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First report of methicillin-resistant Staphylococcus aureus from cage-cultured tilapia (Oreochromis niloticus)

Atyah MA, Zamri-Saad M, Siti-Zahrah A

Vet Microbiol, 2010 Aug 26;144(3-4):502-4.
PMID: 20189324 DOI: 10.1016/j.vetmic.2010.02.004

Swabs from the brain, eyes and kidneys of tilapia from 11 farms were collected for a period of 2 years. They were grown on blood agar before cultures of suspected Staphylococcus aureus were subjected to ABI STAPH Detection Kit and PCR for identification. They were then grown on oxacillin resistance screening agar base (ORSAB) and subjected to PCR using the MRSA 17 kb forward and reverse primers to identify the methicillin-resistant S. aureus (MRSA). A total of 559 isolates of Staphylococcus spp. were obtained, from which 198 (35%) isolates were identified as S. aureus. Of the 198 S. aureus isolated from tilapias, 98 (50%) were identified as methicillin-resistant S. aureus (MRSA). Since global spread of multi-drug-resistant bacteria has increased in the past decade, this new finding in fish should be of concern.

Matched MeSH terms: Fish Diseases/microbiology*
Identification of Vibrio harveyi isolated from diseased Asian seabass Lates calcarifer by use of 16S ribosomal DNA sequencing

Ransangan J, Mustafa S

J Aquat Anim Health, 2009 Sep;21(3):150-5.
PMID: 20043399 DOI: 10.1577/H09-002.1

The grow out of Asian seabass Lates calcarifer in marine net-cages is a popular aquaculture activity in Malaysia. Production of this species is greatly affected by the occurrence of vibriosis, which causes heavy mortality. Generally, young fish are more susceptible; they exhibit anorexia and skin darkening, followed by heavy mortality. The acutely affected older fish may also exhibit bloody lesions around the anus and the base of the fins. Twenty-one bacterial isolates obtained from internal organs (kidney, heart, spleen and liver) of the affected specimens were subjected to phenotypic characterization, testing for antibiotic susceptibility, and 16S ribosomal DNA sequencing. The sequencing result showed that all of the bacterial isolates belonged to Vibrio harveyi. The phenotypic characterization, however, identified 4 of the bacterial isolates as V. harveyi, 16 as V. parahaemolyticus, and 1 as V. alginolyticus. These findings suggest that biochemical features alone cannot be reliably used to identify bacterial pathogens, including V. harveyi, in aquaculture. Antibiotic susceptibility assays showed that some antibiotics, including oxytetracycline, nitrofurantoin, furazolidone, streptomycin, sulfamethoxazole, chloramphenicol, nalidixic acid, and oxolinic acid were effective against V. harveyi. Considering the side effects of these antibiotics, however, their use is not recommended in the aquaculture of Asian seabass.

Matched MeSH terms: Fish Diseases/microbiology*
Efficacy of spray administration of formalin-killed Streptococcus agalactiae in hybrid Red Tilapia

Noraini O, Sabri MY, Siti-Zahrah A

J Aquat Anim Health, 2013 Jun;25(2):142-8.
PMID: 23724958 DOI: 10.1080/08997659.2013.781553

An initial evaluation of spray vaccination was carried out with 60 hybrid Red Tilapia Oreochromis spp., divided into three groups that consisted of 10 fish per group with duplicates. The formalin-killed cells (FKCs) of Streptococcus agalactiae were administered once to group 1 by spray and once daily for five consecutive days to group 2. Group 3 remained as the untreated control group and was sprayed with normal saline. A booster was given twice to all the groups, once at the second week and again at the fourth week after the first vaccination. After this initial evaluation, a challenge study was conducted with 40 tilapia divided into two groups that consisted of 10 fish per group with duplicates. Group 1 was vaccinated with FKCs of S. agalactiae by a single spray administration while group 2 remained as the untreated control group. A booster was given twice using the same protocol as in the initial evaluation. After 6 weeks, fish from one of the duplicate tanks from each of groups 1 and 2 were challenged with pathogenic S. agalactiae by intraperitoneal (IP) injection, while fish in another tank were challenged through immersion. Based on the observations, serum immunoglobulin M (IgM) levels were significantly higher (P < 0.05) in the challenged fish than in the either the preexposed fish or the control group 1 week after the initial exposure. However, no significant differences (P > 0.05) were noted between challenged groups 1 and 2. In addition, no significant differences (P > 0.05) were observed between the frequencies of exposure. The mucus IgM level, however, remained high after each booster until the end of the 8-week study period. Meanwhile, serum IgM levels decreased after the challenge. A higher percentage of survival was noted for fish challenged through immersion (80%) compared with IP injection (70%). These results suggested that single spray exposure was able to induce IgM, which gave moderate to high protection during the challenge study.

