Characterization and Molecular Identification of Bacterial Isolates from Tail and Fin Rot Infected Silver Carp

In order to determine the impact of Valipora campylancristrota Wedl, 1855 (Cestoda, Cryporinchidae) plerocercoids on growth, development, and condition factors of juvenile silver carp (Hypophthalmichthys molitrix Valenciennes, 1844) (age category 0+), two experimental groups of fish were established and maintained under identical conditions in a pond farm. The first group of silver carp was spontaneously infected with the larvae of the causative agent of valiporosis, while the second group of fish was free from this invasion. Results indicated that in April, weight and length of the infected silver carp were 23.5% and 4.4% lower, compared to the healthy fish, and the Fulton's condition factor in the diseased fish was 8.4% lower compared to the non-infected silver carp. When conducting this research in October, it was determined that the weight, length, and condition factor of the silver carp in the first group were 32.0%, 10.3%, and 35.6% lower, respectively, compared to the fish in the second group that were free from cestode larvae. Clinical examination revealed emaciation, reduced muscle mass in the caudal and anal fin regions of the examined fish. Some individuals with valiporosis exhibited spinal deformities and asymmetrical abdominal cavities due to intestinal distension. Post-mortem examinations revealed a distended gallbladder filled with bile, indicative of acute and chronic catarrhal inflammation of the gallbladder mucosa. Qualitative changes in bile were observed: in silver carp infected with valiporosis, the bile was a yellowish-green color and more viscous.

Summary The authors have experimental evidence that the protozoa causing the swimbladder inflammation (SBI) of the common carp (Cyprinus carpio) are indentical with presporogonic stages of Sphaerospora renicola Dykova et Lorn, 1982 parasitizing the renal tubules. Homogenates prepared from the thickened and inflamed swimbladder of naturally infected common carp, when injected into the abdominal cavity of fish, produced renal sphaerosporosis in the infection-free common carp if the homogenates contained the parasites described by Kovacs-Gayer et al. (8). By intraperitoneal injection, the Unidentified Blood Organisms (UBOs) living in the blood of the common carp were transmissible to common carp, from the blood of which they were demonstrable for a long time. However, they were not transformed into Sphaerospora. To other cyprinids (gibel carp, silver carp, grass carp, tench, roach) neither the blood stages nor the swimbladder stages were transmissible from the common carp.

The object of this study was to detect the bacterial species that founded in infected common carps Cyprinus carpio in semi-closed systems in two districts of Basrah province. Bacterial gill disease, skin ulcer disease and intestine inflammatory disease were recorded. In the present study, Aeromonas sobria (38.46) was the predominant species, followed by Citrobacter freundii (29.23), Vibrio cholerae (21.53) and Serratia fonticola (10.76%). Identification of the isolates was carried out depending on the morphology of colony, specific media and identification using VITEK 2 system (Biomerieux- USA). Five antibiotic discs were used for antibiotic sensitivity test by the disk diffusion method. A. sobria was sensitive to Nitrofurantion, V. cholerae was sensitive to Ampicillin, C. freundii was resistance for all antibiotics while S. fonticola was sensitive to Gentamicin. This study represented the first investigation in Iraq on bacterial isolates from semi- closed system.

ABSTRACTThe bacterial genus Vagococcus has been described to include eight recognized species: Vagococcus fluvialis, Vagococcus fessus, Vagococcus salmoninarum, Vagococcus lutrae, Vagococcus elongates, Vagococcus penaei, Vagococcus acidifermentans and Vagococcus carniphilus. In this study, V. carniphilus was isolated from the myocardium, liver, spleen and kidney tissues of diseased crucian carps. After isolation, biochemical, 16S rDNA gene sequence and phylogenetic analysis was performed. Next, the phenotypic and genomic characteristics were analysed using different biochemical tests and antibiotic susceptibility testing. The bacterial isolate was gram-positive. The biochemical testing results indicated that a positive reaction occurred in the tubes containing glycerol, galactose, mannitol, sucrose sorbitol and d-ribose, and a negative reaction with α-galactosidase, β-galactosidase and hydrogen sulphide. A DNA product (∼1500 base pairs) was amplified from 16S rDNA. BLAST analysis revealed that V. carniphilus from crucian carps was highly similar to V. carniphilus isolated from other sources and showed the highest similarity (95%) with reference strains. Additionally, pathological analysis revealed that infected crucian carp tissues (myocardium, liver, spleen and kidney) and mouse tissues (myocardium, lung, liver, spleen and kidney) were severely damaged. In summary, we isolated V. carniphilus from diseased crucian carp, and this isolate induced pathological changes in crucian carp and mice.

