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Isolation and Pathogenicity Determination of Bacillus cereus Associated with Ulcer Formation in African Catfish Clarias gariepinus



Abdelgayed M. Younes, Alkhateib Y. Gaafar, Alaa Eldeen Z. Abu-Bryka, Attia A. Abou Zaid and Ahmed A. Askora
 
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ABSTRACT

Background and Objective: Bacillus cereus is a toxin-producing facultatively anaerobic Gram-positive rods and could cause diseases in aquatic animals. The main objective of this study was to determine the isolation and pathogenicity of Bacillus cereus associated with ulcer formation in African catfish Clarias gariepinus. Materials and Methods: Live moribund African catfish Clarias gariepinus suffered from skin ulceration and hemorrhages were collected from a private fish farm at Kafr El Sheikh Governorate north Egypt, March, 2019. The bacterium isolated from moribund fishes was identified as a Bacillus cereus based on phenotypic, biochemical characteristics and confirmed with 16s rRNA sequence. Results: Experimentally infected fish showed external and internal clinical symptoms typical to those caused by natural infection. The clinical signs of the affected fish showed ulcers on the skin. The severity of ulcers was dose dependent. Antibiotic sensitivity tests showed that the bacterium was sensitive to all antibiotics but cefotaxime. B. cereus isolate was found to be positive for the production of protease, lipase, lecithinase and hemolysin. Conclusion: This study indicated that B. cereus was the causative agent responsible for skin ulceration and disease in African catfish C. gariepinus.

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Abdelgayed M. Younes, Alkhateib Y. Gaafar, Alaa Eldeen Z. Abu-Bryka, Attia A. Abou Zaid and Ahmed A. Askora, 2021. Isolation and Pathogenicity Determination of Bacillus cereus Associated with Ulcer Formation in African Catfish Clarias gariepinus. Asian Journal of Animal Sciences, 15: 10-18.

DOI: 10.3923/ajas.2021.10.18

URL: https://scialert.net/abstract/?doi=ajas.2021.10.18
 
Received: March 15, 2020; Accepted: October 05, 2020; Published: November 14, 2020


Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

North African catfish Clarias gariepinus (Burchell,1822) is known for its high growth rate, resistance to bad water conditions and give excellent meat quality. They are usually polycultured with Nile tilapia in earthen ponds to increase fish output from the same farm and to control undesired reproduction of tilapia1. Fish are susceptible to a wide variety of bacterial pathogens present in normal flora of water. Under stress conditions and water deterioration, these pathogens cause significant economic losses2.

Bacillus cereus is a toxin-producing facultatively anaerobic Gram-positive rods forming a single heat resistant endospore. The endospores are generally oval or sometimes round or cylindrical, very resistant to extremely adverse conditions such as heating, freezing, drying and radiation. B. cereus is ubiquitous in nature because it does not have complex nutrient requirements. The bacteria are widespread in the environment and soils. Thus, processed and non-processed food ingredients are frequently contaminated with B. cereus, such as vegetables, sauces, rice, cereals, fish and dairy products3-5.

B. cereus group also called B. cereus sensu lato, comprises the following closely related species: B. cereus, B. anthracis, B. thuringiensis, B. cytotoxicus, B. mycoides, B. pseudomycoides, B. toyonensis, B. weihenstephanensis and B. Weidmannii based on the similarity in 16S rRNA sequence6. The B. cereus is responsible for causing emetic and diarrhoeal food poisoning in humans. It has also been implicated in wound infections, ocular infection, meningitis, urinary and respiratory tract infections. The pathogenicity ofB. cereus is associated with the production of tissue-destructive toxins and virulence factors. These secreted toxins are enterotoxins especially nonhemolytic enterotoxin, hemolysins, cytotoxins, phospholipases and proteases5-7.

Bacillus species were used extensively in aquaculture practice as probiotics. The most famous examples are B. subtilis, B. Licheniformis and B. cereus. They initiate immunostimulation, besides having adhesion abilities they produce enzymes and vitamins as well as secrete antimicrobial peptides. Thus the application of Bacillus in aquaculture can improve feed utilization, reduce diseases and improve water quality for sustainable aquaculture8,9.

