Impact of Climate Change on Some Seasonal Bacterial Eruptions among Cultured Marine Fishes from Egyptian Coastal Provinces

Climate change is now considered one of the greatest challenges and is expected to have a drastic impact on mariculture. The present study aimed to evaluate the impact of climatic changes on the emergence of bacterial pathogens among cultured marine fish in northern Egyptian provinces. 135 samples of three marine fish species, represented as 45 of Dicentrarchus labrax (700 ±25 g), 45 of Sparus aurata (350 ±25 g)

the total fish produced in Egypt (Mahmoud et al., 2023), and the Deeba Triangle produces a significant portion of this percentage (Eissa et al., 2021).
Deeba Triangle (a triangle region between Damietta and Port Said Province) is currently regarded as one of the most exposed areas to the drastic impact of climate change (Eissa et al., 2021).Climatic changes, including fluctuations in temperature, rainfall patterns and deteriorated water quality are the main triggers of bacterial infection eruptions among the cultured marine fish in these areas (Cianconi et al., 2020).In Egypt, bacterial infections are the main cause of severe mortalities and morbidities in a variety of cultured marine fish and the increase in water temperature in summer has a significant effect on mortality rates, increasing physiological stress and rendering fish more vulnerable to bacterial infection (Kaleem and Sabi, 2021).
Vibriosis is one of the devastating bacterial diseases affecting cultured marine fish, caused by pathogenic Vibrio species, and is regarded as one of the most serious bacterial infections that have zoonotic and financial significance in mariculture (Cascarano et al., 2021).Vibrio alginolyticus and Vibrio parahemolyticus are responsible for mass mortalities among cultured marine fish on many farms throughout the Mediterranean area (Snoussi et al., 2008;Rowley et al., 2014).Vibriosis is one of the diseases that may be severely affected by climatic changes.Vibrios grow efficiently in temperate water (> 15 o C) and at low salinity (<25 ppt) (Vezzulli et al., 2013).Thus, the anticipated rise in temperature and decrease in salinity in temperate regions will affect the epidemiological patterns of vibriosis Climatic changes, such as high water temperatures, especially during summer season, are usually a risk factor for disease outbreaks (Islam et al., 2022).Other environmental stressors, including high salinity, increased ammonia, and physical damage, can all be exacerbated by the climatic changes rendering fish more susceptible to bacterial infections (Mehrim and Refaey, 2023).
The current study aimed to evaluating the impact of climatic changes on the eruption of bacterial diseases among marine fish in the northern Egyptian provinces.

Study area
The study area includes private marine fish farms located in the Deeba Triangle which is bordered by (the Mediterranean Sea to the north, the Damietta Estuary to the west, and Lake Manzala to the south), Shataa Damietta (Damietta governorate) and Ismailia province.The main water supply is coming from the Mediterranean Sea.

Fish Sampling
135 samples of three marine fish species represented as 45 of Dicentrarchus labrax, 45(700 ±25 g), 45 Sparus aurata (350 ±25 g), and 45 Argyrosomus regius (1 kg ±50 g) were collected seasonally from private marine fish farms located in Deeba Triangle, Shataa Damietta (Damietta governorate) and Ismailia province, Egypt.The collected fish were transferred immediately in an insulated icebox to the Lab of Aquatic Animal Medicine and Management, Cairo University.The collected fish were subjected to clinical, postmortem and bacteriological investigations according to Eissa (2016).

Water sampling
Water samples were collected from the fish farms using sterile glass bottles, transported in isolated ice box, and analyzed following the standard procedures described by APHA (2005).Samples were collected in August (summer), and October (autumn).Water samples were physio-chemically analyzed for temperature, dissolved oxygen levels, pH values, and water salinity using a portable multiparameter water quality meter (HI98194, Hanna Instruments) and ammonia using (HI4101, Hanna Instruments).

Bacteriological isolation and identification
Loopfuls from internal organs including (liver, kidney, and spleen) and skin ulcers under complete aseptic conditions were streaked onto tryptic soy agar supplemented with 2% NaCl (TSA, Difco), sheep blood agar with 2% NaCl, and thiosulfate citrate bile salt sucrose agar (TCBS, Oxoid).All inoculated plates were incubated at 28º C for 24-48 h.For purification purposes, single colonies were selected from the plates and re-streaked onto TSA plates with 2% NaCl.Pure colonies were phenotypically identified using Gram staining and Motility test.Conventional biochemical identification was carried out using oxidase, catalase test and sensitivity to a vibriostatic agent (O/129) according to Buller (2004).Furthermore, the biochemical identification of bacterial strains was confirmed using API 20E test (Biomérieux, France), according to the guidelines of the manufacturer.Eventually, the purified strains were kept in tryptic soy broth with 15% (vol/ vol) glycerol and kept at -80° C for further molecular identification.

