Selective Growth of Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus in the Alimentary Tract of a Gastropod, Clithon Retropictus, at a Selected Estuary in Japan.

Self Cite

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...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Kumazawa

Kawasaki

1997

Self Cite

“…Sci. 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].…”

mentioning

confidence: 99%

“…V. parahaemolyticus was detected in its highest level, 10 4 -10 5 colony forming units (cfu)/g, in Clithon retropictus (neritid gastropod), attached-microalgae and muddy sediments of Hashizu and Sada Estuaries in Japan [3,5]. Thermostable direct hemolysin (TDH)-producing (enteropathogenic) vibrios were found to be approximately 20% of V. parahaemolyticus organisms isolated from C. retropictus at Hashizu Estuary, though the occurrence in muddy sediments and water there was 12% and 1%, respectively [5].…”

mentioning

confidence: 99%

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

Velammal

Kato

Miyagi

et al. 2005

Self Cite

ABSTRACT. An estuarine neritid gastropod, Clithon corona, maintained in UV-irradiated recirculating artificial seawater with a salinity of 15 per mil (‰) was found to retain thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus in the gut at significantly higher levels than TDH-non-producing one for at least 14 days. Another estuarine neritid gastropod, C. sowerbianus, was not able to support the preferential survival of TDH-producing organisms. This evidence suggests that, if TDH-producing vibrios are brought to estuaries inhabited by C. corona, repeated ingestion of V. parahaemolyticus by this gastropod could lead to accumulation of TDH-producing vibrios in the estuaries. KEY WORDS: Clithon corona, reservoir, V. parahaemolyticus.J. Vet. Med. Sci. 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. regardless of TDH-productivity [2]. This evidence suggests that V. parahaemolyticus would overwinter in muddy sediments at estuaries, grow there with rising water temperature, be released into the water column and attach to diatom algae, where it would be ingested by juvenile C. retropictus, thereby supporting selective growth of TDH-producing vibrios in the gut.The range of C. retropictus includes many estuaries of the main land of Southern Japan [6], though V. parahaemolyticus-gastroenteritis has been reported throughout the world including Southeast Asia [8]. Two neritid gastropods, C. corona and C. sowerbianus, predominate in the estuaries of the Southwest Islands (subtropical zone) of Japan, where C. retropictus seems to take refuge in freshwater areas above estuaries in competition with these gastropods [10]. A halophile, V. parahaemolyticus, could not survive in the gut of C. retropictus which inhabits freshwater areas. This raises the possibility that C. corona and C. sowerbianus are the reservoirs for TDH-producing strains of V. parahaemolyticus in the estuaries of tropical and sub-tropical zones.In the present study, two TDH-producing and two TDHnon-producing strains of V. parahaemolyticus were investigated. 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 gr...

“…Sci. 63(12): 1257-1261, 2001 An estuarine neritid gastropod, Clithon retropictus, is an important reservoir of an enteropathogenic organism, thermostable direct hemolysin (TDH)-producing strain of Vibrio parahaemolyticus [3,4]. TDH-producing strain D3 was found to survive at a level of 10 4 -10 5 and 10 0 viable units/g for up to 21 days in the alimentary tract of juvenile and adult C. retropictus, respectively, ingested the strain and maintained in ultraviolet (UV)-irradiated recirculating artificial seawater with a salinity of 20‰ at 28°C [5].…”

mentioning

confidence: 99%

Migratory Responses of Hemocytes to Vibrio parahaemolyticus in the Alimentary Tract of an Estuarine Neritid Gastropod, Clithon retropictus.

Kumazawa

Minei

2001

Self Cite

ABSTRACT. Migratory responses of hemocytes to Vibrio parahaemolyticus strain D3 in the alimentary tracts of an estuarine neritid gastropod, Clithon retropictus, and a related marine neritid, Nerita albicilla, were examined under the scanning electron microscope. After ingesting the strain, active responses were seen at the esophagus, stomach and anterior intestine of adult C. retropictus and at the middle and posterior intestines of adult N. albicilla. When the alimentary tracts were isolated from the gastropod and incubated in vitro with strain D3, active response was induced at the most parts of the tract of the adult gastropods and at the stomach and the anterior intestine of juvenile C. retropictus. The responding hemocytes were confirmed to be granulocytes in the semi-thin sections of the tract of adult C. retropictus. The poor hemocyte responses at the middle and posterior intestines of juvenile C. retropictus might support the colonization of the organism there. KEY WORDS: alimentary tract, Clithon retropictus, hemocyte, migration, Vibrio parahaemolyticus.J. Vet. Med. Sci. 63(12): 1257-1261, 2001 An estuarine neritid gastropod, Clithon retropictus, is an important reservoir of an enteropathogenic organism, thermostable direct hemolysin (TDH)-producing strain of Vibrio parahaemolyticus [3,4]. TDH-producing strain D3 was found to survive at a level of 10 4 -10 5 and 10 0 viable units/g for up to 21 days in the alimentary tract of juvenile and adult C. retropictus, respectively, ingested the strain and maintained in ultraviolet (UV)-irradiated recirculating artificial seawater with a salinity of 20‰ at 28°C [5]. However, the strain was eliminated from 2 marine neritid gastropods, Nerita albicilla and Heminerita japonica, maintained in 35‰ artificial seawater within 4 days [8,10]. Hemocytes of adult C. retropictus and adult and juvenile N. albicilla were attracted to strain D3 chemotactically in modified Chernin's balanced salt solution (CBSS), which was enhanced significantly in the presence of the respective molluscan plasma [6,9]. In contrast, hemocytes of juvenile C. retropictus were attracted to strain D3 chemotactically in the presence of C. retropictus plasma but not in the absence of the plasma [6]. From these findings, V. parahaemolyticus was expected to induce active migratory responses of the hemocytes at the epithelia of the alimentary tract in adult and juvenile N. albicilla and adult C. retropictus, but not in juvenile C. retropictus. The present study characterizes the hemocyte response to V. parahaemolyticus in the alimentary tract of juvenile C. retropictus. MATERIALS AND METHODSAdult and juvenile C. retropictus, 12-20 and 5-8 mm in shell height, respectively, were collected at estuaries in Okinawa Island. Adult N. albicilla, 10-15 mm in shell height, was collected at the coral reef of Okinawa Island. C. retropictus and N. albicilla were maintained in UV-irradiated recirculating conditions of fresh water and 35‰ artificial seawater [3,7], respectively, at room temperature for 7 days and confi...