However, some studies reported that Bacillus spp. could cause diseases in aquatic animals. In catfish, Bacillus mycoides was isolated from dorsal ulcer and muscle necrosis of channel catfish Ictaluruspunctatus in Alabama10. The B. cereus was claimed to be the causative agent of stinging catfish Heteropneustes fossilis mass mortality in West Bengal, India11.

Moreover, B. cereus was mentioned as a fish pathogen other than catfish such as, common carp Cyprinuscarpio12, striped bass Morone saxatilis13, farmed European sea bass Dicentrarchus labrax in Greece14 and Egypt15 and White Sea bream Diplodus sargus in Egypt16. Furthermore, B. cereus was isolated from dying Chinese soft shell turtle Pelodiscus sinensis17.

The main objectives of this study were to identify the causative agent responsible for catfish skin ulceration in the farm, to determine the extracellular enzyme production and to evaluate their antibiotic susceptibility profile along with the pathogenic capacity and histopathological effects in diseased fish.

MATERIALS AND METHODS

Fish sampling: Live moribund catfish suffered from skin ulceration and hemorrhages were collected from a private fish farm at Kafr El-Sheikh Governorate in North Egypt, in the period between March-April, 2019. Fish sample was packed into a sterile bag. Sample transported in icebox to the hydrobiology laboratory of the National Research Centre following standard protocol, for isolation of the causative agent(s). The fish was subjected to clinical and postmortem examinations.

Isolation and identification of the causative agent: Infected skin lesions were disinfected with 70% ethyl alcohol to avoid contamination from bond water. Skin ulcer scratched using sterile swabs and inoculated on Tryptone Soya Agar (TSA) (Oxoid), then incubated at 28°C for 24 hrs. The strain was characterized by Gram’s staining. Biochemical tests were performed using API20E according to the manufacturer’s instructions (bioMérieux, France). Then, 20% glycerol stock culture was prepared and stored at -80°C.

For the molecular identification, DNA template was amplified in T100 gradient thermal cycler (Bio-Rad). A total volume of 100 μL PCR reaction mixture containing 50 μL 2×DreamTaq PCR master mix (Thermo Scientific), 20 μg of eubacterial universal primer 63f (5'-CAGGCCTAACACATGCAAGTC-3) and 1387r (5'-GGGCGGWGTGTACAAGGC-3) was used as previously described18. The PCR products were purified with the PurelinkTM Quick Gel Extraction (Invitrogen, USA) and sent for sequencing in both directions. Nucleotide similarity to the closest homolog of the microbes was conducted using BLAST Searching Tool (NCBI). The nucleotide sequence was deposited to GenBank.

Detection of extracellular enzymes and hemolysis activity: Bacillus spp. produce a variety of extracellular enzymes. Lecithinase, lipase and proteolytic activity of B. cereus was conducted on egg-yolk modified medium according to the method described by McClung and Toabe19. A positive lecithinase test is noted by the appearance of a white, opaque, diffuse precipitate into the medium surrounding the colony growth. Lipase broke down free fats present in the egg yolks, causing an iridescent sheen (oil on water) on the surface of the colonies when subjected to the light source. A positive proteolysis test was noted by the development of clear zones in the medium surrounding colonial growth. Furthermore, hemolytic activity was tested on blood agar including 5% defibrinated sheep blood.

Antibacterial susceptibility: Antibiotic susceptibility of the isolate was assessed by the disc diffusion method using Müller-Hinton agar (Oxoid). Different antibiotics used in the test were chloramphenicol (30), novobiocin (10), oxytetracycline (30), ampicillin (10), norfloxacin (10), nalidixic acid (30), streptomycin (10), kanamycin (30), erythromycin (15) and cefotaxime (30) (Oxoid). The diameters of the inhibition zone were measured following 24 hrs of incubation at 28°C according to standard guidelines for result evaluation20.

Experimental infection: To evaluate the pathogenic potential of isolated strain, 25 apparently healthy C. Gariepinus free from any visible skin lesions with an average body weight of 120±20 g were randomly assigned into 5 groups with 5 per group. The fish were acclimatized to 25°C for 2 weeks. Catfish were intraperitoneally injected with 0.1 mL serial 10-fold dilution containing 2.7×105, 106, 107 and 108 CFU mL1. A control fish group was injected with 0.1 mL of 0.85% saline. Fish were not fed throughout the experiment and alterations in the fish were recorded. Re-isolation of the causative pathogen was done on TSA plates.