16S rRNA gene sequencing
Representative purified bacterial isolates were selected for molecular identification by partial sequencing of 16S rRNA gene.DNA was extracted from pure colonies using QIAamp DNA Mini kit (Qiagen) according to the manufacturer's protocol.PCR for 16SrRNA gene was done using the universal primer (16S-F: 5'-AGAGTTTGATCCTGGCTCAG-3') and (16S-R: 5'-GGTTACCTTGTTACGACTT-3'), described by Weisburg et al., (1991).The PCR reaction was performed in a final volume of 25 μL, using 1X PCR mix consisting of 0.25 μM of both primer, 200 mM dNTP, 1 U Taq polymerase and 250 ng genomic DNA.PCR reaction conditions were adjusted as follows: initial DNA denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55°C for 60 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min.
Purified PCR amplicons were sequenced using ABI 3730xl DNA sequencer (Applied Biosystems™) at Sigma.The assembled 16S rRNA genes were compared against other 16S rRNA genes sequences using BLAST ® search in the database of GenBank.

Phylogenetic analysis
The sequences of the 16S rRNA gene of bacterial strains were deposited in GenBank.To calculate genetic distance, the phylogenetic trees were constructed using MEGA X 11 using the neighbor-joining method, and the level of confidence was verified by bootstrap analysis for each branch at 1000 repeats (Kumar et al., 2018).

Histopathological Examination
Autopsy samples were taken from the liver, kidney, spleen, swim bladder and gills of fish fixed in neutral buffered formalin 10%, washed, dehydrated, cleared and embedded in paraffin.The paraffin embedded blocks were sectioned at 4-microns thickness and stained by Hematoxylin and Eosin stain (Bancroft et al., 2013) for histopathological examination by a light microscope (Olympus BX50, Japan).

Clinical and Necropsy findings
Moribund fish showed erratic swimming behaviour and decreased appetite.The common gross pathological lesions recorded were skin haemorrhages, erosions, ulcerations, and detached scales.Necropsy findings of the spleen and kidney revealed severe congestion.The liver was congested and some cases, pale and friable, with an enlarged gall bladder.The gills were congested (Fig. 1).The swim bladders showed congestion, enlargement, thickening, and inflammation of their walls.With the presence of serous to hemorrhagic ascetic fluid.

prevalence of infection among the examined fish
The total number of clinically diseased fish was 71 with 52.6 % prevalence rate.For Sparus aurata, the total number of clinically diseased fish was 27 from 45 with 60% prevalence rate.For Dicentrarchus labrax, the total number of clinically diseased fish was 29 from 45 with 64.5 % prevalence rate.For Argyrosomus regius, the total number of clinically diseased fish was 15 from 45 with 30.3% prevalence rate.The prevalence of infection among the examined fishes (apparently healthy and clinically diseased) is presented in (Table 1).Three different bacterial isolates were identified using conventional and semi-automated biochemical tests.The first identified bacterium was Vibrio alginolyticus that appeared as 2-3 mm smooth yellow colonies on TCBS, and large swarming colonies on TSA + 2% NaCl.The bacterial isolates were gram-negative straight to slightly curved rods, motile, oxidase and catalase positive, and susceptible to vibriostatic agent (O/129).The API E 20 profile of V. alginolyticus isolates is presented in Table 2.
The second identified bacterial pathogen was Vibrio parahaemolyticus that appeared 1-2 mm pin-point green colonies with a dark blue center on TCBS, and small, white, non-swarming on TSA + 2% NaCl.The bacterial isolates were gram-negative short rods, motile, oxidase and catalase positive, and susceptible to vibriostatic agent (O/129).The API E 20 profile of V. parahaemolyticus isolates (Table 2).
The third identified bacterial isolate was identified as Photobacterium damselae subsp.piscicida, that appeared small grayish colonies on TSA + 2% NaCl, and did not grow on TCBS agar.The bacterial isolates were gram-negative bipolar cocobacili short rods, non-motile, non-hemolytic, oxidase and catalase positive, and sensitive to vibriostatic agent (O/129).The API E 20 profile of P. damselae subsp piscicida isolates (Table 2).
Fig. 2: Phylogenetic analysis based on the 16S rRNA gene sequences of V. alginolyticus, V. parahemolyticus and P. damselae subsp.Piscicida isolated in this study.The tree was constructed and analyzed by the neighbor-joining method.

Seasonal prevalence of bacterial isolates in Sparus aurata
Fifty suspected strains were isolated from the clinically diseased fishes.A total of 20 bacterial isolates were retrieved from moribund Sparus aurata.The most prevalent isolated bacterial strains were V. alginolyticus with 50% of the total isolates.Other bacterial pathogens belonging to V. parahaemolyticus, and P. damselae subsp piscicida were also identified with 35%, and 15% of the total isolates, respectively.The seasonal prevalence of bacterial isolates among moribund Sparus aurata of this study (Table 3).