Matched MeSH terms: Fish Diseases/microbiology
Natural Concurrent Infection of Vibrio harveyi and V. alginolyticus in Cultured Hybrid Groupers in Malaysia

Mohamad N, Mohd Roseli FA, Azmai MNA, Saad MZ, Md Yasin IS, Zulkiply NA, et al.

J Aquat Anim Health, 2019 03;31(1):88-96.
PMID: 30536485 DOI: 10.1002/aah.10055

In September 2016, a marine fish farm operator in Selangor, Malaysia, reported a disease outbreak affecting juvenile hybrid groupers (Camouflage Grouper Epinephelus polyphekadion × Tiger Grouper E. fuscoguttatus). The average daily mortality was 120 fish, resulting in a cumulative mortality rate of 29% within 10 d. The affected hybrid groupers displayed lethargy, excessive mucus production, rotten fins, congestion of livers and kidneys, and enlargement of spleens. Microscopically, general congestion of the brains and internal organs was evident. Vibrio harveyi and V. alginolyticus were successfully isolated from the diseased fish. The isolated pathogens were found to be sensitive to oxytetracycline and tetracycline, but resistant towards ampicillin and vancomycin. Experimental infections using the isolated V. harveyi (108 CFU/mL), V. alginolyticus (108 CFU/mL), and concurrent infection by V. harveyi (108 CFU/mL) and V. alginolyticus (108 CFU/mL) in juvenile Asian Seabass Lates calcarifer resulted in 60, 100, and 100% mortality, respectively, within 240 h postinfection. The experimentally infected Asian Seabass demonstrated similar clinical signs and histopathological changes as the naturally infected hybrid groupers. However, concurrently infected fish demonstrated severe clinical signs and histopathological changes compared with single infections. These results suggest that both isolates of Vibrio are pathogenic to fish and responsible for the disease outbreak. However, concurrent infection involving V. alginolyticus and V. harveyi leads to a more devastating impact to the cultured fish. This is the first report of concurrent Vibrio infection in cultured marine fish in Malaysia.

Matched MeSH terms: Fish Diseases/microbiology
Environmental Factors Associated with the Presence of Vibrionaceae in Tropical Cage-Cultured Marine Fishes

Mohamad N, Mustafa M, Amal MNA, Saad MZ, Md Yasin IS, Al-Saari N

J Aquat Anim Health, 2019 06;31(2):154-167.
PMID: 30653742 DOI: 10.1002/aah.10062

This study investigated the environmental factors associated with the presence of Vibrionaceae in economically important cage-cultured tropical marine fishes: the Asian Seabass Lates calcarifer, snapper Lutjanus sp., and hybrid grouper Epinephelus sp. Fish sampling was conducted at monthly intervals between December 2016 and August 2017. The body weight and length of individual fish were measured, and the skin, eye, liver, and kidney were sampled for bacterial isolation and identification. Water physicochemical parameters during the sampling activities were determined, and the enumeration of total Vibrionaceae count was also conducted from water and sediment samples. Nine species of Vibrio were identified, including V. alginolyticus, V. diabolicus, V. harveyi, V. campbellii, V. parahaemolyticus, V. rotiferianus, V. furnissii, V. fluvialis, and V. vulnificus. Photobacterium damselae subsp. damselae was also identified. A total of 73% of the isolated Vibrio belonged to the Harveyi clade, followed by the Vulnificus clade (5.5%) and Cholera clade (0.6%). Highest occurrence of Vibrio spp. and P. damselae subsp. damselae was found in hybrid grouper (72%), followed by Asian Seabass (48%) and snapper (36%). The associations of Vibrio spp. and P. damselae subsp. damselae with the host fish were not species specific. However, fish mortality and fish size showed strong associations with the presence of some Vibrio spp. On average, 60% of the infected cultured fish exhibited at least one clinical sign. Nevertheless, inconsistent associations were observed between the pathogens and water quality. The yearlong occurrence and abundance of Vibrionaceae in the environmental components indicate that they might serve as reservoirs of these pathogens.

