Abstract: Background and Objective: Summer mortality considered a well-established phenomenon affecting cultured tilapia farms allover Egypt in the past 5 years. The present study was undertaken for determining the pathogenic bacteria incorporated in this phenomenon affecting seven tilapia farms using a rapid diagnostic technique. Materials and Methods: Thirty-five clinically diseased Nile tilapia were sampled for determining the clinical, gross internal and pathological lesions followed by isolation and automatic biochemical identification for the recovered isolates using VITEK 2 system. Antibiotic susceptibility test was done for bacterial isolates together with conduction of a treatment trial using oxytetracycline and sulphadiazine-trimethoprim combination. Results: Twenty-eight Enterobacter cloacae, 15 Vibrio cholera and 7 Plesiomonas shigelloides isolate were identified using VITEK 2 system. Eighty-six, 52 and 10% of the recovered isolates were sensitive to oxytetracycline, sulphadiazine-trimethoprim and ampicillin, respectively. Various degenerative changes in posterior kidney, hepatopancreas and fundic region of the stomach were observed during the histopathological examination including congestion, hemorrhage, leucocytic infiltration and necrosis. Ammonia level was ranged between 0.33-0.72 mg L–1 in farms water. In feed administration of oxytetracycline and sulphadiazine-trimethoprim combination at a dose of 50 and 30 mg kg–1 body weight respectively for 10 consecutive days was successful treatment with improving farm water quality. Conclusion: E. cloacae, V. cholera and P. shigelloides were the causative agents of the outbreak affecting the studied farms, high ammonia level was act as a predisposing factor, antibiotic treatment with improving farm water parameters was effective in termination of this disease condition.
INTRODUCTION
Aquaculture is a fast growing sector in Egypt, it supplies the Egyptian market with 1.175 million t of valuable and cheap source of protein in comparison with other animal protein sources. In last few years, despite the expansion in Nile tilapia production, this sector has suffered from a severe annual economic losses estimated by about one billion Egyptian pound as a direct cause of what is known as summer mortality. During this outbreak, affected fish shows the general signs of septicemia during the clinical and postmortem examination1.
Gram-negative bacteria including family Vibrionaceae and the members of family enterobacteriaceae considered pathogenic and opportunistic to cultured fish species including tilapia2,3.
Traditional methods used for biochemical identification of bacterial pathogens are time consuming and laborious, VITEK 2 is a highly accurate automated biochemical bacterial identification system, it provides a rapid identification of clinically important bacterial pathogens which saves a valuable time for fish health manger to give an accurate decision during the early stage of the outbreak when application of treatment is still a good choice in disease control.
Anti-microbials are a valuable tool used in controlling susceptible infectious diseases in farm animals as well as cultured fish4-6. Oxytetracycline is a bacteriostatic antibiotic related to tetracycline group, it acts by inhibition of bacterial protein synthesis through binding with 30 subunit of bacterial ribosome7 it is widely used in aquaculture8. Sulfonamides are bacteriostatic anti-microbials, it act by inhibition of dihydropteroate synthase enzyme that incorporate the Para amino benzoic acid (PABA) in folic acid synthesis, sulfonamides turned to bactericidal when potentiated with trimethoprim which is a potent competitive inhibitor of microbial dihydrofolate reductase enzyme, they work together to prevent bacterial nucleic acid synthesis9, potentiated sulfonamides are successfully used in American and European fish farms for controlling the susceptible bacterial pathogens.
Pond water quality is an essential element for fish culture, water parameters including ammonia, dissolved oxygen and pH can affect fish growth, health and determine the failure or success of the overall culture practices10. High organic matter levels and poor quality of the aquatic environment responsible for high mortality rates during outbreaks11. Exposure to sub- lethal concentration of un-ionized ammonia increase susceptibility to bacterial fish diseases12.
The present study was done to determine the bacterial etiological agents responsible for this outbreak in tilapia farms using a rapid technique (VITEK 2 identification system) together with determination of their sensitivity to anti-bacterial agents, followed by conduction of a treatment trial to act as a guide line for dealing with futuristic disease condition.
MATERIALS AND METHODS
Study area: Samples were taken from seven diseased tilapia farms located at El-Kafr EL-Sharky, Alhamoul district, Kafrelsheikh governorate, Egypt. Studied farms located between longitude 31.287389-31.298390 N and latitude 31.070345-31.075491 E. Fish stoking density in studied farms was 4-5 fish per square meter.
