Aeromonas veronii and ulcerative syndrome in cultured Nile tilapia (Oreochromis niloticus) and their associated factors

This research was carried out to study the bacterial etiology of ulcerative syndrome in Nile tilapia (Oreochromis niloticus) and its associated environmental stressors. A total of 90 live and moribund Nile tilapia showing signs of ulcerative dermatitis were collected from various fish ponds in El-Sharkia Province, Egypt, during the summer seasons from June 2019 to August 2020. Collected fish were subjected to full clinical and postmortem investigation followed by bacteriological and molecular identification of the bacterial agents as well as histopathological examinations. Moreover, water samples from the fish sampling sites were collected and analyzed physiochemically and microbiologically. Furthermore, the pathogenicity of the isolated strains was assessed by challenging apparently healthy Nile tilapia to determine the LD50. Clinically, the examined fish exhibited ulcerative dermatitis as focal or fused large ulcers surrounded by red zones of hemorrhages at different body parts. Bacteriologically, Aeromonas veronii biovar sobria and A. caviae were isolated and identified. Moreover, 16S rRNA gene sequencing and phylogenetic analysis verified the identity of these species. Antibiogram profiling of the recovered Aeromonas sp. isolates showed complete resistance to ampicillin, with high susceptibility to gentamycin, trimethoprim/sulphamethoxazole, and erythromycin. Histopathological examination of the affected fish revealed epidermal necrosis with dermal edema and an influx of inflammatory cells into the dermis and hypodermis. The LD50 of A. veronii biovar sobria and A. caviae was estimated as 1.5 × 107 and 1.5 × 108 CFU/fish; respectively. The water temperature and ammonia concentrations recorded a significant rise (P ≤ 0.05) in July and August compared to June; however, the highest pH levels were recorded during August, followed by July. Also, the total bacterial load significantly increased (P ≤ 0.05). Finally, it was concluded that the improvement of water quality helps sustain the health of farmed fish .


Introduction
Egypt is the biggest aquaculture producer in Africa.Egyptian Nile tilapia (Oreochromis niloticus) represents the most essential aquatic cultured species that sets Egypt as one of the top countries in the tilapia aquaculture industry after China and Indonesia, followed by Thailand, Bangladesh, and the Philippines (FAO 2022).Egypt produced 1,114,265 million tons of tilapia in 2020, which is considered the first fish in Egypt and the second fish all over the globe after carp.In fact, local consumption accounts for almost all of Egypt's tilapia production, and only 28.11 thousand tons of fish production were exported in 2020 (GAFRD 2020).A current value chain investigation of the business shows that cultivated fish value in Egypt is mostly based on tilapia production, with mullet being the second most essential species in private farms (Eltholth et al. 2015).Nevertheless, challenges with infectious disease outbreaks causing great losses, especially bacterial pathogens resulting in high mortalities (Assefa & Abunna 2018;Hassan et al. 2020) that defied maintainable intensification of worldwide aquaculture.
Climate change has both direct and indirect impacts on aquaculture.Directly, it affects changes in water supply, water temperature, and extreme weather events.In addition to changing the frequency and prevalence of bacterial pathogens in aquaculture globally, climate change also distributes bacterial pathogens into new geographic regions, leading to the emergence of emerging infections (Crumlish 2017).Accordingly, protocols are directed toward predictive capability for disease outbreaks by checking the bacterial composition and quantity in tilapia and their rearing condition (Pakingking et al. 2015).For instance, the 2009 motile Aeromonas septicemia (MAS) outbreaks in East Mississippi and West Alabama resulted in fish aquaculture losses for about three million pounds of food-size fish per year.These outbreaks occurred due to an uprising in water temperature that had a significant effect on fish mortality (Rasmussen-Ivey et al. 2016).
Egypt has been facing unexplained mortalities in tilapia fish farms during summer months for the past 3-4 years.Based on the organisms identified from affected fish, several causative agents for ulcerative syndrome (US) have been hypothesized, with the main causes of the disease being considered to be bacteria, viruses, fungi, and parasites (Kamilya and Baruah (2014).Fish producers and the fisheries industry have suffered significant financial losses due to diseases.Contrary to the common belief that the etiological agent(s) of the USA are currently unknown, organisms from the possibly fish-pathogenic genera from the lesions and blood samples of diseased fish, Aeromonas and Pseudomonas, were often recovered, where the most frequently recovered bacteria, including Aeromonas hydrophila and Aeromonas sobria.It is generally known that a variety of Aeromonas species contributed to diseases in aquatic ecosystems (affecting both wild and cultured, freshwater, and marine fish species), mass mortality, and significant financial losses to the aquaculture industry (Hassan et al. 2017).Disease development and progression are influenced by bacterial load, level of environmental degradation, water quality, and fish immune system (Elgendy et al. 2017).The physical, biological, and chemical characteristics of water are all considered.Fish welfare and growth are often impacted.Although the aquaculture environment depends on many different water quality elements, just a few play a crucial role (Abdel-Tawwab et al. 2019).In view of these facts, this study aimed to detect the bacterial causes of the ulcerative syndrome in cultured Nile tilapia and their associated stress factors in different aquaculture ponds in Egypt.

