic metals and metalloids in fish—a review. Environ Sci Pollut Res. 2013;20(4):2133–49. https://doi.org/10.1007/s11356-012-1459-y.

    Article  CAS  Google Scholar 

  39. Abhijith BD, Ramesh M, Poopal RK. Responses of metabolic and antioxidant enzymatic activities in gill, liver and plasma of Catla catla during methyl parathion exposure. J Basic Appl Zool. 2016;77:31–40. https://doi.org/10.1016/j.jobaz.2015.11.002.

    Article  CAS  Google Scholar 

  40. Dorval J, Hontela A. Role of glutathione redox cycle and catalase in defense against oxidative stress induced by endosulfan in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicol Appl Pharmcol. 2003;192(2):191–200. https://doi.org/10.1016/S0041-008X(03)00281-3.

    Article  CAS  Google Scholar 

  41. Lindenstrøm T, Secombes CJ, Buchmann K. Expression of immune response genes in rainbow trout skin induced by Gyrodactylus derjavini Infections. Vet Immunol Immunopathol. 2004;97(3–4):137–48. https://doi.org/10.1016/j.vetimm.2003.08.016.

    Article  PubMed  CAS  Google Scholar 

  42. Liu X, Chen N, Gao X, Zhang Y, Li X, Zhang Y, Bingb X, Huangc H, Zhang X. The Infection of red sea bream iridovirus in mandarin fish (Siniperca chuatsi) and the host immune related gene expression profiles. Fish Shellfish Immunol. 2018;74:474–84. https://doi.org/10.1016/j.fsi.2018.01.020.

    Article  PubMed  CAS  Google Scholar 

  43. Purcell MK, Kurath G, Garver KA, Herwig RP, Winton JR. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) Infection or DNA vaccination. Fish Shellfish Immunol. 2004;17(5):447–62. https://doi.org/10.1016/j.fsi.2004.04.017.

    Article  PubMed  CAS  Google Scholar 

  44. Zhou B, Wang XI, Feng LI, Wang Y, Yang L, Zhen X, Tan W. Mitochondrial activity and oxidative stress functions are influenced by the activation of AhR-induced CYP1A1 overexpression in cardiomyocytes. Mol Med Rep. 2017;16:174–80. https://doi.org/10.3892/mmr.2017.6580.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Zou J, Secombes CJ. The function of fish cytokines. Biology. 2016;5(2):23. https://doi.org/10.3390/biology5020023.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Xu DH, Shoemaker CA, Klesius PH. Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish. FEMS Microbiol Lett. 2012;329:160–7. https://doi.org/10.1111/j.1574-6968.2012.02518.x.

    Article  PubMed  CAS  Google Scholar 

  47. Abdel-Radi S, Rashad MM, Ali GE, Eissa AE, Abdelsalam M, Abou-Okada M. Molecular characterization and phylogenetic analysis of parasitic copepoda; Ergasilus sieboldi isolated from cultured gilthead sea bream (Sparus aurata) in Egypt, associated with analysis of oxidative stress biomarkers. J Parasitic Dis. 2022;46(4):1080–9. https://doi.org/10.1007/s12639-022-01531-0.

    Article  Google Scholar 

  48. Kvach Y, Tkachenko MY, Seifertová M, Ondračková M. Insights into the diversity, distribution and phylogeny of three ergasilid copepods (Hexanauplia: Ergasilidae) in lentic water bodies of the Morava river basin, Czech Republic. Limnologica. 2021;91:125922. https://doi.org/10.1016/j.limno.2021.125922.

    Article  Google Scholar 

  49. Montes MM, Martorelli SR. A bayesian analysis of the parasitic ecology in Jenynsia multidentata (Pisces: Anablepidae). Iheringia Série Zoologia. 2017;107. https://doi.org/10.1590/1678-4766e2017024.

  50. Waicheim MA, Mendes Marques T, Rauque CA, Viozzi G. New species of Ergasilus Von Nordmann, 1832 (Copepoda: Ergasilidae) from the gills of freshwater fishes in Patagonia. Argentina Syst Parasitol. 2021;98(2):131–9. https://doi.org/10.1007/s11230-021-09966-4.