The present study was aimed to find the most common causative agents relates to skin ulceration in most common fish farming (ponds and cages) affects Cyprinus carpio in Wasit province harvested from three different regions. The laboratory isolation and detection of bacteria in this study was carried out in medical laboratory techniques in Middle Technical University and Central Public Health Laboratory in Baghdad from September to December 2020. For this study 240 infected common carp Cyprinus carpio L. were used, 120 samples from cages and 120 samples from ponds. Samples were taken from three regions, which represented the north, central, and south of the governorate. Samples from skin ulcers were taken from affected areas in the body of the fish, skin, gills and fins, then transporting to the laboratory to perform bacterial culture. The present study illustrates that the common causative agent of ulceration in fish skin, gills and fins was bacteria with most detected bacteria were Aeromonas hydrophila (56.7%) in cages and (37.5%) in ponds, followed by Pseudomonas aeruginosa (40.83%) in cages and (35.08%) in ponds, and Citrobacter freundii (41.66%) in cages and (25.8%) in ponds through the months including in this study. In conclusion, the most important pathogenic bacteria related to skin ulceration in both cages and ponds in all three locations in Wasit province were Aeromonas hydrophila, Pseudomonas aeruginosa and Citrobacter freundii. The infection rate was higher in cages compared to ponds because of increase pollution by sewage and organic compound and due to stress condition resulting from higher stoking density.

The present study was conducted to know the culture strategies and fish health and disease problems in pond aquaculture in Mymensingh, Bogra and Pabna districts of Bangladesh. Questionnaire interview and participatory rural appraisal tools like focus group discussion (FGD) were conducted with selected fish farmers. In total 90 farmers were interviewed and 9 FGD sessions were conducted. Most of the respondents practiced carp and pangas polyculture and they had disease problems in their ponds. Prevalence of fish disease varied with cultured species and locations. The most prevalent diseases as reported by the farmers were pop eye (57.78%), ventral reddening (55.55%), tail and fin rot (48.89), hemorrhagic lesion over the body surface (45.56%), dropsy (40%), gill rot (40%), white spot (40%) and epizootic ulcerative syndrome or EUS (33.33%). According to farmers opinion most susceptible species to disease was silver carp, Hypophthalmichthys molitrix (57.78%); followed by mrigal, Cirrhinus cirrhosus (50%); catla, Catla catla (47.78%); sarputi, Barbodes gonionotus (47.78%); rui, Labio rohita (42.22%) and pangas, Pangasianodon hypophthalmus (42.22%). Higher mortality was found in pangas (53.56%). Clinically, pangas, koi and sarputi were found severely affected. Most of the farmers used some treatment measures against disease problems. This study also highlited some fish health management problems in pond aquaculture. Further studies should include identification and characterization of pathogens involved in different types of diseases in pond aquaculture. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19944 J. Bangladesh Agril. Univ. 11(2): 377-384, 2013

Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.

Fisheries play an important role in increasing the Nepalese economy as well as sustaining livelihood of some ethnic groups of our country. With the increased demand of fish, pisciculture have also increased to a great extend. Due to the rise in fish culture, there has been also rise in fish diseases. The study aims to investigate different parasitic, bacterial, fungal diseases in fish and to suggest treatment to control the diseases in four different fish farm of Nepal. A cross- sectional qualitative method was used to collect data from four selected fish farm (Kakani, Trishuli, Begnas, Mirmi) of Nepal. Infected fishes were transferred to the lab in oxygen filled plastic bags and further tested for bacterial, fungal and parasitic infection. The result of the study indicates that Epizootic Ulcerative Syndrome was the most common bacterial-fungal disease that had a significant impact on common carp fish especially in Trishuli, Begnas and Mirmi. Coccidiosis caused by Eimeria spp was found to be a growing problem in rainbow trout farming (Kakani, Nuwakot) infecting intestine, liver, gut and skin causing yellow diarrhea and skin lesions. Trichodina was observed number one problematic parasitic in carp culture not only in government farm like Begnas and Mirmi, but also in commercial farms in most of the fishery areas of the country. Fin rot were more commonly reported during winter months affecting common carp, Silver carp, Bighead carp, Fingerling Fish causing fin erosion in them. Ascites in brood fishes (Trishuli) was noticed which may be due to bacterial infection and due to nutritional deficiency. Besides this, white spot in the gills (a protozoan parasitic disease) was perceived and to control it Neodox with Formalin (150ppm) was used and was found to be very effective. This study also identified some fish health management related problems in fish farms of Nepal such as lack of assistance, poor technical knowledge, and lack of suitable diagnostic laboratory and their proper use.

Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried out.Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media. The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability. Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities. Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities. Maximumactivity of lipase was exhibited by a strain isolated from silver carp. Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut. The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics.

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase-negative, nitrate-negative, catalase-positive and indole-negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β-lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD50 of 1.24 × 105 and 1.88 × 107 cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug-resistant (MDR)-A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.

The potential for aquaculture development in Indonesia, especially the shrimp farming industry, is projected to continue to increase. However, the current growth in aquaculture production is in line with the increasing number of disease outbreaks and can affect the production, profitability, and sustainability of the industry worldwide. Vibriosis is a major challenge in aquaculture that causes fish mortality in large numbers and rapid times. Vibriosis treatment is still limited to the same antibiotics in humans and animals. For this reason, alternative microorganisms that are more specific and selective in inhibiting vibrio are needed, one of which is the exploration of termite nest association bacteria. This study aims to obtain isolates of termite nest association bacteria that are antivibrio. Isolation of bacteria using ISP2 media. Furthermore, antagonistic screening was carried out against V. alginolyticus and V. harveyi bacteria. Isolates performed morphological and biochemical characterization followed by molecular identification with 16s rRNA sequence analysis using universal primer 27F 1492R. Isolation results obtained 15 isolates with varying colors and shapes. Ten isolates are Gram-positive bacteria and 12 isolates have a positive catalase test. Seven isolates have antivibrio activity, namely isolates SR9, SE1, SE4, SE5, SE6, SE7 and SE8. Isolate SE1 showed the best antivibrio activity capable of inhibiting the growth of V. alginolyticus and V. harveyi bacteria with an inhibition zone value of 3.5 cm and 4 cm, respectively. The results of the molecular analysis showed that isolate SE1 has 99.65% similarity with Bacillus amyloliquefaciens strain HY2-1. These bacteria have characteristics that have the potential to be used as probiotics in the aquaculture industry, especially vibrio biocontrol agents.

Abstract. Muhiddin NH, Herawati N, Ramlawati, Hidayah N. 2024. Characterization of antibiotic-producing bacteria from cassava tape fermented by traditional ragi tape. Biodiversitas 25: 3359-3368. This study was carried out to determine the phenotypic and molecular characteristics and identification of antibiotic producing bacterial isolates from cassava tape fermented by traditional ragi tape. The type of research is exploratory, including isolation and characterization of antibiotic-producing bacterial isolates from cassava tape fermented by traditional ragi tape from Bone District and Gowa District, South Sulawesi Province, and Polewali Mandar District, West Sulawesi Province. Bacterial isolation was done using the pour cup method, and an antibiotic test was performed using the Kirby-Bauer method with Escherichia coli and Staphylococcus aureus as indicator bacteria. The isolation process yielded two bacterial isolates that exhibited antibiotic properties, namely isolates Bb1 and Bb2. The phenotypic characterization of the isolate Bb1 revealed an irregular shape, bacillus-cell shape, Gram-positive, the presence of endospores, positive catalase, motile, aerobic, positive citrate, the capacity to ferment glucose, producing acid and acetyl methyl carbinol. Meanwhile, the Bb2 isolate exhibits a round colony shape, bacillus-cell shape, Gram-positive, produces endospores, positive catalase activity, motile, facultatively anaerobic, negative citrate, can ferment glucose and lactose, produces acid and acetyl methyl carbinol. The results of molecular identification demonstrated that bacterial isolate Bb1 exhibited a 99.72% homology level with Bacillus velezensis strain CR-502. In comparison, bacterial isolate Bb2 showed a 99.58% homology level with Bacillus paramycoides strains MCCC 1A04098. The findings indicate that bacterial isolates derived from cassava tape fermented by traditional ragi tape have the potential to be developed into functional foods and novel antibiotic compounds for application in the healthcare sector. This study highlights the importance of phenotypic and molecular characterization in explaining the diversity of microorganisms present in cassava tape as a traditional food fermentation.