Ethical approval
The Suez Canal University Research Ethics Committee, which adheres to the principles of the Canadian Council on Animal Care, authorized this study (2,022,063).

Experimental fish samples
A total of 90, moribund Nile Tilapia with signs of ulcerative dermatitis were randomly collected from different freshwater fishponds in El-Sharkia Province during the summer seasons from June 2019 to August 2020.Simultaneously, a total of 81 subsurface water samples (500 mL) were collected in triplicate from the same fish ponds where the infected fish specimens were collected.Water samples were taken in sterile dark glass bottles with stoppers.Fish and water samples were placed in an insulated ice box with crushed ice and quickly transported to microbiology and the water analysis laboratory at the Fish Farming and Technology Institute (FFTI)-Suez Canal University.

Clinical examination
Collected moribund fish were exposed to clinical and postmortem examination, and specimens from skin lesions were collected and processed for bacteriological, molecular, and histopathological examinations.Clinical examination of diseased Nile tilapia was performed as previously stated by Schäperclaus et al. (1992).Fish were observed for hemorrhages, erosions, ulcers, or other clinical abnormalities in different body parts.

Isolation and phenotypic identification of bacterial pathogens
Immediately after sampling, freshly dead and moribund fish specimens were examined bacteriologically (Austin & Austin 1989).Prior to bacteriological sampling, the external body surfaces of the examined fish were sterilized with 70% ethanol.Loopful from skin ulcers were aseptically enriched in tryptic soy broth, TSB, and incubated for 24 h at 28 °C.For selectively isolating Aeromonas pathogens, loopful from TSB was inoculate on ASA medium (Aeromonas selective agar, selective media for several kinds of Aeromonas sp.) (HIMEDIA, India) then purified on Tryptic Soya Agar (Oxoid, USA).Presumptive cultural and morphological characters of bacterial colonies from the inoculated plates were assessed after being incubated at 28 °C for 24-48 h.Suspected colonies were picked up and subcultured onto nutrient agar (Oxoid, USA) for purification.Isolates were biochemically characterized using Gram staining, oxidase, and catalase.Further confirmation was carried out using API 20E (analytical profile index for Enterobacteriaceae) (API Bio-Merieux, France) (Buller 2004;Eissa 2016).Identified isolates were stored in nutrient broth supplemented with 20% glycerol at − 80 °C freezer for further investigations (Austin & Austin 1989).

Antibiotic susceptibility test for the isolated bacteria
The susceptibility profile of bacterial isolates to antibiotics was determined by the Kirby-Bauer disk diffusion susceptibility method (Bauer et al. 1966) against six antimicrobial agents commonly used in fish farms in the study area.In brief, bacterial suspensions at a concentration equivalent to 0.5 McFarland standard units were spread over MHA plates (Muller Hinton agar) (Difco, Sparks, Maryland, USA).Antimicrobial discs including oxytetracycline (OTC), ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT), gentamycin (CN), erythromycin (ERT), and ciprofloxacin (Cip) (Oxoid, USA) were placed on the plates and incubated overnight at 28 °C in an inverted position.The inhibition zone of each antimicrobial agent was interpreted according to the Clinical and Laboratory Standards Institute M100-S22 Guidelines (CLSI 2018).

Molecular characterization of the recovered isolates
Oligonucleotide primer sequences for 16S rRNA (F27: AGA GTT TGATCMTGG CTC AG and R1492: TAC GGY TAC CTT GTT ACG ACTT, Metabion, Germany) were used to amplify a product size of 1485 bp as previously described (Lagacé et al. 2004).The reaction was performed in 35 cycles using a thermal cycler (Eppendorf, Vapo.protect, Germany) as follows: initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min.The amplified bands were detected using a UV transilluminator (ECX-F15.M, France) after gel electrophoresis using Hangzhou Bioer Technology Co. LTD, GE-100 (China).