Samples: Samples were collected during May and June, 2016, 35 clinically diseased mono-sex (Oreochromis niloticus) were collected as five fish were sampled from each farm. Sampled fish weight was 120±25 g, each moribund fish was placed in a separate plastic bag and transported in ice box to fish diseases lab, National Institute of Oceanography and fisheries, Alexandria branch.
Clinical and gross internal examination: The clinical examination was performed as mentioned by Noga13 for observation of any external abnormality and the gross internal examination was performed as described by Heil14 to determine any gross internal lesions.
Isolation of bacterial pathogen: It was done according to the method described by Aboyadak et al.1 briefly, two samples were taken from each fish (35 sample from internal organs and 35 from external body lesions), samples each sample was inoculated to tryptic soy broth (Oxoid®) and incubated at 35°C for 12 h then cultured on tryptic soy agar (Oxoid®) with incubation at 35°C for 18-24 h.
Automatic biochemical identification for the recovered isolates using VITEK-2 system: A single colony from each morphologically similar colonies was picked up for gram stain procedures that was performed according to the method described by Collins et al.15 to determine the type of cards used in the identification procedures.
Few morphologically identical colonies were collected with a platinum loop from the agar plate and were subjected to automated biochemical identification as described by Aboyadak et al.4.
Histopathological examination: The histopathological examination was performed according to Robert16 tissue specimens from hepatopancreas, posterior kidney and stomach wall was taken, fixed in 10% buffered formalin, dehydrated in ascending grade ethyl alcohol and cleared in xylene, sectioned to 4 μm thickness and mounted over a glass slide then stain with hematoxylin and Eosin (H and E). Stained tissue sections were examined & photographed using Optika microscope with digital camera (Optika, Italy).
Determinate of farm water parameters: Dissolved oxygen and pH were determined using portable dissolved oxygen meter for aquaculture and water proof portable pH meter (HANNA, Italy). Ammonia level was determined using portable ammonia photometer (HANNA, Italy) based on the methods described by APHA17.
Anti-microbial sensitivity test: Agar disk diffusion test was done to determine the sensitivity of recovered bacterial isolates to sulphadiazine-trimethoprim (SXD 25 μg), oxytetracycline (OTC 30 μg) and ampicillin (AM 10 μg) according to the method described by CLSI18.
Drugs used in the treatment trial: Depending on the sensitivity test results, the majority of recovered bacteria isolates were sensitive to oxytetracycline and sulphadiazine-trimethoprim based on this results both drugs were used in treatment trial at a dose 50 and 30 mg kg–1 b.wt., respectively. Oxytetracycline® (oxytetracycline 20%) and Co-Trimazine® (sulphadiazine 10% + trimethoprim 2%), ADWIA, Egypt were used in the treatment trial done in one of the affected fish farms.
Treatment protocol: A daily dose of 470 g from each drug was mixed with 56 kg fish ration using 1 kg fish oil as binder, medicated feed was prepared daily for 10 consecutive days, the given number was calculated based on the stocking density of 16.000 fish/farm (0.4 ha), 1875 kg biomass and 3% daily feeding rate.
Statistical analysis: One sample t-test was used for determine the values of farm water parameters expressed as mean±standard error using MedCalc for windows, version 17.2.2, (MedCalc Software, bvba, Ostend, Belgium, https://www.medcalc.org; 2017).
RESULTS
Results of clinical and gross internal examination: Presence of haemorrhagic skin ulcers of various sizes was the only observed clinical sign while, their were many prominent postmortem lesions as congested internal organs including stomach, intestine, hepatopancreas with enlarged gall bladder filled with thick greenish bile.
Results of automated biochemical identification of bacterial etiological agents: Fifty bacterial isolate were recovered from diseased fish, all of them were Gram-negative. Based on automatic biochemical identification using VITEK 2 system 28, 15 and 7 isolates were identified as Enterobacter cloaca, Vibrio cholera and Plesiomonas shigelloides (Table 1) with a probability estimated by 99, 98 and 99%, respectively. The biochemical characteristics of the recovered isolates was shown in Table 2.
Results of histopathological examination: The histopathological examination of diseased fish posterior kidney tissue revealed the presence of several pathological lesions as darkening and shrinkage of glomerular taughtand increase the Bowman's space. Tubular necrosis and detached tubular epithelium with multifocal interstitial hemorrhages were also cleared (Fig. 1a-c).