    Article  PubMed  Google Scholar 

  51. Walter TC, Boxshall GA. World of Copepods database. World Register of Marine Species. https://www.marinespecies.org/copepoda on 8 March 2022. https://doi.org/10.14284/356.

  52. Ondrăckovà M, Fojtů J, Seifertová M, Kvach Y, Jurajda P. Non-native parasitic copepod Neoergasilus japonicus (Harada, 1930) utilizes non-native fish host Lepomis gibbosus (L.) in the floodplain of the River Dyje (Danube Basin). Parasitol Res. 2019;118(1):57–62. https://doi.org/10.1007/s00436-018-6114-1.

    Article  PubMed  Google Scholar 

  53. Song Y, Wang GT, Yao WJ, Gao Q, Nie P. Phylogeny of freshwater parasitic copepods in the Ergasilidae (Copepoda: Poecilostomatoida) based on 18S and 28S rDNA sequences. Parasitol Res. 2008;102:299–306. https://doi.org/10.1007/s00436-007-0764-8.

    Article  PubMed  CAS  Google Scholar 

  54. Ben Kahla-Nakbi A, Chaieb K, Besbes A, Zmantar T, Bakhrouf A. Virulence and enterobacterial repetitive intergenic consensus PCR of Vibrio alginolyticus strains isolated from Tunisian cultured gilthead sea bream and sea bass outbreaks. Vet Microbiol. 2006;117:321–7. https://doi.org/10.1016/j.vetmic.2006.06.012.

    Article  PubMed  CAS  Google Scholar 

  55. Oldewage WH, Van As JG. Observations on the attachment of a piscine gill parasitic ergasilid (Crustacea: Copepoda). Afr Zool. 1987;22(4):313–7.

    Google Scholar 

  56. Yuniar AT, Palm HW, Walter T. Crustacean fish parasites from Segara Anakan lagoon, Java, Indonesia. Parasitol Res. 2007;100:1193–204. https://doi.org/10.1007/s00436-006-0391-9.

    Article  PubMed  Google Scholar 

  57. Rottmann RW, Francis-Floyd R, Durborow R. The role of stress in fish Disease. Stoneville, MS: Southern Regional Aquaculture Center; 1992.

    Google Scholar 

  58. Svobodová Z. Water quality and fish health. Food and Agriculture Org.; 1993.

  59. Nofal MI, Abdel-Latif HM. Ectoparasites and bacterial co-infections causing summer mortalities among cultured fishes at Al-Manzala with special reference to Water quality parameters. Life Sci J. 2017;14(6):72–83. https://doi.org/10.7537/marslsj140617.11.

    Article  CAS  Google Scholar 

  60. Shameena SS, Kumar S, Kumar K, Raman RP. Role of temperature and co-infection in mediating the immune response of goldfish. Microb Pathog. 2021;156:104896. https://doi.org/10.1016/j.micpath.2021.104896.

    Article  PubMed  CAS  Google Scholar 

  61. Hakalahti T, Karvonen A, Valtonen ET. Climate warming and Disease risks in temperate regions–Argulus coregoni and Diplostomum spathaceum as case studies. J Helminthol. 2006;80(2):93–8. https://doi.org/10.1079/JOH2006351.

    Article  PubMed  CAS  Google Scholar 

  62. Marchiori NC, Gonçalves ELT, Tancredo KR, Pereira-Junior J, Garcia JRE, Martins ML. Effect of water temperature and salinity in oviposition, hatching success and infestation of Aphanoblastella mastigatus (Monogenea, Dactylogyridae) on Rhamdia quelen. Brazilian J Biology. 2015;75:245–52. https://doi.org/10.1590/1519-6984.14014.

    Article  Google Scholar 

  63. Groner ML, McEwan GF, Rees EE, Gettinby G, Revie CW. Quantifying the influence of salinity and temperature on the population dynamics of a marine ectoparasite. Can J Fish Aquat Sci. 2016;73(8):1281–91. https://doi.org/10.1139/cjfas-2015-0444.