Sequencing and phylogenetic analysis
Purified PCR products were sequenced in the forward and/or reverse directions in an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA) using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA, Cat.No. 4336817).Sequences were registered in GenBank under the following accession numbers (OP658967 and OP658968).A BLAST® analysis (Basic Local Alignment Search Tool) (Altschul et al. 1990) was initially performed to establish sequence identity to GenBank accessions.The quality of DNA sequencing chromatograms was checked using Chromas software (Technelysium Pty Ltd, Australia).Trimming was performed when necessary, and the sequences were exported in FASTA format for BLAST search.The phylogenetic tree was constructed using the maximum likelihood method, Tamura-Nei model (Tamura & Nei 1993), and 1000 bootstrap replicates in MEGAX software (Tamura & Nei 1993).Twenty 16S rDNA sequences were used for phylogenetic tree construction.These sequences included two from the current study, 16 from Aeromonas veronii and A. caviae obtained from the GenBank database via BLAST search, and two from Vibrio fluvialis as an outgroup for rooting the phylogenetic tree.

Histopathological examination
Specimens from skin lesions of naturally diseased fish were collected and then fixed immediately in 10% natural buffered formalin for histological examination.The specimens were dehydrated by serial dilution of ethyl alcohol (30, 50, 70, 90%, and absolute), followed by clearing in xylene, then bloked in paraffin wax.The paraffin-embedded tissues were sectioned at 5 µm and stained with hematoxylin and eosin stain according to Bancroft and Gamble (2008).Finally, Canada balsam was used for mounting the stained tissue sections, and the prepared tissue slides were examined under a light microscope (Zisse prime star research microscope, Carl Zeiss, MicroImaging, 37,081, GmbH, Germany) and photography was done using an AxioCam ERc5s digital camera (Zeiss, Germany).

Bacterial strains and in vivo pathogenicity testing
Ten-day median lethal concentration (LD 50 ) was determined (Yonar et al. 2019) by intraperitoneal injection of healthy Nile tilapia with graded doses of both strains, A. veronii biovar sobria and A. caviae, recovered from the diseased Nile tilapia, to determine their pathogenicity.Both strains tested positive for the gelatinase "pathogenicity marker" and were generally resistant to the tested antibiotics.Strains of A. veronii biovar sobria and A. caviae were refreshed on nutrient agar (Oxoid, UK) and incubated at 28 °C for 24 h, then collected in sterile phosphate-buffered saline (PBS), followed by centrifugation of the bacterial suspension at 3000 × g for 15 min at 4 °C.The bacterial pellet was then washed three times in sterile PBS (pH 7.2) before being spectrophotometrically adjusted to three concentrations (1.5 × 10 6 , 1.5 × 10 7 , and 1.5 × 10 8 CFU/mL) to determine LD 50 (in which 50% fish mortality was observed in experimented Nile tilapia).
Seventy apparently healthy Nile tilapia of 90-g mean weight were obtained from FFTI's production unit at Suez Canal University.The fish were acclimated in 200-L glass aquaria, in an aquarium unit, with aerated fresh water at 20 °C for 7 days, then randomly distributed into seven well prepared glass tanks (3 tanks/bacterial strain) and one control tank that supplied with automatic areators, and 20% of the water was exchanged daily by siphoning and substitution with clean dechlorinized water from a storage tank.Prior to injection, fish were sedated with clove oil solution (12.5 mg/L) (Awad et al. 2022).
Ten fish per tank were injected intraperitoneally with 0.2 mL of bacterial culture suspension containing 1.5 × 10 6 , 1.5 × 10 7 , and 1.5 × 10 8 CFU/mL for each bacterial strain, respectively, whereas ten control fish received an identical injection volume of sterile PBS.Injected fish were observed daily for 10 days to detect any disease signs and mortalities.The mortality cause was recognized by re-isolating the challenged strain from the visceral organs of dead fish using Aeromonas selective agar incubated at 28 °C for 24 h (Vasudeva Rao et al. 2006).

