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Transmission and Toxigenic Potential of Vibrio Cholerae in Hilsha Fish (tenualosa Ilisha) for Human Consumption in Bangladesh

Zenat Z. Hossain
Department of Microbiology, University of Dhaka, Dhaka, Bangladesh

Israt Farhana
Department of Microbiology, University of Dhaka, Dhaka, Bangladesh

Suhella M. Tulsiani
Institute of Public Health, University of Copenhagen, Copenhagen, Denmark

Anowara Begum
Department of Microbiology, University of Dhaka, Dhaka, Bangladesh

Peter K. M. Jensen
Institute of Public Health, University of Copenhagen, Copenhagen, Denmark

Abstract/ Executive Summary:

Fish have been considered natural reservoirs of Vibrio cholera, the deadly diarrheal pathogen. However, little is known about the role of fish in the transmission of V. cholera from the Bay of Bengal to the households of rural and urban Bangladesh. This study analyzes the incidence and pathogenic potential of V. cholera in Hilsha (Tenualosa ilisha), a commonly caught and consumed fish that exhibits a life cycle in both freshwater and marine environments in Bangladesh. During the period from October 2014 to October 2015, samples from the gills, recta, intestines, and scale swabs of a total of 48 fish were analyzed. The fish have collected both at local markets in the capital city Dhaka and directly from fishermen at the river. PCR analysis by targeting V. cholerae species-specific comp W gene revealed that 39 of 48 (81%) fish were positive in at least one of the sample types. Real-time PCR analysis demonstrated that the cholera-causing ctx Agene was detected in 20% (8 of 39) of V. cholera e-positive fish. A total of 158 V. cholerae isolates were obtained which were categorized into 35 genotypic groups. Altogether, 25 O1and 133 non-O1/O139 strains were isolated, which were negative for the cholera toxin gene. Other pathogenic genes such as assent/sto, hlyA, chxA, SXT, rtxC, and HA-P were detected. The type three secretion system gene cluster (TTSS) was present in 18% (24 of133) of non-O1/O139 isolates. The antibiotic susceptibility test revealed that the isolates conferred high resistance to sulfamethoxazole-trimethoprim and kanamycin. Both O1and non-O1/O139 strains were able to accumulate fluid in rabbit ileal loops and caused distinctive cell death in HeLa cells. Multilocus sequence typing (MLST) showed clonal diversity among fish isolates with pandemic clones. Our data suggest a high prevalence of V. cholera in Hilsha fish, which indicates that this fish could serve as a potential vehicle for V. cholera transmission. Moreover, the indigenous V. cholera strains isolated from Hilsha fish possess considerable virulence potential despite being quite diverse from current epidemic strains. This represents the first study of the population structure of V. cholera associated with fish in Bangladesh.

Keywords: Fish, Hilsha, Vibrio cholerae, Transmission, Population, Pathogenic potential, Bangladesh

Location: Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Hilsa

Objectives:

In this study, Hilsha fish is analyzed for the first time to be a potential carrier of V. cholerae, and for its role as a risk factor in the transmission of V. cholera to humans.

Methodology:

Sample Collection and Processing: Four Hilsha fish were collected each month for a period of 1 year, from October 2014 to September 2015; two “market fish” from local markets in and around Dhaka and two “fresh fish” which were freshly caught near the bank of the Padma River (for a total of 48 fish). All the fish (mean body weight 765 g) were healthy, with a bright, shiny appearance and natural odor. The fresh fish was bought directly from fishermen at the major landing point (Paturia, approximately 80 km away from Dhaka) early in the morning. The fish were caught on the previous night in the lower Padma River between Shariatpur and Chandpur. Each fish was collected in individual sterile collection bags and transported to the University of Dhaka laboratory within 4 h of the collection in a cool box maintaining cold condition. Four samples were aseptically taken from each fish:—two slits of fish gills, gut, rectum and an outer swab of scales in phosphate-buffered saline (PBS). For the fish collected from the local market(fish stored on ice), ice samples where the fish were kept frozen were also collected in sterile zip-lock sample collection bags. A total of 8 and 10 samples each of fresh-caught fish and local market fish were analyzed each month for 1 year, for a final total of 96 and 120 samples. Approximately 6 gm of gill, gut and rectum samples were enriched in 60 mL of Alkaline Peptone Water (APW) (1 L distilled H2O, 10 gL−1peptone, 10gL−1sodium chloride; pH 8.5). One mL of PBS outer swab and storage ice water were transferred to 9 mL of APW for enrichment. All of the samples were incubated at 37⁰C for 24 h. Bacterial Strains A total of 158V. cholera strains were isolated by using conventional cultural media TCBS (Thiosulfate citrate bile-salts sucrose agar). Species identification of all the strains was further confirmed by standard biochemical assays and V. cholera species-specificompWgene target PCR (Nandi et al., 2000; Choopun et al., 2002; Huq et al., 2012). Molecular Characterization of V. cholerae Isolates Genomic DNA from the isolates was extracted by the boiler template method described earlier. Serological assays and PCR targeting the rfb sequences specific for O1 and O139 serogroups were used for further subtyping of all V. cholera isolates. PCR was performed to detect the virulence and regulatory genes ofV.choleraeO1/O139 and non O1/O139 (Supplementary). Total DNA samples that were detected as V. choler a-positive were further analyzed for the presence of cholera toxin gene(ctxA) and their sequences of O1 and O139 serogroups. PCR reactions were performed by using the protocol described previously in the section Total DNA Extraction and Detection of V. cholerae by Polymerase Chain Reaction. Realtime PCR to detectctxAgene was performed by following the previously published protocol (Blackstone et al., 2007). Positive and negative controls used in PCR experiments are listed in supplementary. Antibiotic Susceptibility AssayAntibiotic susceptibility of theV. cholera strains were conducted by agar disk diffusion method using commercial disks (Oxoid, UK). The strains were tested for Tetracycline (30μg), Sulfamethoxazole-trimethoprim (25μg), Chloramphenicol (30μg), Kanamycin (30μg), Neomycin (30μg) according to the standard guidelines of Clinical and Laboratory Standards Institute (CLSI) (Patel et al., 2014). The zone standards for Enterobacteriaceae were used when there were no established breakpoint interpretive criteria for V. cholerae. E. coliATCC25922 was used as a quality control strain. The experiment was done in duplicate. Toxicity Assay Nine V. cholera strains were studied including 6 V. choleraeO1 and 3 non O1/O139 serogroups for analyzing pathogenic potential on established animal models and human cancer cell line. Multilocus sequence typing (MLST) method was used to determine the nucleotide changes in housekeeping genes of these 9 isolates compared to the existing database (see section MLST below). Tissue Culture Assay Culture supernatants of V. cholera strains were tested for cytotoxicity in the HeLa cell-line (human cervical carcinoma cell line). Following previous protocol, the cell-free culture supernatants were prepared by centrifugation and filtration through a 0.22-μm-pore size filter unit (Millex-GS; Millipore Corp., Bedford, Mass; Sharma et al., 1998).HeLa cells were grown as monolayers in Dulbecco’sModified Eagles’ medium (DMEM) (Thermo Fisher Scientific, USA) containing 1% penicillin-streptomycin (1:1) and 0.2% gentamycin and 10% fetal bovine serum (FBS). Cells (4.4×104/400μl) were seeded onto 24-well plates and incubated overnight at 37⁰C in a humidified 5% CO2 atmosphere. Thereafter, 100μl of the culture supernatant sample was added each well. Cytotoxicity was examined under an inverted light microscope (Olympus, Japan) after 24 h of incubation. TheuninoculatedThe uninoculatedTrypticase soy broth and V. choleraeO1 El TorN16961were used as a negative and positive control. Duplicate wells were used for each sample.

Source of Information: Frontiers in Microbiology | www.frontiersin.org1February 2018 | Volume 9 | Article 222

Article Link (Online Journal): https://doi.org/10.3389/fmicb.2018.00222

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, as cases of cholera in Bangladesh continue to occur, new transmission dynamics and their potential influence on virulence should be monitored. This study presents new data on the prevalence of Vibrio cholerae in Hilsha fish, and the possibility of an alternative route of transmission to households (as opposed to drinking water) in Bangladesh. The spectrum of the V. cholerae population isolated from Hilsha fish samples was highly heterogeneous, based on genotypic profile analyses. Nevertheless, the Vibrio cholerae isolates lacked cholera toxin, yet in vitro and in vivo activity showed the disease potential of the isolates. Despite the presence of the cholera toxin gene in Hilsha fish samples, isolation of toxigenic strains was not successful. Still, it demands close monitoring of the coastal catch of Hilsha fish for cholera transmission and public health awareness to minimize the health risk posed by non-cholera Vibrio serogroups.

Knowledge Management: Journal