    Article  Google Scholar 

  64. Mathews PD, Patta AC, Gama GS, Mertins O. Infestation by Ergasilus Coatiarus (Copepoda: Ergasilidae) in two amazonian cichlids with new host record from Peru: an ectoparasites natural control approach. CR Biol. 2018;341(1):16–9. https://doi.org/10.1016/j.crvi.2017.12.001.

    Article  Google Scholar 

  65. Eaves AA, Ang KP, Murray HM. Occurrence of the parasitic copepod Ergasilus labracis on Threespine sticklebacks from the south coast of Newfoundland. J Aquat Anim Health. 2014;26(4):233–42. https://doi.org/10.1080/08997659.2014.938871.

    Article  PubMed  Google Scholar 

  66. Murray HM, Ang KP. The effects of Local Environmental conditions and the emergence of Young of the year on the Regional distribution, prevalence, and intensity of Ergasilus labracis (Copepoda) parasitic on three-spined stickleback (Gasterosteus aculeatus) from. Can Comp Parasitol. 2018;85(1):1–12. https://doi.org/10.1654/1525-2647-85.1.1. the Bay d’Espoir/Hermitage Bay Region of Newfoundland.

    Article  Google Scholar 

  67. Thompson FL, Iida T, Swings J. Biodiversity of vibrios. Microbiol Mol Biol Rev. 2004;68(3):403–31. https://doi.org/10.1128/mmbr.68.3.403-431.2004.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Huang Z, Yu K, Fang Y, Dai H, Cai H, Li Z, Kan B, Wei Q, Wang D. Comparative genomics and transcriptomics analyses reveal a unique environmental adaptability of Vibrio fujianensis. Microorganisms. 2020;8(4):555. https://doi.org/10.3390/microorganisms8040555.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Sanches-Fernandes GM, Sá-Correia I, Costa R. Vibriosis outbreaks in aquaculture: addressing environmental and public health concerns and preventive therapies using gilthead seabream farming as a model system. Front Microbiol. 2022;13:904815. https://doi.org/10.3389/fmicb.2022.904815.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Abdallah FB, Ellafi A, Lagha R, Kallel H, Bakhrouf A. Virulence gene expression, proteins secreted and morphological alterations of Vibrio parahaemolyticus and Vibrio alginolyticus in response to long-term Starvation in seawater. Afr J Microbiol Res. 2011;5(7):792–801. https://doi.org/10.5897/AJMR10.653.

    Article  Google Scholar 

  71. Di Pinto A, Ciccarese G, Tantillo G, Catalano D, Forte VT. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J Food Prot. 2005;68(1):150–3. https://doi.org/10.4315/0362-028x-68.1.150.

    Article  PubMed  Google Scholar 

  72. Jiang K, Xu L, Su Y, Wang Y, Guo Z, Xu H, Gao F, Feng J. Analysis of antibiotic resistance spectrum of Vibrio harveyi strains isolated from maricultured fish in the South China Sea during 2012–2014. South China Fish Sci. 2016;12:99–107. https://doi.org/10.3969/j.issn.2095-0780.2016.06.013.

    Article  Google Scholar 

  73. Zhao S, Ma L, Wang Y, Fu G, Zhou J, Li X, Fang W. Antimicrobial resistance and pulsed-field gel electrophoresis typing of Vibrio parahaemolyticus isolated from shrimp mariculture environment along the east coast of China. Mar Pollut Bull. 2018;136:164–70. https://doi.org/10.1016/j.marpolbul.2018.09.017.

    Article  PubMed  CAS  Google Scholar 

  74. Halliwell B, Gutteridge JMC. Free radicals in Biology and Medicine. Oxford: Oxford University Press; 1999.

    Google Scholar 

  75. Di Giulio RT, Washburn PC, Wenning RJ, Winston GW, Jewell CS. Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environ Toxicol Chem. 1989;8:1103–23. https://doi.org/10.1002/etc.5620081203.