Water parameters analysis
On-site, the fish pond's water temperature, salinity, and dissolved oxygen were measured by portable instruments according to manufacturer guidelines.The pH levels were measured by a pH meter according to manufacturer guidelines.The ammonia concentration was measured colorimetrically using the available kit's protocol (Kenworthy et al. 1982).
After serially diluting water samples, 1 mL was transferred in triplicate into a sterile glass Petri dish.Then, 15 mL of nutrient agar medium was added to each plate, mixed, and then dried out.The plates were incubated at 35 °C for 48 h.After the incubation period, the number of produced colonies per plate of the same dilution was counted, and the total viable count was detected, as previously described by Crites et al. (2010).

Statistical analysis
All values were expressed as mean ± standard error (SE).Statistical differences among the means of water parameters were determined using one-way analysis of variance (ANOVA) for all water parameters using IBM SPSS 25 for windows and Duncan's multiple range test (Duncan 1955).The level of significance was detected at a probability level of 0.05 (P ≤ 0.05).

Clinical signs with postmortem findings of naturally diseased fish
Diseased Nile tilapia were recorded during the summer season and collected with an average body weight of 70 ± 3 g and a total body length of 12.5 ± 2 cm.Naturally infected fish with serious skin lesions were lethargic and inappetence, swimming near the surface of the water with darkness in their skin.The examination revealed focal and/or diffused scale-loss with ulceration over the body surface (Fig. 1a).Diseased fish exhibited large ulcers surrounded with red zone at the peduncle (Fig. 1b) and head regions (Fig. 1c) accompanied by erosion in all fins.
Internally, the postmortem lesions revealed distention of the abdomen with reddish-yellow ascetic fluid and hemorrhage with congestion in visceral organs.The gall bladder was distended with bile, and the intestinal tract was hemorrhagic and inflamed.Furthermore, congested gills were also observed.

Microbiological identification of the isolated bacteria
Phenotypically, 39 Aeromonas isolates were retrieved from the investigated fish samples.On Aeromonas base agar, red to pink colonies were seen, while creamy-white circular, smooth pure convex colonies up to 3 mm in length were observed on tryptic soy agar.Isolates appeared as rod, straight, short Gram-negative rods, and they were oxidase-positive, catalase-positive, and motile.Using the API20 E system, the identity of the retrieved isolates matched the standard biochemical criteria for A. veronii biovar sobria and A. caviae with codes 3,566,527 and 2,066,777, respectively.

Molecular confirmation of the isolated bacteria and sequencing
PCR amplification of the universal bacterial 16S rRNA gene generated 1485-bp specific band characteristic for all tested isolated strains (Fig. 2).This was confirmed by conducting a BLAST search of the isolate sequences against those in the GenBank database.Based on our phylogenetic analysis, Aeromonas veronii biovar sobria was assigned to the same cluster of other Aeromonas veronii strains biovar sobria from shellfish samples and tilapia (MW720982 and EF61963, respectively) and with MF370515 from India and MH532482 from China (Fig. 3).Similarly, Aeromonas caviae existed in the same cluster of other Aeromonas caviae strains as confirmed by phylogenetic analysis and BLAST search, where the identity percent ranged from 99.3 to 99.6% in the Aeromonas caviae sequences located in the same cluster.

Histopathological examination
Histopathological analysis of the tissue specimens from the ulcerative lesion of the infected Nile tilapia revealed dermal edema with a large influx of neutrophils into the dermis beside epidermal necrosis accompanied by inflammatory cellular infiltration in the hypodermis (Fig. 4a).Moreover, the damaged epidermis exhibited spongiosis and partial lifting from  the underlying thickened basement membrane together with clumping of melanin granules in the underlying dermal melanocytes beside few inflammatory cell infiltrations (Fig. 4b).
Additionally, the hypodermal tissue showed focal hyaline degeneration and mononuclear cells infiltration accompanied with congestion in hypodermal blood vessels beside melanocytes proliferation (Fig. 4c).The underlining musculature displayed infiltration of mononuclear inflammatory cells as well as proliferation of the bacterial colonies between the muscle bundles that showed intermuscular edema with focal hyaline degeneration and necrosis (Fig. 4d).

Pathogenicity test
The tested strains, A. veronii biovar sobria and A. caviae, were used to determine the LD 50 for Nile tilapia.Both strains were shown to be pathogenic and caused observable symptoms of infection and mortality in challenged fish.After injection, typical signs of septicemia, such as cutaneous hemorrhage and erosions, as well as hemorrhages in visceral organs, were noticed.The LD 50 of the two strains, A. veronii biovar sobria and A. caviae, were estimated as 1.5 × 10 7 and 1.5 × 10 8 CFU/fish, respectively, and they were re-isolated from the dead fish.