The animal experiment was carried in compliance with the National Research Centre Animal Care Committee and performed the following regulations and guidelines for the care and use of animals in research.

Histopathological examination: After complete necropsy of the fish, fresh tissue specimens, skin lesions, hepatopancreas, posterior kidney, spleen, gills and brain were collected from different experimental groups, fixed in Davidson’s fixative, dehydrated in ascending grades of alcohol, cleared with xylene, embedded in paraffin, sectioned at 5 μm thickness, stained by hematoxylin-eosin (H and E) cover-slipped then visualized by Light Microscope (Olympus BX43)21.+

RESULTS

Clinical signs of naturally infected fishes: Naturally infected C. gariepinus revealed abnormal behavior, skin ulceration, bulging eyes (Fig. 1a), vent protrusion, abdominal distention. Internal lesions include enlargement and congestion of kidney and liver with an accumulation of fluid in the body cavity.

Bacterial characterization and identification: Morphological features coupled with biochemical profile revealed that the pathogenic isolate was motile, long bacilli arranged in pairs or long chains, endospore-forming, Gram-positive bacterium belonging to the Bacillus family. Colonies were large, white, flat with irregular perimeters and 2-5 mm in diameter. Biochemical tests revealed that B. cereus was positive to oxidase, catalase, Voges Proskauer, hydrolyzed carbohydrates, reduced nitrate to nitrite and negative to indole.

The 16S rRNA was partially sequenced and the isolate was presumptively identified as B. cereus and submitted to the GenBank database under the accession number MT084573. The blasting of 16S rRNA sequencing result demonstrated that it shared 99.76% homology with the nearest homologous species B. cereus and B. anthracis.

Extracellular enzymes and hemolysis activity: The results for the production of extracellular enzymes by the Bacillus isolate indicated that B. cereus isolate was found to be positive for the production of protease, lipase. In addition, B. cereus isolate was positive for lecithinase activity, which causes cell lysis by disrupting the membrane. Furthermore, B. cereus was β-hemolytic on blood agar.

Antimicrobial sensitivity testing: Antibiotic susceptibility tests indicated that the isolate was sensitive to all the used antibiotics except cefotaxime as shown in Table 1.

Experimental infection: In the experimental infection, LD50 value of B. cereus isolate was determined as 2.7×106 CFU mL1.

Table 1:
Antimicrobial sensitivity testing of the isolated strain of Bacillus cereus
Image for - Isolation and Pathogenicity Determination of Bacillus cereus Associated with Ulcer Formation in African Catfish Clarias gariepinus

Image for - Isolation and Pathogenicity Determination of Bacillus cereus Associated with Ulcer Formation in African Catfish Clarias gariepinus
Fig. 1(a-b): Gross external lesions in Clarias gariepinus
  (a) Naturally infected specimen and (b) Experimentally challenged specimen injected with B. cereus 2.7×108 CFU mL1, 1a-white arrows: Multiple small sized shallow skin ulcers, 1a-black arrow: Hemorrhagic exophthalmia, 1b-white arrows: Extensive deep ulceration along the fish trunk, 1b-white arrow head: Diphtheritic like membrane formation, 1b-black arrows: Distinctive multifocal fin erosions

Experimentally infected fish showed external and internal clinical symptoms similar to those caused by natural infection in farm. Some signs and symptoms were observed such as skin depigmentation, hemorrhagic spots of skin, gills and fins, bulging eyes, swollen abdomens and abnormal behavior. The skin hemorrhagic spots welled gradually to form ulcerative lesions. The size of ulcer was dose-dependent. The small dose produced small ulcers, which were self-limited and completely healed within 2 weeks from infection. The ulcers of higher doses were observed to grow in size and underlying muscles become eroded (Fig. 1b). Finally, severely ulcerated fish died within first 3 days of infection. The internal lesions included ascites, oedema, hepatic congestion, enlarged spleen and posterior kidney and vent protrusion. To confirm the causative agent, B. cereus re-isolated from the skin lesions and other internal organs of experimentally infected fish.