Hepatopancreas of affected fish was also severely affected, hepatic cell death, vacuolar degeneration, pyknosis and karyorrhexis were clear at the cellular level, at the tissue level inflammation observed by leucocytic infiltration, necrosis and congestion of hepatic parenchyma together with thrombus formation in many blood vessels was also noticed (Fig. 1d-g).
The fundic region of the diseased fish stomach was inflamed manifested in presence of leucocytic infiltration between lamina propria and submucosa, destruction and necrosis of fundic gland was observed in some samples while destruction and detachment of columnar epithelial lining the mucous membrane was dominant in the majority of examined samples (Fig. 1h-j).
| Table 1: | Recovered bacterial isolates from diseased Oreochromis niloticus |
| Fig. 1(a-j): | Posterior kidney of naturally infected Oreochromis niloticus with obvious degenerative changes including, degenerated glomeruli (DG) manifested by darkening, shrinkage of glomerular taughtand increasing Bowman's space, presence of necrotic areas (N), multi focal haemorrhage (H) and degenerated renal tubules with detached tubular epithelium and obliterated renal tubules (DT), hemosiderin deposition (HC). X = 400. d-g) hepatopancreas of naturally infected Oreochromis niloticus showing loss of tissue architecture with presence of necrotic foci (N) and leucocytic infiltration (LI), X = 400, (d). Severely congested blood sinusoids (CS), necrotic areas (N), vacuolar degeneration (VD), pyknotic nucleus (arrow), swollen hepatocytes with pale cytoplasm in different degrees of vacuolar degeneration (HC), X = 400, (e). Enlarged distended blood vessels with multiple thrombi (TH), X = 40, (f). Ballooning degeneration of hepatocytes, rarefied cytoplasm (HC), Karyorrhexis (K), vacuolar degeneration (VD) X = 1000, (g). h-j) Stomach (fundic region) of naturally infected Oreochromis niloticus showing leucocytic infiltration (arrows) between lamina propria and submucosa, X = 100, (H). Large necrotic area (N) with destruction of fundic glands (FG) X = 100, (i). Destruction and detachment of epithelial lining the folded mucosa covering the fundic gland (black arrows) X = 100, (j) |
| Table 2: | Biochemical characters of Enterobacter cloacae, Vibrio cholera and Plesiomonas |
Results of anti-microbial sensitivity test for recovered isolates: Eighty-six, 52 and 10% of recovered isolates were sensitive to oxytetracycline, sulphadiazine-trimethoprim and ampicillin, respectively. Most of recovered E. cloacae and all P. shigelloides isolates were sensitive to oxytetracycline and sulphadiazine-trimethoprim but resist ampicillin on the other hand nearly all V. cholera isolates were sensitive to oxytetracycline but none of them were sensitive to sulphadiazine-trimethoprim and few isolates were sensitive to ampicillin (Table 3).
Results of the treatment trial: Daily mortality was ranged between 55-300 fish/day before treatment, that equals 0.35-1.87% of the stoked fish, then it had been decreased to 0.15% at the 7th day post treatment (Table 4) and ceased 1 day after the last dose.
| Table 3: | Antibiotic susceptibility of recovered bacterial isolates |
| S: Susceptible, I: Intermediate susceptible, R: Resistant | |
| Table 4: | Daily fish mortality before and after treatment |
| Table 5: | Dissolved oxygen, pH and ammonia level in affected farms water |
Results of tested water parameters: Dissolved oxygen level and pH value of tested water samples were within the acceptable range for tilapia culture as oxygen level was 5.45±0.32 mg L–1 and pH was 7.72±0.18 and their range didn't change before and after treatment while the recorded ammonia level was over the permissible limit in all tested water samples as it was ranged between 0.33-0.72 mg L–1 (Table 5). During the treatment trial, partial daily change of 30% bond water helped in decreasing ammonia level to 0.08 mg L–1 that augmented the treatment trial.
DISCUSSION
Bacterial fish diseases are the major cause of a huge economic losses in aquaculture all over the world, Egypt suffered badly in the past 5 years from summer mortality in Oreochromis niloticus farms1.
