ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 23 : 76– 80 DOI: 10.24200/jams.vol23iss1pp76-80 Reveived 13 Mar 2018 Accepted 7 Oct 2018 Bacterial infection in farmed barramundi juveniles, Lates calcarifer Buthaina Al-Khaziri, Azhar Al-Busaidi, Adil Al-Sulimani, Najla Al-Mandhari,*Gilha Yoon *1Dr. Gilha Yoon( ) ghyoon@squ.edu.om. Department of Marine Science and Fisheries, College of Agricultural and Marine Sciences, Sul- tan Qaboos University, P.O. Box 34, Al-Khod 123, Sultanate of Oman Introduction Barramundi, Lates calcarifer is a catadromous fish of the family Latidae and the order of Per-ciformes. This species is widely distributed from the Indo-West Pacific region to Papua New Guinea and Northern Australia. Barramundi has been one of the most important cultured finfish in Asian countries. This fish has become the second highest potential species for freshwater aquaculture after tilapia In the Sultanate of Oman. Due to the recent introduction of Barramundi aquaculture in Oman, there has been an interest to study this species. However, as any other cultured fish species; a significant economic damage has been reported in farmed barramundi due to various infectious pathogenic bacteria around the world. Bacterial infections of several species are being iden- tified as the major cause of losses in the aquaculture in- dustry (Bromage et al. 1999). A Gram-positive coccus of the species Lactococcus garvieae has been recorded as a zoonotic pathogen that has been identified as one of the major causative agents of aquaculture pathogenic prob- lems worldwide (Chen et al. 2001). The Gram-negative Aeromonas sobria has also been identified as a primary pathogenic causative agent of many fish species (Jura- jMajtán et al. 2012). Many of modern and effective drugs and antibiotic are synthesised from traditional folk of medicine (Na- tarajan et al., 2003). Therapeutic efficacy of many differ- ent plants has been discovered in the traditional med- icine practices (Al Maqbool et al.1985). The healing attribution of henna leaves has been known for centu- ries and it is a subject of scientific studies (Azaizeh et al. 2003). This plant is scientifically known as Lawsonia inermis that belongs to the family Lythraceae (Abdulmo- neim 2007). Henna leaves contain lawsone (2-hydroxy-1, 4-Napthoquinone) components that are red orange in colour. (Abdulmoneim 2007). The chemical data of the henna leaves prompted to investigate its antibacterial دراسة العدوى البكتريية ألمساك الرباموندي املستزرعة بثينة اخلزيري وأزهار البوسعيدي وعادل السليماين وجنالء املنذري وجيلها يوين Abstract. In 2016, Barramundi, Lates calcaifer juveniles imported from Thailand to the aquaculture station at Sultan Qaboos University exhibited remarkable external clinical signs of losing scales, severe muscle necrosis, and ascites. Fin rot was also observed in the diseased fish. The mortality reached more than 90% and it was assumed that some bacterial species were associated with the diseased fish. The objective of the present study was to investigate the infection route of disease, revealing the causative agent and finding the most effective antibiotic treatment. The suspected pathogen vehicle was mosquito larvae in the outlet of the culture tank as bacterium was isolated inside the mosquito larvae. Pri- mary, traditional phenotypic tests and the vitek test confirmed that the bacteria were pathogenic Aeromonas sobria and Lactococcus garvieae. Eight of the most commonly used antibiotics in the aquaculture industry was used for antibiotic susceptibility test. It showed that that Gentamycin was the most effective antibiotic while the most effective environ- mentally friendly source was effort of henna, Lawsonia inermis, at a concentration of 10%. Keywords: Barramundi, Lates calcaقifer, mortality, antibiotic, Aeromonas sobria, Lactococcus garvieae, Henna. املســتخلص: عام 2016، أظهرت أمساك الرباموندي املســتوردة من تايالند إىل حمطة اإلســتزراع الســمكي يف جامعة الســلطان قابوس عالمات ســريرية خارجيــة ملحوظــة كفقــدان الزعانــف، خنــر العضــالت احلــاد، واالستســقاء. كمــا لوحــظ تــآكل الزعانــف يف األمســاك املريضــة. بلغــت نســبة الوفيــات أكثــر مــن 90% وقــد أفــرض