Water quality analysis
Water parameters along the investigated period are demonstrated in Table 2.During the investigated months, the water temperature significantly increased (P ≤ 0.05) in July and August compared to June, while the water salinity and dissolved oxygen levels remained constant.Furthermore, the pH levels recorded the highest values during August, followed by July.A significant increase in water ammonia concentrations was significantly raised (P ≤ 0.05), especially in July and August compared to June.The total bacterial load was also significantly increased (P ≤ 0.05) in the same months.

Discussion
Diseases related to bacterial pathogens are listed as one of the major challenges threatening the sustainability of the aquaculture industry worldwide.An extensive range of bacterial infections may infect fish, where most of them are opportunistic and disease makers when fishes are physiologically out of balance, nutritionally insufficient, or were under other stressors, i.e., poor water quality, overstocking (Noga 2010), with Aeromonas spp.being one of the most common incriminated agents.
During summer seasons, Nile tilapia suffer from severe outbreaks in various freshwater aquaculture farms, where affected fish exhibit red sores on the skin along both body sides.This problem is not only responsible for financial losses due to fish mortalities, but also, these sores reduce the marketability of survivor fish.Clinically, in the studied farms, infected fish appear to be losing their activity, off food, and swimming near the water surface, with dark skin beside hemorrhagic fins, tail rot, and skin ulcers.These common clinical signs were also commonly observed in recent studies (Baumgartner et al. 2017;El Asely et al. 2020;Abdelsalam et al. 2021).Furthermore, Hassan et al. (2017) added that infected fish clinically exhibited dullness and complete absence of the reflex, whereas grossly fish showed ulcerations, detached scales with pale skin patches, hemorrhages over the body surface, and in some cases, abdominal dropsy developed.