In the present study VITEK 2 system helped in the rapid detection of pathogenic bacteria affecting cultured tilapia. Twenty-eight E. cloacae isolate were identified, E. cloacae considered a potential pathogen infecting many cultured fish species including Pangasianodon hypophthalmus, Mugil cephalus, tilapia nilotica and red hybrid tilapia19-22. Several members of family vibrionaceae are fish pathogens, in the present study 15 V. cholera isolate was determined among the causative agents of current outbreak, recently few reports supporting this result and identified V. cholera from diseased tilapia1,23,24. The present study restricted seven isolates as P. shigelloides25,26 also isolated it from clinically diseased O. niloticus and Clarias gariepinus, Ozturk and Altinok27 reported several outbreaks in many fish species infected with this pathogen.
Hemorrhagic skin ulcers was the only clinical sign recorded, while the found gross postmortem lesions were congested internal organs, these observations were in accordance with that recorded by Austin and Austin2, El-Barbary28and Hassan et al.29 regarding bacterial infection of O. niloticus. In harmony with this research results23,25,30 mentioned nearly similar clinical and postmortem signs in O. niloticus infected with E. cloacae, V. cholera and P. shigelloides, respectively.
Histopathological examination is an excellent tool in understanding the diseases pathogenesis as well as the development of clinical signs and postmortem lesions that induced by bacterial pathogens. Posterior kidney tissues of naturally infected fish showed degenerated glomeruli and renal tubules, presence of necrotic areas and multi focal haemorrhage similar finding were mentioned, demonstrating the destructive effect of different bacterial infections on fish renal system. Aly et al.31 observed marked tubular degeneration in kidneys and glomerular atrophy with hematopoietic tissue depletion in O. niloticus experimentally infected with E. cloacae, El-Sharaby et al.32 described congested glomerular capillaries, necrosis in renal tubular epithelium in kidney of O. niloticus naturally infected with Vibrio species. In agreement with current research results Liu et al.26 observed kidney injury during the histological examination of O. niloticus challenged with P. shigelloides previously isolated from diseased cultured tilapia33 and also found necrotic lesions in the kidney of naturally infected farmed O. niloticus.
Hepatopancreas of affected fish showed loss of tissue architecture with presence of necrotic foci and congestion, hepatocytes showed vacuolar degeneration, nuclear pyknosis and karyorrhexis that indicating the severe affection of hepatic tissue at the cellular level, liver is the most important organ in metabolism and detoxification mechanism, liver dysfunction is predisposing to increase the susceptibility to environmental pollutant and bacterial toxins that makes fish more suffering under bacterial infection. In accordance with our findings, Aly et al.31 recorded presence of coagulative necrosis in hepatocytes and vacuolar degeneration with focal necrosis associated with E. cloacae infection. El-Sharaby et al.32elucidated congested hepatic sinusoids, mononuclear cell infiltrations, associated with Vibrio species infected. In harmony with current research results26 as they observed liver injury during the histological examination of diseased cultured tilapia infected with P. shigelloides.
Destruction and detachment of epithelial lining the fundic mucosa together with destruction of fundic glands and mononuclear cell infiltration, indicating the destructive effect of the bacterial infection on fish digestive system, it may also pointed to the probability of involvement in this disease pathogenesis as gastric mucosa may act as a porter of entry of pathogenic bacteria.
The recorded clinical signs, gross internal lesions and histopathological alterations were occur as a sequel of the inflammatory reaction which started with leucocytic infiltration after that congestion and edema followed by necrosis, this inflammatory reaction was initiated by bacterial invasion together with production of their virulence factors including enzymes, toxins, adhesins and anti-phagocytic factors.
E. cloacae possess many virulence factors contributing to its pathogenicity, Albesa et al.34 identified leukotoxic toxin and hemolytic toxin from E. cloacae, they described their roles in induction of oxaditive stress. Michalska and Gospodarek35 reported that E. cloacae has many virulence factors including lipopolysaccharide endotoxin, type1 fimbriae, capsula and they also recorded the ability of E. cloacae to produce enterobactin, yersiniabactin, aerobactin and bacteriocins. Enterobacter species has adhesinthat responsible for adhesions to cells and tissues36.
Austin and Austin2 mentioned that V. cholerae among highly virulent bacterial fish pathogens, the extracellular enzymes of V. cholera including proteases and purified vibrio proteases are toxic to fish37. Laviad-Shitrit et al.38 recorded the presence of zonula occludens toxin, hemolysin haemagglutinin/protease and outer membrane protein producing gens from V. cholera isolates recovered from tilapia. The V. cholera has specific surface receptors used for binding to connective tissues as collagen, fibrinogen and fibronectin39.