Matched MeSH terms: Fish Diseases/microbiology*
Possible transmission routes of Vibrio spp. in tropical cage-cultured marine fishes

Nurliyana M, Amal MNA, Zamri-Saad M, Ina-Salwany MY

Lett Appl Microbiol, 2019 Jun;68(6):485-496.
PMID: 30834548 DOI: 10.1111/lam.13146

This study investigates the possible transmission routes of Vibrio spp. in a tropical cage-cultured marine fishes. Samplings of cultured Asian seabass, red snapper, hybrid grouper, wild fish, trash fish, fish fry, water and sediment samples were conducted from December 2016 to August 2017. All fish were dissected in situ and swabs were taken aseptically from the skin, eye, liver and kidney for bacterial isolation and identification. Bacterial isolation and identification from water, sediment and trash fish were also made. A total of 261 Vibrio spp. isolates recovered from the cultured, wild and fry fish, as well as from the sediment and water of the farm environment were analysed. Sequences of the pyrH gene were used to investigate the degree of relatedness and possible transmission routes existing between the isolated Vibrio spp. The population tree revealed the existence of selected Vibrio spp. that possibly transmitted between the newly introduced fish fry and wild fish into the cultured fish, while water also might possibly serves as natural transmission medium of certain Vibrio spp. in this fish farm. SIGNIFICANCE AND IMPACT OF THE STUDY: The source of transmission of Vibrio spp. into farmed marine fish remains unclear. This study highlights the possible transmission routes of Vibrio into cage-cultured marine fishes via newly introduced fish fry and wild fish. Understanding the routes of transmission of Vibrio spp. might help in controlling the disease in the near future.

Matched MeSH terms: Fish Diseases/microbiology
First Report of Achlya oblongata Infection in Freshwater-Reared Asian Seabass Lates calcarifer

Lau LM, Hatai K, Fukano H, Kurata O

J Aquat Anim Health, 2018 12;30(4):239-244.
PMID: 29710400 DOI: 10.1002/aah.10018

In September 2014, a freshwater oomycete was first isolated from Asian Seabass Lates calcarifer fry that were reared in freshwater at a fish hatchery in Sabah, Malaysia. A fungal strain was isolated from infected fry by using glucose yeast extract (GY) agar. From morphological identification, the strain belonged to the genus Achlya based on the mode of zoospore release. Molecular phylogenetic analysis of the internal transcribed spacer region sequences from the strain showed high similarity (99-100%) to Achlya oblongata. The isolate was able to grow on GY agar incubated at 15-35°C, in GY broth adjusted to pH 3.0-11.0, and in up to 1.0% NaCl. This is the first report of Achlya infection in freshwater-reared Asian Seabass in Malaysia.

Matched MeSH terms: Fish Diseases/microbiology*
Detection of Tilapia Lake Virus in Egyptian fish farms experiencing high mortalities in 2015

Nicholson P, Fathi MA, Fischer A, Mohan C, Schieck E, Mishra N, et al.

J Fish Dis, 2017 Dec;40(12):1925-1928.
PMID: 28590067 DOI: 10.1111/jfd.12650
Matched MeSH terms: Fish Diseases/microbiology
Fulltext Virulence-associated genes and antibiotic resistance patterns of Vibrio spp. isolated from cultured marine fishes in Malaysia

Mohamad N, Amal MNA, Saad MZ, Yasin ISM, Zulkiply NA, Mustafa M, et al.