Histopathological findings: Microscopic examination of tissue specimens revealed different lesions with various degrees of pathological harm in comparison to non-infected fish tissues (Fig. 2a-t). Skin lesions (ulcers) of all treated fish showed also various degrees of congestion, haemorrhages, oedema, mononuclear cell infiltrations and fibrous granulation tissue at the periphery of the ulcerative tissue, with prominent necrosis inside the ulcer either in dermal tissue layers or in the underlying musculature, the degree of harm is progressive with increasing the injected bacterial doses (Fig. 2a, e, I, m, q).

Image for - Isolation and Pathogenicity Determination of Bacillus cereus Associated with Ulcer Formation in African Catfish Clarias gariepinus
Fig. 2(a-t): Micrographs of various tissues of Clarias gariepinus
 
(a) Normal skin tissue, (b) Normal hepatic tissue with multifocal activation of MMCs, (c) Normal renal tissue with multifocal activation of MMCs, (d) Normal splenic tissue with some scattered pigmented macrophages, (e) Normal skin tissue with mild sub-epidermal oedema, (f) Normal hepatic tissue, (g) Renal tissue showing mild tubular vacuolar degeneration with mild mononuclear cell infiltration in interstitial tissue, (h) Splenic tissue with mild depletion of red pulp with appendance of scattered pigmented macrophages, (i) Skin tissue with severe congestion (C) and Hemorrhages in the sub-epidermal area with hyperplasia of fibrous connective tissue, (j) Hepatic tissue showing diffuse vacuolar degeneration, (k) Renal tissue showing oedema and mild tubular vacuolar degeneration with focal tubular necrosis, (l) Splenic tissue with severe degree of congestion (C), (m) Skin tissue with hyperplasia of fibrous connective tissue and necrosis of underlying muscular tissue (*), (n) Hepatic tissue showing diffuse vacuolar degeneration, (o) Renal tissue showing oedema and moderate diffuse tubular vacuolar degeneration with multifocal tubular and interstitial necrosis with mononuclear cell infiltration in interstitial tissue, (p) Splenic tissue showing congestion (C), Depletion of both red and white pulp and multifocal fibrosis, (q) Skin tissue with congestion and hyperplasia of fibrous connective tissue and necrosis of both fibrous connective tissue (*) and the underlying muscular tissue, (r) Hepatic tissue showing diffuse vacuolar degeneration and multifocal necrosis and fibrosis (*), (s) Renal tissue showing oedema and moderate diffuse tubular vacuolar degeneration with multifocal tubular and interstitial necrosis (*) with mononuclear cell infiltration in interstitial tissue, (t) Splenic tissue showing depletion of both red and white pulp (*) and multifocal fibrosis with some scattered pigmented macrophages, H and E. bar = 50 μm, (a-d) Control negative group, (e-h) group-2 (2.7×105 CFU mL1), (i-l) group-3 (2.7×106 CFU mL1), (m-p) group-4 (2.7×107 CFU mL1) and (q-t) group-5 (2.7×108 CFU mL1)

Hepatopancreas of all treated fish showed various degrees of congestion in main blood vessels, hemorrhages, oedema with some vacuolar degeneration and necrosis in hepatocytes which are replaced with fibrous connective tissue in higher injected doses (Fig. 2b, f, j, n, r). The posterior kidney also revealed increasing tissue pathology with the gradual increase in the injected bacterial doses in all treated fish groups, the lesions varied between congestion, oedema and minor vacuolar degenerative changes to reach higher degrees of tubule-glomerular and interstitial necrosis infiltrated with mononuclear cells and haemorrhages (Fig. 2c, g, k, o, s). Spleen in lower doses showed signs of hypraemia and congestion while in higher doses it turned to show severe degrees of depletion of both white and red pulps replaced by fibrous tissue (Fig. 2d, h, l, p, t). Gills were not affected by lower injected doses, while in higher doses hyperplasia of malpighian cells lining the base of secondary gill lamellae supervenes. Finally, brain tissue was not affected in all treated groups, except that the highest injected dose causes moderate degree of congestion of meningeal blood vessels. The lesions scoring in different groups as well as the degrees of damage are summarized in Table 2.