Hospital septic surfaces represent critical reservoirs for persistent microbial contamination and may contribute to the spread of opportunistic pathogens in healthcare environments. Enzyme-producing bacteria colonizing such surfaces can enhance environmental persistence and resistance to routine sanitation procedures. This study aimed to investigate the diversity, enzymatic potential, and phylogenetic relationships of bacteria isolated from hospital septic surfaces using morphological, biochemical, and molecular approaches. Environmental samples were collected from selected hospital septic surfaces, and bacterial isolates were obtained using standard microbiological techniques. Morphological and biochemical characterization was performed using conventional assays, including catalase, protease, lipase, and amylase activity tests. Genomic DNA was extracted from purified isolates, and the 16S rRNA gene region was amplified and sequenced. Sequence similarity analysis was performed using BLAST, and phylogenetic relationships were constructed using MEGA11 software based on the Maximum Likelihood method with bootstrap analysis. A total of 28 bacterial isolates were initially obtained, and genomic DNA was extracted from all isolates; however, reliable sequencing data suitable for molecular identification were successfully obtained for 26 isolates. A total of 26 bacterial isolates were successfully identified from hospital septic environments. Catalase and protease activities were widely observed among isolates, while lipase and amylase activities varied between species. Molecular identification based on 16S rRNA gene sequencing revealed the predominance of bacterial species belonging to the genera Paenibacillus, Bacillus, and Staphylococcus. Several isolates, including Paenibacillus glucanolyticus and Staphylococcus haemolyticus, showed high sequence similarity (99-100%) with reference strains and formed well-supported phylogenetic clusters. Molecular identification and phylogenetic analysis were performed using 26 successfully sequenced isolates. The presence of diverse enzyme-producing bacterial populations on hospital septic surfaces highlights their potential role in environmental persistence and contamination. These findings emphasize the importance of continuous environmental surveillance and improved sanitation strategies to reduce the risk of microbial dissemination in healthcare settings.

Asian sea bass (Lates calcarifer), commonly known as barramundi, is an economically important food fish inhabiting coastal and brackish waters. In Sri Lanka, demand for commercial and recreational fishing has increased due to its mild flavor and culinary versatility. However, disease outbreaks pose significant challenges to aquaculture production, particularly in cage culture systems. This study investigated the causative agent of mass mortality events in Asian sea bass farms in Trincomalee, Sri Lanka. Fish exhibiting clinical signs, including skin ulcers, fin rot, exophthalmia, and lethargy, were collected for examination. Samples from the kidney, liver, and spleen were cultured on Tryptic Soy Agar (TSA) and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar at 37 °C for 24-48 hr. Isolates produced small to medium, round, convex colonies on TSA and green colonies on TCBS. Biochemical characterization revealed Gram-negative, oxidase-positive, catalase-positive, motile rods that fermented glucose without gas production. Molecular identification using 16S rRNA polymerase chain reaction confirmed the pathogen as Vibrio harveyi with 99.44% similarity to strain CP014038.2. The sequence was deposited in GenBank (accession number OR351233). Histopathological examination revealed tubular injury, necrotic epithelial cells, and macrophage-lymphocyte infiltration in liver and kidney tissues. This study confirms that Vibrio harveyi is strongly associated with Vibriosis, causing mass mortality in Asian sea bass cage culture in Trincomalee. These findings provide essential baseline data for developing effective disease prevention and control strategies in Sri Lankan aquaculture systems.

Phenotypic and genotypic characterization of Aeromonas jandaei involved in mass mortalities of cultured Nile tilapia, Oreochromis niloticus (L.) in Brazil