Seasonal prevalence of bacterial isolates in Dicentrarchus labrax
A total of 20 bacterial isolates were isolated from diseased Dicentrarchus labrax.The most prevalent isolated bacterial strains were V. alginolyticus with 50% of the total isolates.Other bacterial strains belonging to V. parahaemolyticus, and P. damselae subsp piscicida were also identified with 30%, and 20% of the total isolates, respectively.The seasonal prevalence of bacterial isolates among diseased Dicentrarchus labrax of this study (Table 4).

Seasonal prevalence of bacterial isolates in Argyrosomus regius
A total of 10 bacterial isolates were retrieved from moribund Argyrosomus regius.The most prevalent isolated bacterial strains were V. alginolyticus with 40% of the total isolates.Other bacterial strains belonging to V. parahaemolyticus, and P. damselae subsp piscicida were also identified with 30%, and 30% of the total isolates, respectively.The seasonal prevalence of bacterial isolates among moribund Argyrosomus regius of this study (Table 5).

Histopathological findings
Histopathological examination of Gilthead seabream hepatic tissues showed focal haemorrhages were detected in the parenchyma associated with dilatation and congestion of the portal vein (Fig. 3 a) as well as dilatation and congestion of the central vein with activation of melanomacrophage centre (Fig. 3 b & C). .Concerning kidneys, there was activation of the melanomacrophage centre with severe diffuse vacuolar degeneration of tubular lining epithelium (Fig. 3 d) and lymphoid proliferation in between the tubules (Fig. 3

e).
Spleen showing activation of melanomacrophage center in between the follicles (Fig. 3 f).Histopathological examination of meagre swim bladder revealed inflammatory cells infiltration and fibrosis in the bladder wall (Fig. 5 a) associated with congestion in the blood vessels (Fig. 5 b).liver, there was hyperplasia of collagen fibers (Fig. 5 c) Vacuolar degeneration of hepatocytes (Fig. 5d).kidneys, there were sever dilatation and congestion in the blood vessels between the tubules and glomeruli (Fig. 5 e).Concerning Spleen, there was ischemia in the pulps (Fig. 5 f).

DISCUSSION
Climate change is now considered one of the biggest challenges facing the national, regional, and global societies.Fisheries represent a significant industry in the Egyptian national income (Abd El Tawab et al., 2018).In aquaculture economy, marine fish represent the main investment options for northern coastal fishermen (GFARD, 2020).There are varieties of environmental stressors, such as improper water quality characteristics and increasing water temperature that might affect marine fish.In mariculture, these stressors, particularly declining water quality characteristics in Deeba Triangle, could promote the spread of infectious diseases, resulting in outbreaks in Egyptian mariculture In the present study, stressful aquatic environments, such as low dissolved oxygen, elevated water temperatures, inconsistently high salinity and a remarkably high increase in un-ionized ammonia, together with severe climatic conditions, have staggeringly compromised the fish immune status and increased their vulnerability to infection by ubiquitous bacteria, including Vibrio spp.and highly invasive bacteria (Photobacterium spp.) (Rebl et al., 2020).There were similarities between our results and those described by Abdelaziz et al. (2013), who recorded an abrupt increase in un-ionized ammonia, water temperature, and salinity during recurrent episodes of mortalities in European seabass and gilthead seabream.
In the current study, a total of 135 marine fishes were collected from several private marine farms located in Deeba Triangle, Shataa Damietta (Damietta governorate), and Ismailia province.In respect to the prevalence of infection, the total number of infected fish was 71 from 135 with a 52.6 % prevalence rate.These results nearly agreed with those of El-Gendy (2013) who recorded 44.1% prevalence rate among infected fish.In contrast, our result was slightly lower than that of Moustafa et al.
(2010) who recorded 69.9% prevalence rate among infected fish.This variation in prevalence may be attributed to differences in sampling season, isolation locations, and species diversity.
The disease outbreaks in fish may be linked to the suppression of fish immune system.Such immune-suppression is positively correlated with severe climatic conditions such as an abrupt rise in water temperature and a subsequent decrease in dissolved oxygen (Abdelaziz et al., 2013) (2016).This can be explained that the higher temperatures as a result of severe climatic change reduced immunity and decreased resistance to disease, so fish became more vulnerable to septicemic infection.
The histopathological examination of various organs of moribund fishes revealed that the liver showed focal hemorrhages in the parenchyma associated with severe congestion of blood vessels and severe degeneration in the hepatocytes.While kidneys showed mononuclear cell infiltration around renal tubules, diffuse vacuolar degeneration of tubular lining epithelium, severe dilatation and congestion in the blood vessels between the tubules and glomeruli.Spleen showed focal proliferation of

CONCLUSION
Vibriosis caused by V. parahaemolyticus and V. alginolyticus and photobacteriosis caused by Photobacterium damselae subspecies piscicida are significant bacterial diseases affecting cultured marine species.Vibriosis and Photobacteriosis eruptions are triggered with hot climate with consequent deterioration of pond's water quality.Local and systemic immunities are key factor in progression of these bacterial eruptions.Thus, enhancing immunity will decisively minimize the possibilities of such bacterial eruptions.