DISCUSSION

The African catfish (C. gariepinus) (Siluriformes: Clariidae) has increasing economic importance because of its extensive use in polycultured with Nile tilapia in earthen ponds in Egypt. Its main role in this farming system is to control the unwanted fish reproduction in the grow-out ponds by devouring tilapia hatchlings. Earthen pond type culture system depends mainly on increasing the natural food availability by the addition of organic fertilizers such as livestock manure, with the increase in water pollution and high organic loads in fish ponds, fish skin and gills are usually attacked by waterborne bacteria22,23. The B. cereus is common in soil and different environment and has the capacity to spread easily3. In this study, the causative agent was identified to be Gram-positive B. cereus bacterium based on its characteristic and 16s rRNA sequence analysis.

Similar to these results, some studies reported that Bacillus sp. could cause diseases in catfish, B. mycoides was isolated from dorsal ulcer and necrotic musculature in an epizootic of disease in cultured channel catfish (Ictaluruspunctatus)10. The B. cereus was isolated during mass mortality of stinging catfish (Heteropneustes fossilis) in West Bengal, India. The affected fish showed numerous ulcers on the skin which gradually increase in size to form ulcerative dermatitis11. Moreover, Bacillus spp. is considered as pathogens of fish other than channel catfish. Pychynski et al.12 reported that B. cereus and B. subtilis can cause gill arch necrosis, branchionecrosis, in common carp (Cyprinus carpio). B. Cereus retrieved from apparently healthy striped bass (Morone saxatilis) was lethal to other healthy individuals when injected at 107 CFU/fish13. Furthermore, Bacillus spp. are common bacterial fish pathogens affecting European sea bass Dicentrarchus labrax aquaculture in Greece14. Moreover, a highly lethal B. cereus pathogen was isolated from dying Chinese softshell turtle (Pelodiscus sinensis)17.

In a previous study,4 Staphylococcus epidermidis,2 B. cereus and one Pseudomonas stutzeri isolates were identified as the causative agents responsible for mortalities in European sea bass (Dicentrarchus labrax) in Egypt15. Also, in another study15 S. epidermidis and11 B. cereus bacterial isolates were obtained from septicemic cases in a hatchery broodstock of white sea bream (Diplodus sargus) in Egypt16.

In this study, the experimentally injected fish showed symptoms of lethargy, skin discoloration, superficial hemorrhagic ulcers, deep ulcers that reached the underlying muscle and tail erosions. Some fishes showed hemorrhages on the fins and vent, exophthalmia and congested internal organs. These clinical signs and postmortem findings were approximately similar to the previously recorded findings in B. cereus infections in white seabream16 and stinging catfish11.

The present study indicated that B. cereus isolate was sensitive to antibiotic used but cefotaxime. This finding was in line with previous reports that B. cereus is sensitive to the most antibiotics especially chloramphenicol, ciprofloxacin and tetracycline11,16. The results obtained in this study and others showed that B. Cereus infection in aquaculture could be effectively controlled by antibiotic addition.

Bacillus spp. produce a variety of extracellular enzymes such as protease, lipase, lecithinase, gelatinase, hemolysins, enterotoxins, cytotoxins, phospholipases responsible for food spoilage and their pathogenesis in human5,6,24. In this study, B. cereus isolate was found to be positive for the production of protease, lipase, lecithinase and hemolysin. Similarly, Ozdemir and Arslan24 found that all Bacillus spp. isolated from retail fish and ground beef secreted protease, lipase, gelatinase and DNase. While 69.2% were positive for lecithinase activity.

CONCLUSION

This study confirmed that B. cereus was responsible for skin ulceration and disease in African catfish C. gariepinus. Fish experimentally re-infected confirmed that the isolate was considered the causative agent of the condition. The pathogen could be controlled using antibiotics. The production of extracellular enzymes participated in B. cereus pathogenicity and tissue damage.

SIGNIFICANCE STATEMENT

This study will help the researchers to uncover the causative agent(s) of African catfish C. gariepinus ulcerative dermatitis in Egypt, with revealing of more detailed pathological changes observed later after experimental challenge using such pathogen(s).

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