    Article  Google Scholar 

  76. Nunes B, Caldeira C, Pereira JL, Gonçalves F, Correia AT. Perturbations in ROS-related processes of the fish Gambusia holbrooki after acute and chronic exposures to the metals copper and cadmium. Environ Sci Pollut Res. 2015;22(5):3756–65. https://doi.org/10.1007/s11356-014-3580-6.

    Article  CAS  Google Scholar 

  77. Almeida JA, Barreto RE, Novelli ELB, Castro FJ, Moron SE. Oxidative stress biomarkers and aggressive behavior in fish exposed to aquatic cadmium contamination. Neotrop Ichthyol. 2009;7:103–8. https://doi.org/10.1590/S1679-62252009000100013.

    Article  Google Scholar 

  78. Mozhdeganloo Z, Heidarpour M. Oxidative stress in the gill tissues of goldfishes (Carassius auratus) parasitized by Dactylogyrus spp. J Parasit Dis. 2014;38(3):269–72. https://doi.org/10.1007/s12639-013-0239-z.

    Article  PubMed  CAS  Google Scholar 

  79. Xu SL, Wang DL, Jia CY, Jin S, Wang CL, Zou X. Effects of Vibrio alginolyticus Infection on immune-related enzyme activities and ultrastructure of Charybdis japonica gills. Aquaculture. 2013;396:82–8. https://doi.org/10.1016/j.aquaculture.2013.02.042.

    Article  CAS  Google Scholar 

  80. Kar B, Mohanty J, Hemaprasanth KP, Sahoo PK. The immune response in rohu, Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) Infection: kinetics of immune gene expression and innate immune response. Aquac Res. 2015;46(6):1292–308. https://doi.org/10.1111/are.12279.

    Article  CAS  Google Scholar 

  81. Saurabh S, Mohanty BR, Sahoo PK. Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) Infection. Vet Parasitol. 2011;175(1–2):119–28. https://doi.org/10.1016/j.vetpar.2010.10.001.

    Article  PubMed  CAS  Google Scholar 

  82. Grasso V, Padilla D, Bravo J, Román L, Rosario I, Acosta B, Vega B, et al. Immunization of sea bream (Sparus aurata) juveniles against Photobacterium damselae subsp. piscicida by short bath: Effect on some pro-inflammatory molecules and the Mx gene expression. Fish Shellfish Immunol. 2015;46(2):292–6. https://doi.org/10.1016/j.fsi.2015.06.030.

    Article  PubMed  CAS  Google Scholar 

  83. Reyes-Becerril M, López-Medina T, Ascencio-Valle F, Esteban MÁ. Immune response of gilthead sea bream (Sparus aurata) following experimental Infection with Aeromonas hydrophila. Fish Shellfish Immunol. 2011;31(4):564–70. https://doi.org/10.1016/j.fsi.2011.07.006.

    Article  PubMed  CAS  Google Scholar 

  84. Poisa-Beiro L, Dios S, Montes A, Aranguren R, Figueras A, Novoa B. Nodavirus increases the expression of Mx and inflammatory cytokines in fish brain. Mol Immunol. 2008;45(1); 218–25. https://doi.org/10.1016/j.molimm.2007.04.016.

  85. Benhamed S, Guardiola FA, Martínez S, Martínez-Sánchez MJ, Pérez-Sirvent C, Mars M, Esteban MÁ. Exposure of the gilthead seabream (Sparus aurata) to sediments contaminated with heavy metals down-regulates the gene expression of stress biomarkers. Toxicol Rep. 2016;26(3):364–72. https://doi.org/10.1016/j.toxrep.2016.02.006.