The P. shigelloides is normal inhabitant micro-organism in the aquatic environment40, many virulence factors have been incorporated in pathogenicity including enterotoxin41, cytotoxin lipopolysaccharides42 and cholera-like toxins43, Gonzalez 44 enumerated the virulence factors associated with P. shigelloides, of them adhesion, invasiveness, enterotoxin, cytolysins, haemolysin and elastin.
Sensitivity test cleared that most E. cloacae strains were sensitive to oxytetracycline and sulphadiazine-trimethoprim which is coincided with observation of previous study45,46, who recorded the sensitivity of E. cloacae isolated from several fish species to tetracyclines and sulfa-trimethoprim and resistance to ampicillin, Davin-Regli and Pages47 mentioned that E. cloacae is naturally resistant to ampicillin and Conceicao et al.48 related the natural resistance of E. cloacae to ampicillin to β-lactamase production and proved it through identification of new chromosomal AmpC β-lactamase, these observations were in harmony with current research results.
All the recovered P. shigelloides strains were sensitive to both oxytetracycline and sulphadiazine-trimethoprim and resistant to ampicillin which come in harmony with the findings described by Matsuyama et al.49 that indicated its sensitivity to tetracycline and trimethoprim and resistance to ampicillin, Stock and Wiedemann50 also recorded susceptibility of P. shigelloides from aquatic environment to tetracycline, trimethoprim-sulfamethoxazole while it resist amoxicillin.
On the other hand, nearly all V. cholera isolates were sensitive to oxytetracycline but they were resistant to sulphadiazine-trimethoprim and ampicillin, in accordance with the pervious stusy51, who recorded the sensitivity of V. cholera isolated from tilapia to tetracycline and resistance to ampicillin, sensitivity to tetracycline and resistance to sulfa-trimethoprime52.
Oxytetracycline and sulphadiazine-trimethoprim combination decreased the mortality rate that indicated its successfulness in termination of bacterial infection in the affected farm which can also reflect the synergistic effect of trimethoprim to both oxytetracycline and sulphadiazine. Bondad-Reantaso et al.5 recorded that oxytetracycline is the most frequently used antibiotic in aquaculture which reflects its value as a treatment. Potentiated sulphonamides are among the commonly used antibacterials in fish farming53-55.
Ammonia level in the bond water of infected farms was ranged between 0.33-0.72 with a mean value 0.52±0.05 mg L–1 which is much higher than acceptable normal value (0.06 mg L–1). Un-ionised ammonia (NH3) is toxic to fish10, increased ammonia over the acceptable limit act as a predisposing factor for outbreaks, ammonia act as stress factor lead to impairment of disease response56. High ammonia level decreased the fish immunity and subsequently increase disease susceptibility12. In current study increased ammonia level may be due to decreased bond water change. Partial daily change (about 30%) of bond water help in decreasing ammonia level to 0.08 mg L–1 which is much acceptable than previous value. Current work finding suggested that increased ammonia level is play an important role in occurrence and progression of this disease as high ammonia level decrease the fish immunity turned it more susceptible to bacterial infection. Dissolved oxygen level was ranged between (4.85-6.8) with mean value (5.45±0.32) mg L–1 which considered acceptable for tilapia culture (4-5) mg L–1. The pH value was ranged between (7.02-8.13), the mean pH value was (7.72±0.18) which considered in the optimum rage for tilapia culture as described by El-Sayed10.
CONCLUSION
Advanced diagnostic techniques as VITEK 2 system was valuable for rapid identification of Enterobacter cloacae, Vibrio cholera and Plesiomonas shigelloides responsible for occurrence of the outbreak hits tilapia farms at Kafrelsheikh. These bacteria considered opportunistic pathogens responsible for inducing serious diseases specially under stressful condition as high ammonia level. Medicated feed contains oxytetracycline and sulphadiazine-trimethoprim combination at a dose of 50 and 30 mg kg–1 body weight, respectively for ten consecutive days was effective in termination of this disease condition together with improving farm water quality.
SIGNIFICANCE STATEMENT
This article identified twenty-eight Enterobacter cloacae, 15 Vibrio cholera and 7 Plesiomonas shigelloides isolate as the actual causes of the outbreak affecting tilapia farms and considered the high ammonia level as a predisposing cause, it also proves the efficacy of oral antibiotic administration using oxytetracycline and sulphadiazine-trimethoprim combination at a dose of 50 and 30 mg kg–1 b.wt., respectively for ten consecutive days in termination of infection delete together with improving farm water quality.