Fig. 3 :
Fig.3: photomicrograph, Gilthead seabream (a) liver showing focal hemorrhages with dilatation and congestion of the portal vein (black arrow) (H&EX200).(b) as well as dilatation and congestion of the central vein (black arrow) and (c) activation of melanomacrophage center (black arrows) (H&EX200).(d) kidneys showing diffuse vacuolar degeneration of tubular lining epithelium and activation of melanomacrophage center (black arrows) (H&EX200).e) higher magnification of the previous photo showing lymphoid proliferation in between the tubules (black arrow) (H&EX400).f) Spleen showing activation of melanomacrophage center in between the follicles (black arrows) (H&EX400).
melanomacrophage center.The presence of histopathological alterations in the internal organs clearly explained the septicemic nature of Vibrio and photobacterium infections.The obtained results are consistent with Mladineo et al., (2006) and Ragab et al., (2022) who found the same alteration in different fish.The lesions in the hemopoietic tissue including kidney and spleen indicated that, the Vibrio and Photobacterium infection may suppress the immune system and give a chance to infection with other bacteria.The lesions in the splenic cells and the blood vessels may indicate the effect of the bacterial toxins on the endothelial lining of the blood vessels (Hassan et al., 2015).The tissue damage in various organs may also be attributed to the production of bacterial extracellular products, which reveal a critical role in the pathogenesis of vibriosis and photobacteriosis (Eissa et al., 2018; Aly et al., 2020; Ragab et al., 2022).

Peĉur Kazazić et al., 2019; Bagni, 2021; Colloca and Cerasi, 2021).
The disease is characterized by the existence of several white nodules on the surface of the internal organs (particularly the kidney and spleen) (

Labella et al., 2019; Morick et al., 2023).
The eruptions of photobacteriosis are known to arise in summer, and severe mortalities typically happen when water temperatures rise above 18-20 o C.Under this temperature, fish can harbour the pathogen as a subclinical infection for extended periods of time (

Table 1 :
Prevalence of infection among the examined fishes (apparently healthy and clinically diseased)

Table 2 :
Phenotypic and biochemical characteristics of the retrieved isolates

Table 3 :
Seasonal prevalence of recovered isolates from moribund Sparus aurata

Table 4 :
Seasonal prevalence of recovered isolates from moribund Dicentrarchus labrax

Table 5 .
Seasonal prevalence of recovered isolates from moribund Argyrosomus regius

.
Immunologically, the aforementioned climatic changes are well known to have a significant impact on adaptive, innate and cellular immune responses (

Cascarano et al., 2021).
The clinical examination of the moribund fishes revealed the presence of skin haemorrhages, erosion, and ulcers.These clinical signs are predominantly noticed in septicemic bacterial infections, such as Vibrio and Photobacterium species (

Moustafa et al., 2010; Essam et al., 2016). While
the postmortem examination of the necropsied fishes revealed clear signs of septicemia manifested by congestion in the kidney, abdominal ascites, gill congestion, an enlarged liver with an engorged gall bladder, and splenomegaly.Our findings were in concordance with those reported by Abdelaziz

Essam et al., 2016; Eissa et al., 2018; Patel et al., 2018; Khalil and Abdel-latif,2022).
Notably, the API 20 E system is usually used to investigate the biochemical reactions of bacterial strains.However, several molecular methods have been designed for accurate and quick identification of infectious bacteria in cultured and wild fishes (

Essam et al. (2016) Eissa et al. (2020) and Eissa et al. (2021), who
used 16S rRNA gene to identify Vibrio and Photobacterium strains from moribund fishes.In the current study, three different bacterial pathogens have been isolated from moribund fishes.The isolated bacteria were Vibrio alginolyticus, Vibrio parahaemolyticus and Photobacterium damselae subsp.piscicida.These bacterial pathogens are highly recognizable fish bacteria that infect cultured marine fish and have been linked to mass mortalities in farmed seabass (Dicentrarchus labrax), seabream (Sparus aurata) and meagre (Argyrosomus regius) in numerous Egyptian regions (