    Article  CAS  Google Scholar 

  86. Zhang J, Yao J, Wang R, Zhang Y, Liu S, Sun L, Jiang Y, Feng J, Liu N, Nelson D, Waldbieser G, Liu Z. The cytochrome P450 genes of channel catfish: their involvement in Disease defense responses as revealed by meta-analysis of RNA-Seq data sets. Biochim Biophys Acta. 2014;1840(9):2813–28. https://doi.org/10.1016/j.bbagen.2014.04.016.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  87. Chu P, He L, Zhu D, Huang R, Liao L, Li Y, Zhu Z, Wang Y. Identification, expression and functional characterisation of CYP1A in grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol. 2019;95:35–43. https://doi.org/10.1016/j.fsi.2019.10.022.

    Article  PubMed  CAS  Google Scholar 

  88. Santos P, Peixoto D, Ferreira I, Passos R, Pires P, Simões M, et al. Short-term Immune responses of Gilthead Sea bream (Sparus aurata) juveniles against Photobacterium damselae subsp. piscicida. Int J Mol Sci. 2022;23(3):1561. https://doi.org/10.3390/ijms23031561.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  89. Gomez D, Sunyer JO, Salinas I. The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. Fish Shellfish Immunol. 2013;35(6):1729–39. https://doi.org/10.1016/j.fsi.2013.09.032.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  90. Lazado CC, Caipang CM. Mucosal immunity and probiotics in fish. Fish Shellfish Immunol. 2014;39(1):78–89. https://doi.org/10.1016/j.fsi.2014.04.015.

    Article  PubMed  CAS  Google Scholar 

  91. Morshed SM, Lee TH. The role of the microbiome on fish mucosal immunity under changing environments. Fish Shellfish Immunol. 2023;139:108877. https://doi.org/10.1016/j.fsi.2023.108877.

    Article  PubMed  CAS  Google Scholar 

  92. Mathieu-Bégné E, Blanchet S, Rey O, Toulza E, Veyssière C, Manzi S, Lefort M, Scelsi O, Loot G. A longitudinal survey in the wild reveals major shifts in fish host microbiota after parasite Infection. Mol Ecol. 2023;32(11):3014–24. https://doi.org/10.1111/mec.16901.

    Article  PubMed  Google Scholar 

  93. Mathieu-Bégné E, Blanchet S, Rey O, Scelsi O, Poesy C, Marselli G, Loot G. A fine‐scale analysis reveals microgeographic hotspots maximizing Infection rate between a parasite and its fish host. Funct Ecol. 2022;36(2):380–91. https://doi.org/10.1111/1365-2435.13967.

    Article  Google Scholar 

  94. Reynolds LA, Finlay BB, Maizels RM. Cohabitation in the intestine: interactions among helminth parasites, bacterial microbiota, and host immunity. J Immunol. 2015;195(9):4059–66. https://doi.org/10.4049/jimmunol.1501432.

    Article  PubMed  CAS  Google Scholar 

  95. Abdelhalim AI, Lewis JW, Boxshall GA. The life-cycle of Ergasilus sieboldi Nordmann (Copepoda: Poecilostomatoida), parasitic on British freshwater fish. J Nat Hist. 1991;25(3):559–82. https://doi.org/10.1080/00222939100770361.

    Article  Google Scholar 

  96. Kabata Z. Crustacea as enemies of fishes. In: Snieszko SF, Axelrod HR, editors. Diseases of fishes, Book 1. Jersey City, PA: TFH Publishers; 1970.

    Google Scholar 

  97. Lui A, Manera M, Giari L, Mulero V, Dezfuli BS. Acidophilic granulocytes in the gills of gilthead sea bream Sparus aurata: evidence for their responses to a natural Infection by a copepod ectoparasite. Cell Tissue Res. 2013;353(3):465–72. https://doi.org/10.1007/s00441-013-1627-5.

    Article  PubMed  CAS  Google Scholar 

  98. Dong HT, Taengphu S, Sangsuriya P, Charoensapsri W, Phiwsaiya K, Sornwatana T, Khunrae P, et al. Recovery of Vibrio harveyi from scale drop and muscle necrosis Disease in farmed barramundi. Lates calcarifer in Vietnam Aquaculture. 2017;473:89–96. https://doi.org/10.1016/j.aquaculture.2017.02.005.