أن عــدوى بكترييــة كانــت الســبب يف هــذه الوفيــات. اهلــدف مــن هــذه الدراســة هــو البحــث يف طريقــة العــدوى البكترييــة، كشــف العامــل املســبب هلــا، وإجيــاد املضــاد احليــوي األكثــر فعاليــة. الناقــل املشــتبه هلــذه العــدوى هــي يرقــات البعــوض املوجــودة يف خــزان تفريــغ امليــاه مــن أحــواض اإلســتزراع الســمكي، حيــث مت عــزل البكترييــا مــن داخــل الريقــات. أكــدت التحليــالت التقليديــة األوليــة، وحتليــل vitek أن البكترييــا املســببة للمــرض هــي Aeromonas sobria وLactococcus garvieae . مت اســتخدام مثانيــة أنــواع مــن املضــادات احليويــة األكثــر اســتخداما يف االســتزراع الســمكي يف اختبــار احلساســية للمضــادات احليويــة. أظهــرت الدراســة أن Gentamycin كان املضــاد احليــوي األكثــر فعاليــة ضــد البكترييــا املســتخلصة، يف حــن أن أوراق احلنــاء Lawsonia inermis هــي املضــاد احليــوي الصديــق للبيئــة بركيــز %10. الكلمات املفتاحية: الرباموندي، ليتس كالكريفر، املضادات احليوية، إيروموناس سوبريا، الكتوكوكوس غارفيي، احلناء. 77Research Article Al-Khaziri, Al-Sulimani, Al-Mandhari,Yoon activity. As disease prevention and control is fundamental to the success of aquaculture, therefore, the present study aimed to investigate the infection route of diseased Bar- ramundi, to reveal the causative agent that causes mass mortality and to find the most effective antibiotic treat- ment and finally to screen environmentally-friendly treatment methods derived from Omani herbs. Materials and Methods Different tests like primary, traditional phenotypic and experimental challenge tests were applied to identify the pathogenic vehicles that cause such a mass Barramundi mortality. An antibiotic susceptibility test was also ap- plied to find the most effective treatment. Fish history of samples In August 2016, Barramundi juveniles were imported from Thailand to the aquaculture station at Sultan Qa- boos University. There was an observed increase in the morbidity and mortality of the fish, where more than 90% of fish died in that period. The fish showed remark- able external clinical signs of scales losing, severe muscle necrosis, ascites, and fin rot (Figure 1, a and b). Isolation of bacteria The suspected pathogen vehicle (mosquito larvae) were collected from the outlet of the culture tank and grouped according to their different life stages. The bacteria were isolated from each stage and cultured in Tryptone Soya Agar (TSA). The cultured bacteria were incubated at 37oC for 24 hours. Each colony of the cultured bacteria was cultured again in separated TSA plates. The plates were incubat- ed at 37oC for 24 hours. Identification of bacteria Primary test Gram Staining was used for the isolated bacteria to iden- tify the group of bacteria; whether they were gram-pos- itive or negative. Secondary test A 3% KOH Solution was used for a primary test to divide the groups of bacteria into gram positive and gram neg- ative. A Densichek Plus was used to measure the density of the bacteria used: a saline solution was added to the samples to make the density between 0.03 to 0.5 mg/ml. An identification machine (Vitec Ltd.) was used to iden- tify the bacterial species, within 24 hours automated. Table 1. The eight antibiotics used and their average inhibition zone for the Aeromonas sobria species. ANTIBIOTICS Disk potency (µm) Diameter of zone of inhibition (mm) Average inhabited zone (mm) Susceptible Intermediate Resistant Oxytetracycline (OT 30) 30 ≥ 23 19-22 ≤ 18 24 ± 0.1 Ciprofloxacin (CIP 5) 5 ≥ 21 16-20 ≤ 15 24 ± 0.1 Erythromycin (E 15) 15 ≥ 21 16-20 ≤ 15 25 ± 0.2 Chloramphenicol (C 30) 30 ≥ 21 18-20 ≤ 17 21 ± 0.1 Gentamycin (CN 10) 10 ≥ 15 13-14 ≤ 12 22 ± 0.2 Tetracyclin (TE 30) 30 ≥ 28 25-27 ≤ 24 25 ± 0.2 Nitrofurantoin (F 300) 300 ≥ 17 15-16 ≤ 14 21 ± 0.1 Ampicillin (A 10) 10 - - - 0 Figure 1. Remarkable external clinical signs ofthe infect- ed fish: scale loss, severe muscle necrosis ad fin rot. 78 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Bacterial infection in farmed Barramundi juveniles, Lates calcarifer Experimental challenge test To identify the causative agent of the mass mortality, an experimental challenge test was performed by infecting healthy Barramundi juveniles by the suspected