Table 2 Physicochemical and microbiological parameters of the examined water samples
The recommended range was assessed according to Boyd and Tucker (2012) for Temp: 22-32, pH: 6.5-8.5, DO: 5-7, and ammonia < 0.1 Data are expressed as mean ± SE; standard error.Different superscript letters in the row indicate a significant difference (P< 0.05)
Based on phenotypic characterization and API20 biochemical identification, the isolated bacterial pathogens from diseased fish under investigation were presumptively identified as A. veronii and A. caviae.Similarly, Aeromonas spp.were isolated from diseased O. niloticus in fish farms showing high outbreaks from April to October (El Asely et al. 2020).
Antimicrobial sensitivity screening of Aeromonas spp.revealed complete resistance to ampicillin with considerable resistance to oxytetracycline (38%).Similarly, previous studies documented high resistance of Aeromonas spp. to ampicillin, oxytetracycline, and β-lactam agents (El-Son et al. 2019;Fauzi et al. 2021).However, in another study, Aeromonas strains were sensitive to oxytetracycline (Samal et al. 2014).The drug resistance developed by Aeromonas spp. is mainly due to the location of resistance genes in transposons, such as plasmids, transposons, and integrons, which can facilitate their rapid spread between bacteria (Romero et al. 2012).
Lately, genetic analysis of numerous bacterial agents implicated in fish deaths has grown significantly in relevance, either to discover new organisms in a specific location or to validate the morphological and biochemical categorization of the pathogens that cause infection (Abdelsalam et al. 2021).In this study, phenotypic and biochemical identification confirmed the identity of Aeromonas spp.by 16S rRNA gene sequence analysis.The 16S rRNA gene sequences were virtually identical to their related bacteria (> 99%).In aeromonads, this gene helps classify organisms into various divisions (Gordon et al. 2007).Aeromonas veronii biovar sobria and A. caviae were isolated and identified from the diseased fish.
Histopathologically, ulcerative lesions from infected Nile tilapia revealed dermal edema with a large influx of neutrophils into the dermis beside epidermal necrosis accompanied by inflammatory cellular infiltration in the hypodermis.Moreover, evidence of bacterial colonies within the dermis was also seen.The damage in the epidermis displayed as spongiosis and partial lifting from the underlying thickened basement membrane together with clumping of melanin granules in the underlying dermal melanocytes, besides a few inflammatory cell infiltrations.Moreover, epidermal hyperplasia was also noticed.Furthermore, the hypodermal tissue showed focal hyaline degeneration and mononuclear cell infiltration accompanied by congestion in hypodermal blood vessels besides melanocyte infiltration.
Additionally, the underlining musculature displayed infiltration and proliferation of mononuclear inflammatory cells as well as the bacterial colony between the muscle bundles that displayed intermuscular edema with focal hyaline degeneration in addition to Zenker's necrosis.Similar observations have been recently reported (Dinçtürk and Tanrıkul 2021;Aly et al. 2020;Laharia and Patel 2020).These tissue alterations might be attributed to releasing bacterial extracellular toxins such as proteases, cytotoxins, and hemolysins that have significant enzymatic activity against fish tissues (Austin and Austin 2016).
Mass tilapia mortality during the summer months, leading to socio-economic losses, might be attributed to a number of reasons and has recently presented a significant challenge for the tilapia farming sector (Reda et al. 2022).Not only infectious causes but also some environmental factors trigger this problem.Poor water quality is one of the main environmental stressors for fish, which alters their physiological responses and disease resistance (Schreck and Tort 2016).In this study, water quality degradation was noted.Water temperature ranged from 24 to 32.05 ℃, dissolved oxygen ranged from 7.2 to 8.25 mg/L, and the pH was alkaline (ranged from 7.6 to 8.28) with a rise in ammonia (ranged from 0.5 to 1.07 mg/L), and the total bacterial load that reached 4.56 × 10 3 CFU/ml.Nevertheless, the temperature, pH, and ammonia levels were above the recommended limits.This increase may be, in turn, a major factor not only in decreasing fish immunity barriers, making them more susceptible to bacterial diseases, but also in increasing the multiplication of pathogenic bacteria in the water that becomes more virulent and facilitates diseases development (Wedemeyer 1996;Plumb & Hanson 2010).Moreover, the probability of total ammonia being transformed into poisonous unionized ammonia at an alkaline pH increases with higher water temperatures, increasing the danger of gill injury as well as opportunistic bacterial infection (Abu-Elala et al. 2016).
During the investigated months, the water temperature reached a significant raise (P ≤ 0.05) in July and August compared to June, while the water salinity and dissolved oxygen levels remained constant.Furthermore, the pH levels recorded the highest values during August, followed by July.As well, a significant increase in water ammonia concentrations was significantly raised (P ≤ 0.05), especially in July and August compared to June.Moreover, the total bacterial load was also significantly amplified (P ≤ 0.05) during the same months.Similarly, in a recent study, most of the mass tilapia mortality was recorded in 2019, between mid-July and mid-October, when temperatures were high (ranging from 27 to 32.5 ℃) (Eissa et al. 2015;Reda et al. 2022).This rising water temperature speeds up infection transmission, increasing disease prevalence and epidemics (Harvell et al. 2002).

Conclusion
This study highlights the incrimination of A. veronii biovar sobria and A. caviae isolated strains in the summer mortalities and ulcerative syndrome in Nile tilapia.Their pathogenicity is confirmed by biochemical, molecular, and in vivo tests.Dermatitis with epidermal necrosis and inflammatory cells influx were the histopathological lesions that characterized this infection among damaged ulcerative fish skin.To minimize the health hazard, antibiotics should be used carefully in aquaculture to prevent the spread of antibiotic resistance within aquatic ecosystems.Water quality practices, in particular, should be closely followed since rising water temperatures and alkalinity enhance bacterial growth and multiplication, increasing the water's bacterial load and virulence as well as increasing ammonia toxicity.
The incidence of US was increased since all changes in these parameters are likely to stress out cultured fish.Finally, sustaining the health of farmed fish would be made possible by applying water quality improvement methods and appropriate biosecurity practices.

Fig. 1
Fig. 1 Pathological lesions in naturally diseased Nile tilapia, Oreochromis niloticus, showing (a) ulcers (black arrow) surrounded with red zone, (b) ruptured eye (white arrow) and ulcers in head and caudal peduncle, and (c) fin rot (arrowhead) with ulcers in the head region and body (black arrow)

Fig. 2 Fig. 3
Fig. 2 Agarose gel electrophoresis of 16S rRNA of Aeromonas sp. from naturally infected Nile tilapia.L, molecular DNA marker; P, positive control; N, negative control

Table 1
Antibiogram of Aeromonas sp. from Nile tilapia