    Article  Google Scholar 

  99. Mohamad N, Amal MNA, Yasin ISM, Saad MZ, Nasruddin NS, Al-saari N, Minog S, Sawabe T. Vibriosis in cultured marine fishes: a review. Aquaculture. 2019;512:734289. https://doi.org/10.1016/j.aquaculture.2019.734289.

    Article  Google Scholar 

  100. Thatcher VE. Amazon fish parasites. Sofia-Moscow: Pensoft Publishers; 2006.

    Google Scholar 

  101. Bush AO, Lafferty KD, Lotz JM, Shostak AW. Parasitology meets ecology on its own terms: Margolis. Revisit J Parasitol. 1997;1:575–83. https://doi.org/10.2307/3284227.

    Article  Google Scholar 

  102. Jorgensen JH, Turnidge JD. Susceptibility test methods: dilution and disk diffusion methods. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of clinical microbiology. ASM Press; 2007. pp. 1152–72. https://doi.org/10.1128/9781555817381.ch71.

  103. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing (26th ed.). CLSI supplement M100S. ; 2016. (ISBN 1-56238-924-6).

  104. Dai L, Lu LM, Wu CM, Li BB, Huang SY, Wang SC, Qi YH, Shen JZ. Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006. FEMS Microbiol Lett. 2008;286:178–83. https://doi.org/10.1111/j.1574-6968.2008.01272.x.

    Article  PubMed  CAS  Google Scholar 

  105. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. https://doi.org/10.1016/s0076-6879(84)05016-3.

    Article  PubMed  CAS  Google Scholar 

  106. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–8. https://doi.org/10.1016/0003-2697(79)90738-3.

    Article  PubMed  CAS  Google Scholar 

  107. Campos-Sánchez JC, Mayor-Lafuente J, Guardiola FA, Esteban MÁ. In silico and gene expression analysis of the acute inflammatory response of gilthead seabream (Sparus aurata) after subcutaneous administration of carrageenin. Fish Physiol. Biochem. 2021;47(5):1623–43. https://doi.org/10.1007/s10695-021-00999-6.

    Article  CAS  Google Scholar 

  108. Salmerón C, Riera-Heredia N, Gutiérrez J, Navarro I, Capilla E. Adipogenic gene expression in Gilthead Sea Bream Mesenchymal Stem cells from different origin. Front Endocrinol (Lausanne). 2016;22(7):113. https://doi.org/10.3389/fendo.2016.00113.

    Article  Google Scholar 

  109. Bancroft JD, Gamble M, editors. Theory and practice of histological techniques. Elsevier health sciences; 2008.

  110. Livak KJ, Schmittgen HD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–8. https://doi.org/10.1006/meth.2001.1262.

    Article  PubMed  CAS  Google Scholar 

  111. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ; 2021. URL https://www.R-project.org/.

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Authors and Affiliations

  1. Department of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

    Mahmoud Abou-Okada

  2. Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

    Maha M. Rashad & Ghada E. Ali

  3. Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

    Shimaa Abdel-Radi

  4. Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

    Azza Hassan

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MA: Conceptualization (lead), Formal Analysis, Methodology (equal), Investigation (lead), Supervision, Writing – Original Draft (lead), Editing final draft. MMR and GEA: Methodology (equal), Investigation (supporting), Writing – Original Draft (supporting). SA: Methodology (supporting), Writing – Original Draft (supporting). AH: Conceptualization (supporting), Methodology (equal), Writing – Original Draft (supporting). All authors read and approved the final manuscript.

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All fish experimental protocols were conducted in accordance with the ARRIVE 2.0 standards (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC), Cairo University). All methods were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines and regulations, Cairo University.

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Abou-Okada, M., Rashad, M.M., Ali, G.E. et al. Oxidative stress, gene expression and histopathology of cultured gilthead sea bream (Sparus aurata) naturally co-infected with Ergasilus sieboldi and Vibrio alginolyticus. BMC Vet Res 19, 277 (2023). https://doi.org/10.1186/s12917-023-03840-9

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