Selective Survival of a Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus in the Alimentary Tract of a Juvenile Estuarine Gastropod(Clithon retropictus).

Abstract

ABSTRACT. Juvenile estuarine gastropods (Clithon retropictus), maintained in ultraviolet ray-irradiated recirculating artificial seawater with a salinity of 20 at 28°C, preserved thermostable direct hemolysin (TDH)-producing strain D-3 of Vibrio parahaemolyticus at a level of 10 4 -10 5 colony forming units per gram (cfu/g) and TDH-non-producing strains N-18 and R-13 at a level of 10 1 -10 2 cfu/g in the alimentary tract for at least 21 days after ingestion. In adults, the numbers of the three strains decreased to a level of 10 0 cfu/g within 21 days under the same conditions. This evidence supports our recent observations that TDH-producing strains increased to a high level in the summer months in the presence of high levels of TDH-non-producing strains in the alimentary tract of juvenile C. retropictus at estuaries in Japan. Lymnaea stagnalis to Trichobilharzia ocellata [14]. Also, hemocytes of juvenile L. stagnalis were shown to be less differentiated in morphology and less active in a lysosomal enzyme, peroxidase, than those of adults [1]. Based on these findings, the present study was done to confirm that juvenile C. retropictus could preserve TDH-producing strains at higher levels than adults and that juveniles could preserve TDH-producing strains at higher levels than TDHnon-producing strains. MATERIALS AND METHODSGastropod: Adult and juvenile C. retropictus, 3.0-4.8 g and 0.2-0.9 g weight, respectively, were maintained in aquaria with tap water at 28°C for one week and confirmed non-detectable for V. parahaemolyticus before experiments.Bacteria: V. parahaemolyticus strains D-3 (TDHp r o d u c i n g ; n a l i d i x i c a c i d -r e s i s t a n t ) , N -1 8 (TDH-non-producing; nalidixic acid-resistant) and R-13 (TDH-non-producing; rifampicin-resistant) [9] were grown in Trypticase Soy Broth (Difco, U.S.A.) containing 1% NaCl at 37°C for 8 hr with shaking. The cells were washed with 20 artificial seawater [9] and suspended at approximately 10 6 cfu/ml in the artificial seawater. Inoculation: A hundred gastropods were incubated in 100 ml of the bacterial suspension containing a single strain in 20 artificial seawater at 25°C for 24 hr with aeration. Then they were washed with distilled water to remove the strains attached to the surface and maintained in aquaria with 30 l of UV-irradiated, recirculating, 20 artificial seawater at 28°C for up to 21 days. Cultured diatom algae, Navicula strain F-1 [2], were fed to the gastropods throughout the study. Every 10-15 gastropods were sacrificed at appropriate time intervals to obtain homogenates of the alimentary tracts attached to mid-gut Thermostable-direct hemolysin(TDH)-producing strain of Vibrio parahaemolyticus is a potential pathogen of human gastroenteritis. The strain was detected at the highest level of 10 5 colony forming units per gram (cfu/g) from a neritid gastropod (Clithon retropictus) at a site of the Hashizu estuary in Japan where juveniles, less than 1.0 g weight, were predominant in a C. retropictus community. It was not, however, detected at sit...

An Estuarine Neritid Gastropod, Clithon corona, a Potential Reservoir of Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus

“…1), suggesting that TDH-producing strains would accumulate in estuaries where C. corona resides, through repeated passages of the strains in the gut of C. corona. In our previous studies, a TDH-producing strain, D3, was found to survive in the gut of a juvenile estuarine neritid gastropod, C. retropictus, at a level 100 times higher than TDH-non-producing strain R13 [7]. Based on this evi- dence, TDH-producing strains were suspected to accumulate in estuaries containing juvenile C. retropictus [7].…”

“…The TDH-producing strains, D5 (O3:K6) and D12 (O3:K6), are nalidixic acid-resistant derivatives of clinical strains, while TDH-non-producing strains, R15 (O8:K41) and R13 (untypable), are rifampicin-resistant derivatives of environmental strains. The bacteria were grown overnight in Heart Infusion Broth (Eiken) supplemented with 3% NaCl at 37°C, washed with artificial seawater (ASW) [7] with a salinity of 35‰, and resuspended at 10 6 cfu/ml in ASW.The two neritid gastropods, C. corona and C. sowerbianus, were collected from Benoki and Ohura rivers in Okinawa, Japan. Adult and juvenile specimens, 9-13 mm and 5-7 mm in shell height, respectively, were cleansed of halophilic organisms of the gut by placement in recirculating freshwater at room temperature for one week.…”

“…67(8): 833-835, 2005 Vibrio parahaemolyticus is an autochthonous estuarine organism that can cause gastroenteritis in man after consumption of contaminated raw fish and shellfish [1,5,8] Another field survey at Sada Estuary found that TDH-producing vibrios grew in the gut of juvenile C. retropictus from a non-detectable level to 2.0 × 10 3 cfu/g (approximately 15% of V. parahaemolyticus organisms) in a twomonth period between July and September 1991 [3]. TDHproducing vibrios survived at a level of 10 4 -10 5 cfu/g maintained in UV-irradiated recirculating artificial seawater with a salinity of 20‰, while TDH-non-producing (non-pathogenic) vibrios survived there at a level of only 10 1 -10 2 cfu/ g [7]. V. parahaemolyticus can attach to estuarine diatom algae, Navicula spp.…”

“…TDHproducing vibrios survived at a level of 10 4 -10 5 cfu/g maintained in UV-irradiated recirculating artificial seawater with a salinity of 20‰, while TDH-non-producing (non-pathogenic) vibrios survived there at a level of only 10 1 -10 2 cfu/ g [7]. V. parahaemolyticus can attach to estuarine diatom algae, Navicula spp.…”

“…The TDH-producing strains, D5 (O3:K6) and D12 (O3:K6), are nalidixic acid-resistant derivatives of clinical strains, while TDH-non-producing strains, R15 (O8:K41) and R13 (untypable), are rifampicin-resistant derivatives of environmental strains. The bacteria were grown overnight in Heart Infusion Broth (Eiken) supplemented with 3% NaCl at 37°C, washed with artificial seawater (ASW) [7] with a salinity of 35‰, and resuspended at 10 6 cfu/ml in ASW.…”