Studies On The Detection Of Norovirus In Marine Environment And The Bioremediation Of Culture Water

Norovirus (NV) contamination and antibiotics residues are the new safetyproblems in marine products. (1) NV is a major cause of viral acute gastroenteritisworldwide, and transmission through water and shellfish is one of the major exposureroutes. Since only a few viral particles are typically present in environment, sensitiveand standardize detection methods for monitoring and controlling norovirus areurgently needed. (2) The real cause of antibiotics residues in marine animal is overuseof antibiotics to control diseases resulted from the bad water quality, thusbioremediation is a key to solve this safety problem. Continuous studies have beendone on the above two problems with results as follows:1. A new procedure for the concentration of nonoviruses from water samples hasbeen developed. This procedure (calcium flocculation method) uses the followingsteps: virus flocculation formed by treatment with CaCl₂ and Na₂HPO₄, virus releaseby sodium citrate dissolution, and virus re-concentration by ultrafiltration. Whenreverse transcription (RT)-PCR was performed after the procedure, the overalldetection sensitivity for seeded noroviruses in a one liter water sample was as low as 1RT-PCR unit. The sensitivity showed in this approach was at least 5-fold-higher thanthe current method with its three steps of adsorption-elution-concentration.2. A standardized method for detection norovirus in shellfish, the conventionalRT-PCR and real-time RT-PCR have been established and have been promulgated byGeneral Administration of Quality Supervision, Inspection and Quarantine of thePeople's Republic of China (AQSIQ) as SN/T 1635-2005. The method involves viral extraction using pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation,tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads,i.e. GPTT procedure. JV12/JV13 primers are used for conventional RT-PCR, while inreal-time RT-PCR, primers COG1F, COG1R, probes RING1(a)-TP, RING1(b)-TP areused for Gâ… noroviruses detection, and primes COG2F, COG2R, RING2-TP are usedfor Gâ…¡noroviruses detection.3. The NV survey results showed that NV was detected in water samples (1/15)and shellfish (Meretris meretris) samples (1/20), an meaningful relationship may existbetween shellfish contamination and aquaculture activities, the norovirus in clamsmay result from improper discharge of human sewage.4. A bacterium "W1" with high activity of bioremediation was isolated fromfishing port water by using of a new enrichment method which adds 1 g/L NaHCO₃and 3 g/L CH₃COONa to the water samples directly. On the basis of studies of themorphological and characteristics of this organism, W1 was identified as a stain ofphotosynthetic bacteria (PSB), Rhodopuesdomonas pulstris.5. Quality control of PSB was carried with a developed method of"semi-solidtube method" using R medium as photosynthetic bacteria (PSB) colony-countingmedium. The R medium was derived from a serial orthogonal experiments, and itwas set as: NaHCO₃ 1.0 g, CH₃COONa 3.0 g, yeast extract 2.0 g, K₂HPO₄ 0.5,Fe-EDTA 0.005 g, agar 8 g, NH₄Cl 1.0 g, MgCl·6H₂O 0.2 g, NaCl 5.0 g, dH₂O 1000ml. This medium possesses the advantages of accurate, rapid and simple for PSBtesting and determining. Valid cells in the W1 inoculant was confirmed to 2.8×10⁹cfu/ml.6. The bioremediation of W1 was realized by its valid cells. W1 promoted thegood recycle of carbon and nitrogen via its anabolism and denitrification. W1 cannotinhibit other microorganisms such as normal aerobic bacteria, vibrio bacteria andvirus (bacteriaphage f2). Estimated with MIA (microbial inhibition assay), it wasconfirmed produces no antibiotic. The bioremediation activity of W1 was investigated by water quality control tests, the results showed that the water quality could beeffectively controlled by the W1 inoculant, the NH₄⁺-N content of shrimp (Penaeusvannamei) pond water was beneath 0.6 mg/L, CODer was reduced 29%, DO contentwas increased 27% and the pH value was adjusted to 7.7-7.8 in the test period. Thus,the test pond water was remedied by W1 well, and the shrimp yield was 6.25% morethan the reference pond. While immobilized W1 was used in shellfish (Meretrismeretrix) culture farm, it showed good functions of water quality control and growingpromotion.No medicine can be used to treat the acute gastroenteritis caused by NVnowadays, meanwhile serious food safe problems is arising being of abuse uses ofantibiotics in marine culture. The rapid, simple and effective detection method for NVestablished in this study may used in the contamination survey of marine products,and this is very important for cutting viruses transmitting as well as controlling andprevention of the acute gastroenteritis which caused by NV, and the achievements ofthis study may also be a valuable reference to the detection and control of otherfoodborne viruses. In addition, a bioremediation for marine culture could be usablevia revealing the bioremediation mechanism of W1, this provides a feasible way tosolve the antibiotic residues problem, and it would be great beneficial to keep themarine products safe and keep the mariculture development sustainable.