Record Details

Quantification of Human Pathogenic Vibrios in Seafood Using Molecular Techniques

KrishiKosh

View Archive Info
 
 
Field Value
 
Title Quantification of Human Pathogenic Vibrios in Seafood Using Molecular Techniques
 
Creator Sadananda Acharya B
 
Contributor Indrani Karunasagar
Shamsundar, B.A.
Krishna Bhatt
Siddappaji
Maragal, M.M.
 
Subject bacteria, diseases, biological phenomena, sampling, irrigation, genes, pcr, seafoods, environment, toxins
 
Description Ph.D. Thesis
Vibrios are autochthonous to estuarine and marine environment and therefore are found in fish and shellfish harvested from such waters. V. cholerae, V. parahaemolyticus and V. vulnificus are the most important human pathogenic Vibrios transmitted to human being through consumption of seafood. Hence, studies are needed with reference to their prevalence, levels, seasonal variation and influence of environmental parameters in tropical waters. In the present study as many as 100 seafood samples comprising of oysters, clams, mussels, shrimps and fish collected along the west coast of India for three successive years were analysed for these pathogens.
The results showed that V. cholerae prevalence was 26% by conventional methods and 10% by polymerase chain reaction (PCR). One sample was positive for ctx (cholera toxin) gene in enrichment lysate. No choleragenic V. cholerae could be found in any of the samples. Hence, the toxigenic V. cholerae is present in a very low level in seafood in Mangalore coast.
V. parahaemolyticus showed 91%, 89% and 82% prevalence by traditional method, colony hybridization and PCR respectively. Total V. parahaemolyticus load ranged from undetectable to 2.0 x 106 cfu/g meat. Further, the incidences of tdh gene bearing V. parahaemolyticus was 05% and 07% by colony hybridization and PCR respectively. Similarly, the incidences of trh gene bearing V. parahaemolyticus was 15.8% and 21% by colony hybridization and PCR respectively. The level of tdh and trh bearing V. parahaemolyticus ranged from undetectable to 2.0x103 cfu/g meat. The results showed that there is a high load of both non-toxigenic and toxigenic V. parahaemolyticus in west coast seafood. No pandemic potential strains of V. parahaemolyticus were isolated from any of the 100 samples tested. Though pandemic strains like O3:K6 are not found in the sample, high load of toxigenic strains is a matter of concern.
V. vulnificus showed 70%, 69% and 83% prevalence by traditional method, colony hybridization and PCR respectively. Quantification of the pathogen in seafood samples revealed its range from undetectable level to 4.75x104 cfu/g meat. The results revealed a very high load of V. vulnificus for seafood samples in west coast.
Present study demonstrates the use of colony hybridization in quantifying total and pathogenic V. parahaemolyticus using alkaline phosphatase labeled oligonucleotide tlh, tdh and trh probes more rapidly and specifically. Further vvhA probe hybridization was found to be a good molecular DNA based technique for quantification of V. vulnificus in seafood samples or processed seafood before export. Polymerase chain reaction to detect tdh and trh gene in enrichment lysates of seafood sample is useful in screening seafood samples for the presence of such genes. Further, vvhA two step nested PCR is highly sensitive in identifying V. vulnificus from enrichment lysate and can very well be used for screening seafood samples for the presence of this pathogen.Salinity was found to be playing a major role than temperature in determining V. parahaemolyticus and V. vulnificus counts in the west coast. Higher levels of V. parahaemolyticus were witnessed with higher water salinity during summer months. In the case of V. vulnifcus, when salinity increased above 30 ppt, the pathogen was not detected in any sample. V. vulnificus were found abundantly during monsoon months when salinity was lower. Faecal coliform counts were found to have no effect onteh density of V. parahaemolyticus and V. vulnificus in our coastal waters. Upon characterization of V. vulnificus isolates, it was found that 90% of the isolates carried newly discovered rtxA toxin gene. Further, simple repeat sequence analysis of the same isolates showed high rate of heterogeneity in the population of V. vulnificus and absence of rtxA toxin gene was associated with low copy number of repeat sequences. To conclude, the results of the present study provides valuable insight into the prevalence and distribution of pathogenic Vibrio spp. The study further elucidates the effect of physico-chemical parameters on their abundance in coastal waters and seafood of Karnataka coast. The study highlights the usefulness of application of molecular techniques such as colony hybridization and polymerase chain reaction (PCR) in enumeration and detecting pathogenic Vibrio spp. in seafood and ensuring its quality and safety. Polymerase chain reaction to detect tdh and trh gene in enrichment lysates of seafood sample is useful in screening seafood samples for the presence of such genes. Further, vvhA two step nested PCR is highly sensitive in identifying V. vulnificus from enrichment lysate and can very well be used for screening seafood samples for the presence of this pathogen. Salinity was found to be playing a major role than temperature in determining V. parahaemolyticus and V. vulnificus counts in the west coast. Higher levels of V. parahaemolyticus were witnessed with higher water salinity during summer months. In the case of V. vulnifcus, when salinity increased above 30 ppt, the pathogen was not detected in any sample. V. vulnificus were found abundantly during monsoon months when salinity was lower. Faecal coliform counts were found to have no effect onteh density of V. parahaemolyticus and V. vulnificus in our coastal waters. Upon characterization of V. vulnificus isolates, it was found that 90% of the isolates carried newly discovered rtxA toxin gene. Further, simple repeat sequence analysis of the same isolates showed high rate of heterogeneity in the population of V. vulnificus and absence of rtxA toxin gene was associated with low copy number of repeat sequences. To conclude, the results of the present study provides valuable insight into the prevalence and distribution of pathogenic Vibrio spp. The study further elucidates the effect of physico-chemical parameters on their abundance in coastal waters and seafood of Karnataka coast. The study highlights the usefulness of application of molecular techniques such as colony hybridization and polymerase chain reaction (PCR) in enumeration and detecting pathogenic Vibrio spp. in seafood and ensuring its quality and safety.
 
Date 2016-07-04T14:14:41Z
2016-07-04T14:14:41Z
2007-09-15
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/68407
 
Language en
 
Format application/pdf
 
Publisher Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar