Reverse transcription-PCR assay for the rapid detection of viable Vibrio cholerae from fresh and processed shrimp

Viable Vibrio cholerae in seafood was rapidly detected by the Reverse transcription-PCR (RT-PCR) assay developed by targeting mRNA ctxA gene without any pre-enrichment. The PCR product size of the selected gene was 308 bp, which aided in the identification of V. cholerae. This RT-PCR assay was rapid, as it detected V. cholerae from fresh shrimp tissue within 5 min on bio-inoculation when the cell count was 1,000,000 cells. The pathogen was detected in 5 h when the load was 110 cells. The assay even detected the pathogen in shrimp that has been cooked for 10 min, frozen for 30 days and even in dried shrimp. Other food-borne pathogens like Salmonella, Staphylococcus aureus and Listeria monocytogenes were not detected by this assay. The developed RT-PCR assay is thus specific and rapid in detecting the viable Vibrio cholerae in seafood without any pre-enrichment.

Administration of Phyllanthus niruri to control IMNV (myonecrosis infectious virus) infection white shrimp Litopenaeus vannamei

<p>ABSTRACT</p><p><br />Infectious myonecrosis (IMN) disease is a major disease in Indonesia shrimp farming. The disease is caused by infectious myonecrosis virus (IMNV). Currently, treatment and drug has not been obtained to control the virus. This research was conducted to determine the effect of Phyllanthus niruri extract in white shrimp (Litopenaeus vannamei) against IMNV infection. Healthy shrimp was given P. niruri extract 20 mg/kg of feed for seven days and after that the shrimp was challenged by orally with IMNV infected shrimp tissue. The positive control was given feed without P. niruri extract and challenged with IMNV infected shrimp tissue, while negative control was not challenged with IMNV infected shrimp tissue. IMNV infection gave a significantly different effect on survival rate. In the shrimp P. niruri previously (86.7%) gave higher survival rate compared to shrimp without P. niruri (66.67%). Survival rate of negative control was 93.33%. IMNV clinical signs in general was white necrotic areas in striated muscles. Histological examination showed that cell necrosis appeared on the mussel tissues. In conclusion the addition of P. niruri to the commercial feed can give the survival rate of shrimp better when challenged with IMNV.<br />Keywords: IMNV, Phyllanthus niruri, Litopenaeus vannamei</p><p><br />ABSTRAK</p><p><br />Penyakit infectious myonecrosis (IMN) merupakan penyakit utama pada budidaya udang di Indonesia. Penyakit ini disebabkan oleh infectious myonecrosis virus (IMNV). Saat ini, belum diperoleh cara dan obat untuk mengendalikan virus IMNV. Penelitian ini dilakukan untuk mengevaluasi pengaruh immunostimulan tepung meniran (Phyllanthus niruri) yang diberikan melalui pakan pada udang vaname (Litopenaeus vannamei) yang diinfeksi IMNV. Udang vaname yang sehat diberi pakan yang mengandung meniran dengan dosis 20 mg/kg pakan selama tujuh hari dan kemudian diuji tantang secara oral dengan memberikan jaringan udang yang telah terinfeksi IMNV. Udang kontrol positif dilakukan dengan memberi pakan komersial tanpa penambahan meniran yang kemudian diuji tantang dengan memberi makan jaringan udang yang terinfeksi IMNV, sedangkan udang kontrol negatif tidak diuji tantang dengan jaringan yang terinfeksi IMNV. Hasil menunjukkan bahwa kelompok udang yang diberi pakan mengandung meniran mempunyai kelangsungan hidup (86,67%) lebih tinggi dibandingkan dengan udang yang tidak diberi pakan mengandung meniran (66,67%) ketika diuji tantang dengan IMNV. Kontrol negatif yang tidak diberi pakan mengandung meniran dan tidak diuji tantang dengan IMNV memberikan kelangsungan hidup 93,33%. Gejala klinis yang ditunjukkan adanya infeksi IMNV terlihat dengan adanya otot putih pada ruas tubuh udang. Pengamatan histopatologi menunjukkan adanya nekrosis pada sel-sel di jaringan otot udang. Sebagai kesimpulan dapat dinyatakan bahwa penambahan meniran pada pakan komersial dapat meningkatkan kelangsungan hidup udang ketika terjadi infeksi IMNV.<br /><br />Kata kunci: IMNV, Phyllanthus niruri, Litopenaeus vannamei</p>

Multiresidue Determination of Fluoroquinolones in Shrimp by Liquid Chromatography-Fluorescence-Mass Spectrometryn

An efficient multiresidue method for analysis of fluoroquinolones in shrimp has been developed in which quantitation by fluorescence and confirmation by Multiple Stage Mass Spectrometry (MSn) is achieved simultaneously. In this method, shrimp tissue is extracted with ammoniacal acetonitrile and the extract is defatted and then evaporated. After dissolution in basic phosphate buffer, fluoroquinolones in the extract are separated by liquid chromatography and quantitated, taking advantage of their intense fluorescence. Eluate from the fluorescence detector enters the MSn, which allows for confirmation by monitoring ratios of 2 prominent product ions in the MS3 or MS2 spectrum. Using this method, 8 fluoroquinolones have been analyzed in shrimp samples fortified at 10, 25, 50, or 100 ppb levels. Recoveries for desethyleneciprofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin ranged from 75 to 92%, with relative standard deviation values of &lt;6%. The limits of quantitation ranged from 0.1 to 1 ng/g. Enrofloxacin and ciprofloxacin were also successfully determined in enrofloxacin-incurred shrimp using this method.

Determination and Confirmation of Identities of Flumequine and Nalidixic, Oxolinic, and Piromidic Acids in Salmon and Shrimp

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5,10,20,40, and 80 ng/g. Average recoveries and relative standard deviations (RSOs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.