BMC Vet Res, 2019 May 28;15(1):176.
PMID: 31138199 DOI: 10.1186/s12917-019-1907-8

BACKGROUND: Vibriosis is an important bacterial disease of cultured marine fishes worldwide. However, information on the virulence and antibiotic resistance of Vibrio spp. isolated from fish are scarce. This study investigates the distribution of virulence associated genes and antibiotic resistance patterns of Vibrio spp. isolated from cage-cultured marine fishes in Malaysia.
RESULTS: A total of 63 Vibrio spp. isolated from 62 cultured marine fishes in various geographical regions in Peninsular Malaysia were analysed. Forty-two of the isolates (66.7%) were positive for all chiA, luxR and vhpA, the virulence genes produced by pathogenic V. harveyi. A total of 62 Vibrio isolates (98%) had tlh gene of V. parahaemolyticus, while flaC gene of V. anguillarum was detected in 43 of isolates (68%). Other virulence genes, including tdh, trh, hlyA and toxRvc were absent from any of the isolates. Multiple antibiotic resistance (MAR) was exhibited in all strains of Harveyi clade, particularly against ampicillin, penicillin, polypeptides, cephems and streptomycin. The MAR index ranged between 0.06 and 0.56, and 75% of the isolates have MAR index of higher than 0.20. Host species and geographical origin showed no correlation with the presence of virulence genes and the antibiotic resistance patterns of Vibrio spp.
CONCLUSIONS: The study indicates that majority of Vibrio spp. isolated from cultured marine fishes possess virulence genes, but were not associated with human pathogen. However, the antibiotics resistance is a real concern and warrants ongoing surveillance. These findings represent an updated knowledge on the risk of Vibrio spp. to human health, and also provides valuable insight on alternative approaches to combat vibriosis in cultured fish.

Matched MeSH terms: Fish Diseases/microbiology*
Quorum sensing is required for full virulence of Vibrio campbellii towards tiger grouper (Epinephelus fuscoguttatus) larvae

Noor NM, Defoirdt T, Alipiah N, Karim M, Daud H, Natrah I

J Fish Dis, 2019 Apr;42(4):489-495.
PMID: 30742313 DOI: 10.1111/jfd.12946

The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4 days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V. campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.

Matched MeSH terms: Fish Diseases/microbiology*
Metabolite profiling of Epinephelus fuscoguttatus infected with vibriosis reveals Omega 9 as potential metabolite biomarker

Nurdalila AA, Mayalvanan Y, Baharum SN

Fish Physiol Biochem, 2019 Jun;45(3):1203-1215.
PMID: 30915615 DOI: 10.1007/s10695-019-00633-6

In this study, we report the starvation effect and vibriosis infection on a tropical fish, the tiger grouper (Epinephelus fuscoguttatus). The tiger groupers were infected with Vibrio vulnificus for 21 days. Gas chromatography-mass spectrometry combined with multivariate analysis was used to assess the variation in metabolite profiles of E. fuscoguttatus. Metabolite productions in infected fishes were significantly influenced by fatty acid production. The Omega 9 (ω-9) was abundant under the challenged conditions compared to Omega 3 (ω-3) and Omega 6 (ω-6). A total of six fatty acids from the ω-9 group were detected in high concentration in the infected fishes compared to the control groupers. These metabolites are Oleic acid, Palmitoleic acid, 6,9-Octadecenoic acid, 8,11-Eicosadienoic acid, cis-Erucic acid and 5,8,11-Eicosatrienoic acid. The production of ω-9 differed significantly (p ≤ 0.001) in the challenged samples. The detected ω-9 compounds were quantified based on three different extraction techniques with Supelco 37-component FAME mix (Supelco, USA). The highest concentration of ω-9 groups compared to the other fatty acids detected is 1320.79 mg/4 g and the lowest is 939 mg/4 g in challenged-starved; meanwhile, in challenged-fed, the highest concentration detected is 1220.87 mg/4 g and the lowest is 917.25 mg/4 g. These changes demonstrate that ω-9 can be used as a biomarker of infection in fish.

Matched MeSH terms: Fish Diseases/microbiology*
Fulltext Effect of Excoecaria agallocha on non-specific immune responses and disease resistance of Oreochromis niloticus against Streptococcus agalactiae

Laith AA, Mazlan AG, Effendy AW, Ambak MA, Nurhafizah WWI, Alia AS, et al.

Res Vet Sci, 2017 Jun;112:192-200.
PMID: 28499213 DOI: 10.1016/j.rvsc.2017.04.020

The current study was designed to evaluate the effects of Excoecaria agallocha leaf extracts on immune mechanisms and resistance of tilapia, Oreochromis niloticus, after challenge with Streptococcus agalactiae. Fish were divided into 6 groups; groups 1-5 fed with E. agallocha leaf extracts at 10, 20, 30, 40 and 50mgkg(-1) level, respectively. Group 6 were fed without extract addition and acted as control. E. agallocha extracts were administered as feed supplement in fish diet for 28days and the hematological, immunological, and growth performance studies were conducted. Fish were infected with S. agalactiae at a dose of 15×105CFUmL(-1) and the total white blood cell (WBC), phagocytosis and respiratory burst activities of leukocytes, serum bactericidal activity, lysozyme, total protein, albumin, and globulin levels were monitored and mortalities recorded for 15days post infection. Results revealed that feeding O. niloticus with 50mgkg(-1) of E. agallocha enhanced WBC, phagocytic, respiratory burst, serum bactericidal and lysozyme activities on day 28 pre-challenge and on 3rd, 6th, 9th, 12th and 15th day post-challenge as compared to control. Total protein and albumin were not enhanced by E. agallocha diet. E. agallocha increased the survival of fish after challenge with S. agalactiae. The highest mortality rate (97%) was observed in control fish and the lowest mortality (27%) was observed with group fed with 50mgkg(-1) extract. The results indicate that dietary intake of E. agallocha methanolic leaf extract in O. niloticus enhances the non-specific immunity and disease resistance against S. agalactiae pathogen.

Matched MeSH terms: Fish Diseases/microbiology*
Fulltext Typology of interventions for antimicrobial use and antimicrobial resistance in aquaculture systems in low- and middle-income countries

Garza M, Mohan CV, Brunton L, Wieland B, Häsler B

Int J Antimicrob Agents, 2022 Jan;59(1):106495.
PMID: 34896577 DOI: 10.1016/j.ijantimicag.2021.106495

Indiscriminate antimicrobial use (AMU) in aquaculture to treat and prevent diseases is common and can lead to the emergence of antimicrobial-resistant micro-organisms, potentially impacting public health and connected ecosystems. This study aimed to develop a typology to classify and characterise interventions to reduce AMU in aquaculture and identify points of action. Seventeen aquaculture and animal health professionals in Asian and African countries were interviewed to gather information on characteristics of interventions in different contexts to develop a typology. Seven types of interventions were defined: (i) legislation and regulations; (ii) industry rules and standards; (iii) voluntary instruments; (iv) commercial technology and alternatives to antimicrobials; (v) on-farm management; (vi) learning and awareness-raising; and (vii) activities with co-benefits. Types were based on intervention function, scope of implementation, implementer, compulsion, strength of the intervention, AMU/antimicrobial resistance (AMR) objective and stakeholder to influence. For each type, examples were described and discussed. The most common interventions to address AMU and AMR were legislative and regulatory frameworks and voluntary instruments, including National Action Plans. Interventions addressing AMU/AMR specifically were scarce. Other interventions focused on indirect effect pathways to AMU and AMR reduction aiming to improve good aquaculture practices, disease prevention and improved management. Monitoring and evaluation of these interventions were found to be rare, only present for interventions driven by development projects and international agencies. The presented typology of existing strategies and interventions addressing AMU/AMR in aquaculture systems can guide evaluation of AMR-sensitive interventions that promote responsible AMU, and informs the design and implementation of future interventions.

Matched MeSH terms: Fish Diseases/microbiology*
Whole-Genome Sequencing of Pseudomonas koreensis Isolated from Diseased Tor tambroides

Kho CJY, Lau MML, Chung HH, Chew IYY, Gan HM

Curr Microbiol, 2023 Jun 25;80(8):255.
PMID: 37356021 DOI: 10.1007/s00284-023-03354-5

Unlike environmental P. koreensis isolated from soil, which has been studied extensively for its role in promoting plant growth, pathogenic P. koreensis isolated from fish has been rarely reported. Therefore, we investigated and isolated the possible pathogen that is responsible for the diseased state of Tor tambroides. Herein, we reported the morphological and biochemical characteristics, as well as whole-genome sequences of a newly identified P. koreensis strain. We assembled a high-quality draft genome of P. koreensis CM-01 with a contig N50 value of 233,601 bp and 99.5% BUSCO completeness. The genome assembly of P. koreensis CM-01 is consists of 6,171,880 bp with a G+C content of 60.5%. Annotation of the genome identified 5538 protein-coding genes, 3 rRNA genes, 54 tRNAs, and no plasmids were found. Besides these, 39 interspersed repeat and 141 tandem repeat sequences, 6 prophages, 51 genomic islands, 94 insertion sequences, 4 clustered regularly interspaced short palindromic repeats, 5 antibiotic-resistant genes, and 150 virulence genes were also predicted in the P. koreensis CM-01 genome. Culture-based approach showed that CM-01 strain exhibited resistance against ampicillin, aztreonam, clindamycin, and cefoxitin with a calculated multiple antibiotic resistance (MAR) index value of 0.4. In addition, the assembled CM-01 genome was successfully annotated against the Cluster of Orthologous Groups of proteins database, Gene Ontology database, and Kyoto Encyclopedia of Genes and Genome pathway database. A comparative analysis of CM-01 with three representative strains of P. koreensis revealed that 92% of orthologous clusters were conserved among these four genomes, and only the CM-01 strain possesses unique elements related to pathogenicity and virulence. This study provides fundamental phenotypic and genomic information for the newly identified P. koreensis strain.

Matched MeSH terms: Fish Diseases/microbiology
Putative apolipoprotein A-I, natural killer cell enhancement factor and lysozyme g are involved in the early immune response of brown-marbled grouper, Epinephelus fuscoguttatus, Forskal, to Vibrio alginolyticus

Low CF, Shamsudin MN, Chee HY, Aliyu-Paiko M, Idrus ES

J Fish Dis, 2014 Aug;37(8):693-701.
PMID: 24304156 DOI: 10.1111/jfd.12153

The gram-negative bacterium, Vibrio alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing brown-marbled grouper aquaculture, causing fish farmers globally to suffer substantial economic losses. The objective of this study was to investigate the proteins involved in the immune response of brown-marbled grouper fingerlings during their initial encounter with pathogenic organisms. To achieve this objective, a challenge experiment was performed, in which healthy brown-marbled grouper fingerlings were divided into two groups. Fish in the treated group were subjected to intraperitoneal injection with an infectious dose of V. alginolyticus suspended in phosphate-buffered saline (PBS), and those in the control group were injected with an equal volume of PBS. Blood samples were collected from a replicate number of fish from both groups at 4 h post-challenge and analysed for immune response-related serum proteins via two-dimensional gel electrophoresis. The results showed that 14 protein spots were altered between the treated and control groups; these protein spots were further analysed to determine the identity of each protein via MALDI-TOF/TOF. Among the altered proteins, three were clearly overexpressed in the treated group compared with the control; these were identified as putative apolipoprotein A-I, natural killer cell enhancement factor and lysozyme g. Based on these results, these three highly expressed proteins participate in immune response-related reactions during the initial exposure (4 h) of brown-marbled grouper fingerling to V. alginolyticus infection.

Matched MeSH terms: Fish Diseases/microbiology
Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans

Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al.

Talanta, 2013 Dec 15;117:312-7.
PMID: 24209346 DOI: 10.1016/j.talanta.2013.09.016

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.

Matched MeSH terms: Fish Diseases/microbiology
Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology

Freeman MA, Yokoyama H, Osada A, Yoshida T, Yamanobe A, Ogawa K

J Fish Dis, 2011 Jun;34(6):445-52.
PMID: 21545438 DOI: 10.1111/j.1365-2761.2011.01255.x

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.

Matched MeSH terms: Fish Diseases/microbiology*
Fulltext Dissemination of streptococcal pyrogenic exotoxin G (spegg) with an IS-like element in fish isolates of Streptococcus dysgalactiae

Abdelsalam M, Chen SC, Yoshida T

FEMS Microbiol Lett, 2010 Aug 1;309(1):105-13.
PMID: 20528946 DOI: 10.1111/j.1574-6968.2010.02024.x

The Lancefield group C alpha-hemolytic Streptococcus dysgalactiae ssp. dysgalactiae (GCSD) causes systemic granulomatous inflammatory disease and high mortality rates in infected fish. Superantigen and streptolysin S genes are the most important virulence factors contributing to an invasive streptococcal infection. PCR amplification revealed that all strains isolated from moribund fish harbored the streptolysin S structural gene (sagA). GCSD fish isolates were PCR negative for emm, speA, speB, speC, speM, smeZ, and ssa. However, the size of the streptococcal pyrogenic exotoxin G (spegg) locus, a superantigen, in positive S. dysgalactiae fish and pig strains was variable. The ORF of the spegg locus of 26 GCSD fish strains and one GCSD pig strain was inserted with IS981SC. Interestingly, the ORF of the spegg locus of two fish strains of GCSD collected in Malaysia was inserted with an IS981SC-IS1161 hybrid IS element. The hybrid IS element was found in all of the GCSD fish isolates and one GCSD pig through PCR screening. Although no insertion sequence (IS) was detected in the spegg locus of S. dysgalactiae ssp. equisimilis (GCSE) strains, a five-nucleotide deletion mutation was detected in the ORF of the spegg locus of one GCSE strain at the supposed site of IS981SC insertion, resulting in a frameshift mutation.

Matched MeSH terms: Fish Diseases/microbiology*
Comparison of four antibiotics with indigenous marine Bacillus spp. in controlling pathogenic bacteria from shrimp and Artemia

Banerjee S, Devaraja TN, Shariff M, Yusoff FM

J Fish Dis, 2007 Jul;30(7):383-9.
PMID: 17584435

Use of antibiotics for the control of bacterial diseases in shrimp culture has caused several adverse impacts to the industry. This has resulted in the search for alternative environment friendly approaches to overcome bacterial infections. This study was conducted to investigate the use of beneficial bacteria as an alternative to antibiotics. Ten pathogenic bacterial species isolated from shrimp, Penaeus monodon, and Artemia cysts were tested for susceptibility to indigenous marine Bacillus subtilis AB65, Bacillus pumilus AB58, Bacillus licheniformis AB69 and compared with oxytetracycline, chloramphenicol, gentamicin and bacitracin, which are common antibiotics used in Asian aquaculture. The Bacillus spp. were isolated from the local marine environment for bioremediation use in shrimp hatcheries and were proven to reduce total ammonium nitrogen. The pathogenic bacterial isolates were 90% susceptible to B. subtilis AB65, 70% susceptible to B. pumilus AB58 and B. licheniformis AB69 and 100% susceptible to oxytetracycline, chloramphenicol and gentamicin but only 40% to bacitracin. Two representative isolates of the vibrio group, Vibrio alginolyticus VaM11 and Vibrio parahaemolyticus VpM1, when tested for competitive exclusion by a common broth method using the marine Bacillus spp., showed decreased viable counts from 10(8) to 10(2) cfu mL(-1). The results suggest that the action of the marine bacteria appears to be significant in protecting the host shrimp against pathogenic bacteria. In addition to the alternative use of antibiotics, the selected marine bacteria had additional bioremediation properties of reducing ammonia.

Matched MeSH terms: Fish Diseases/microbiology
Transmission of Streptococcus agalactiae from a hatchery into a newly established red hybrid tilapia, Oreochromis niloticus (L.) × Oreochromis mossambicus (Peters), farm

Amal MN, Zamri-Saad M, Siti-Zahrah A, Zulkafli AR

J Fish Dis, 2013 Aug;36(8):735-9.
PMID: 23347250 DOI: 10.1111/jfd.12056
Matched MeSH terms: Fish Diseases/microbiology

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