bacteria extracted from the mosquito larvae. The fish were kept and treated under the same conditions as the cultured ones, where the control fish were treated in a similar manner without bacteria. The infected fish were exam- ined for the clinical signs of disease. Treatments Susceptibility test of commercial antibiotics The antibiotic susceptibility test was carried out using eight of the most commonly used antibiotic in the aqua- culture industry (Oxytetracycline, Ciprofloxacin, Eryth- romycin, Chloramphenicol, Gentamycin, Tetracyclin, Nitrofurantoin, and Ampicillin). ISO-Sensitest Agar (Oxoid) was used in this test. The diameter of the inhi- bition zones of the antibiotics were measured (mm) and compared with the antibiotic standard table from Oxoid. Environmentally-friendly treatment Alcohol based extraction was applied for two different Omani herbs (henna, Lawsonia inermis and Zamuta). The henna and Zamuta leaves were grounded and mixed with 100% ethanol for about 24 hours. The extracts were filtered to separate any solids and mixed with ISO agar plates with 5 different concentrations of 10%, 5%, 2.5% 1% and 0.5%, respectively. A pure culture of the bacteria was diluted with PBS (phosphate- buffered saline) and inoculated in each plate. The plates were incubated at 37oC for 24 hours. The same concentrations of the hen- na extract were tested for their antibacterial activity us- ing the spectrophotometer device. Results Identification of bacteria The isolated pathogenic bacteria were Aeromonas sobria and Lactococcus garvieae. These bacteria have been re- ported to cause loss of scales, severe muscle necrosis, and ascites or to cause fish mortalities in some cases. Experimental challenge test The challenge test was applied to fulfil the causative agent of the mortality and compare the clinical signs of the killed fish. The fish died within 24 hours after infec- tion, and they displayed darkened, and with some losing scales, severe muscle necrosis, and fin rots. In the con- trol fish tank, no mortality occurred. Antibiotic test Commercial antibiotics Tables 1 and 2 show the results of the eight different an- tibiotics used for the two species of bacteria (Aeromonas sobria, and Lactococcus garvieae). The most effective antibiotic for the Lactococcus garvieae and Aeromonas sobria was Gentamycin (CN 10) with an average inhab- ited zone of 22 ±0.2 mm and 24 ± 0.3 mm respectively. Generally, both Lactococcus garvieae and Aeromonas sobria were resistant against Ampicillin (A 10). Henna extract showed more bio-activity against bacteria in at the concentrations of 10% and 5%, while it shows low bio-activity at the concentration of 2.5% and no activi- ty against bacteria at the lower concentrations. For the Zamuta extracts, the bio-activity was low at the concen- tration of 10%, while it shows no activity at the lower concentrations (Fig. 2). Discussion Table 2. The eight antibiotics used and their average inhibition zone for the Lactococcus garvieae species. ANTIBIOTICS Disk Potency µm Diameter of zone of inhibition (mm) Average inhabited zone (mm) Susceptible Intermediate Resistant Oxytetracycline (OT 30) 30 ≥ 23 19-22 ≤ 18 25 ± 0.1 Ciprofloxacin (CIP 5) 5 ≥ 21 16-20 ≤ 15 23 ± 0.1 Erythromycin (E 15) 15 ≥ 21 16-20 ≤ 15 26 ± 0.1 Chloramphenicol (C 30) 30 ≥ 21 18-20 ≤ 17 21 ± 0.2 Gentamycin (CN 10) 10 ≥ 15 13-14 ≤ 12 24 ± 0.3 Tetracyclin (TE 30) 30 ≥ 28 25-27 ≤ 24 27 ± 0.2 Nitrofurantoin (F 300) 300 ≥ 17 15-16 ≤ 14 20 ± 0.1 Ampicillin (A 10) 10 - - - 0 79Research Article Al-Khaziri, Al-Sulimani, Al-Mandhari,Yoon Bacterial identification and experimental chal- lenge test Both pathogenic Aeromonas sobria and Lactococcus garvieae have been reported to infect and kill fish in the aquaculture industry (Chen et al. 2001, Majtán et al. 2012). In both cases the infected fish from the farm and from the challenge test, the clinical signs are similar. It also assumed that the pathological findings are similar within the visceral cavity. Lactococcus garvieae infection in fish causes lesions in the vascular endothelium, hem- orrhages, swollen abdomens, and accumulation of as- citic fluid that mainly affect the spleen, liver, brain, gut, kidney, and heart (Vendrell et al. 2006). The pathogenic Aeromonas sobria has been reported to cause hemor- rhages and bleeding of skin lesions (Majtán et al. 2012). Aeromonas sobria and Lactococcus garvieae may act as a primary pathogen and a potential risk factor for Bar- ramundi with mosquito larvae acting as potential reser- voir for the pathogens. Antibiotic test Commercial antibiotics Both of the isolated bacteria only showed resistance against ampicillin, whilst Gentamycin was the best at killing or inhibiting the growth of these bacteria. Environmentally-friendly Alcoholic based ex- traction Henna plant has been known for their healing attribute that considered as an active agent against bacteria and fungi (Abdulmoneim 2007). Since henna shows a high biological antimicrobial activity, it can be used for any microbial infection without any side effects or bac- terial resistance (Abdulmoneim 2007). Henna leaves contain glycosidic compounds like Lawsone (2-hy- droxy-1,4-naphthoquinone) which also known as hen- notannic acid, gallic acid, α-d-Glucose and tannic acid (Ostovari 2009). These compounds have been also proved for their effective antimicrobial activity against human bacteria (Abdulmoneim 2007). However, Zamo- ta leaves did not show high antimicrobial activity against the isolated bacteria. Conclusion It is suggested that Aeromonas sobria and Lactococcus garvieae can act as a primary pathogen for Barramundi that cause a risk of mass mortality. The clinical signs for both bacterial species were lesions in the vascular en- dothelium, hemorrhages, swollen abdomens, accumula- tion of ascitic fluid, and bleeding from skin lesions. The most effective antibiotic against these pathogenic bacte- ria was Gentamycin while henna extract from screened herbs proved to be also highly effective against these bacteria. Acknowledgement The authors would like to thanks to IG/AGR/FISH/16/01, Sultan Qaboos University for financial support of this study. Refrences Abdulmoneim MA. 2007. Evaluation of Lawsonia iner- mis Linn. (Sudani Henna) Leaf Extracts as an Antimi- crobial Agent. Research Journal of Biological Scienc- es 2 (4), 419-423. Almaqbool AZ, Bashir, AK, Farouk, A, Salih, AKM. 1985. Antimicrobial activity of certain Sudanese plants used in folkloric medicine screening for anti- Figure 2. The bio-activity for both a henna, (Lawsonia inermis) and b (zamuta) extracts against bacteria at different concen- trations. 80 SQU Journal of Agricultural and Marine Sciences, 2018, Volume 23, Issue 1 Bacterial infection in farmed Barramundi juveniles, Lates calcarifer microbial activity (IV). Fitoterapia, 56: 331. Azaizeh H, Fulder S, Said K, Khalil O. 2003. Ethno-Bo- tanical knowledge of local Arab practitioners in the Middle Eastern region. Fitoterapia, 74: 98-108. Barramundi. (November 16 2017). Retrieved November 18, 2017, from https://en.wikipedia.org/wiki/Barra- mundi. Bromage ES, Owens L. 2002. Infection of barramundi Lates calcarifer with Streptococcus iniae: effects of different routes of exposure. Discipline of Micro- biology and Immunology, James Cook University, Townsville, Queensland 4811, Australia, Diseases of aquatic organisms 52, 199–205. Hittino EC. 1999. Actococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus my- kiss: similar, but different diseases. Diseases of Aquat- ic Organisms 36, 227–231. Majtán J, Černy J, Ofúkaná A, Takáč P, Kozánek M. 2012. Mortality of therapeutic fish Garra rufa caused by Aeromonas sobria. Asian Pacific Journal of Tropical Biomedicine, 2(2), 85-87. Natarajan MR, Lalithankumar D. 1987. Leaf extracts of Lawsonia inermis as antifungal agent. Current Sci- ence 56:1021-1022. Ostovaria A, Hoseinieha SM, Peikaria M, Shadizade- hb SR, Hashemia SJ. 2009. Corrosion inhibition of mild steel in 1 M HCl solution by henna extract: A comparative study of the inhibition by henna and its constituents (Lawsone, Gallic acid, α-d-Glucose and Tannic acid). Corrosion Science, 51(9), 1935-1949. Vendrell D, Balcázar LJ, Ruiz-Zarzuela I, de Blas I, Gi- ronés O, Múzquiz JL. 2006. Lactococcus garvieae in fish: A review. Comparative Immunology, Microbiol- ogy and Infectious Diseases, 29 (4), 177-198.