Epidemiology and pathogenesis of Vibrio vulnificus

Microbes and Infection

Volumn 2, Issue 2, 2000, Pages 177-188

  Strom, Mark S ^a   Paranjpye, Rohinee N ^a  

^a NORTHWEST FISHERIES SCIENCE CENTER   (United States)

Author keywords

Bacterial pathogenesis; Cytokine induction; Invasive disease; Iron; Oysters; Pili; Toxins

Indexed keywords

BACTERIAL POLYSACCHARIDE; BACTERIAL TOXIN; ENTEROTOXIN; HEMOLYSIN;

BACTERIAL VIRULENCE; BACTERIUM PILUS; CLINICAL FEATURE; ENVIRONMENTAL FACTOR; FOOD CONTAMINATION; GASTROENTERITIS; GENE EXPRESSION REGULATION; GEOGRAPHIC DISTRIBUTION; GRAM NEGATIVE INFECTION; HOST SUSCEPTIBILITY; HUMAN; IRON METABOLISM; LIFE CYCLE; NONHUMAN; OYSTER; PRIORITY JOURNAL; REVIEW; SEPTICEMIA; STRAIN DIFFERENCE; TOXIN SYNTHESIS; VIBRIO VULNIFICUS; WOUND INFECTION;

BACTERIA (MICROORGANISMS); NEGIBACTERIA; OSTREIDAE; VIBRIO VULNIFICUS;

EID: 0034100450     PISSN: 12864579     EISSN: None     Source Type: Journal    
DOI: 10.1016/S1286-4579(00)00270-7     Document Type: Review

References (109)

1. Bonner J.R., Coker A.S., Berryman C.R., Pollock H.M. Spectrum of Vibrio infections in a Gulf Coast community. Ann. Intern. M. 99:1983;464.
2. Hlady W.G. Vibrio infections associated with raw oyster consumption in Florida, 1981-1994. J. Food Prot. 60:1997;353.
3. Morris J.G. Jr,. Vibrio vulnificus - a new monster of the deep? Ann. Intern. M. 109:1988;261.
4. Morris J.G. Jr, Black R.E. Cholera and other Vibrioses in the United States. N. Engl. J. M. 312:1985;343.
5. Mackenzie C.L.J., Burrell V.G.J., Rosenfield A., Hobart W.L. The History, Present Condition, and Future of the Molluscan Fisheries of North and Central America and Europe. Volume 1. Atlantic and Gulf Coasts. NOAA Technical Report NMFS 127. Vol. 1:1997;234. US Department of Commerce.
6. Blake P.A., Merson M.H., Weaver R.E., Hollis D.G., Heublein P.C. Disease caused by a marine Vibrio. Clinical characteristics and epidemiology. N. Engl. J. M. 300:1979;1.
7. Hollis D.G., Weaver R.E., Baker C.N., Thornsberry C. Halophilic Vibrio species isolated from blood cultures. J. Clin. Microbiol. 3:1976;425.
8. Dalsgaard A., Frimodt-Moller N., Bruun B., Hoi L., Larsen J.L. Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark, Eur. J. Clin. Microbiol. Infect. Dis. 15:1996;227.
9. Hoi L., Larsen J.L., Dalsgaard I., Dalsgaard A. Occurrence of Vibrio vulnificus biotypes in Danish marine environments. Appl. Environ. Microbiol. 64:1998;7.
10. Maxwell E.L., Mayall B.C., Pearson S.R., Stanley P.A. A case of Vibrio vulnificus septicaemia acquired in Victoria. Med. J. Aust. 154:1991;214.
11. Myatt D.C., Davis G.H. Isolation of medically significant Vibrio species from riverine sources in south east Queensland. Microbios. 60:1989;111.
12. Shapiro R.L., Altekruse S., Hutwagner L., Bishop R., Hammond R., Wilson S., Ray B., Thompson S., Tauxe R.V., Griffin P.M. The role of Gulf Coast oysters harvested in warmer months in Vibrio vulnificus infections in the United States, 1988-1996. J. Infect. Dis. 178:1998;752.
13. Veenstra J., Rietra P.J., Coster J.M., Slaats E., Dirks-Go S. Seasonal variations in the occurrence of Vibrio vulnificus along the Dutch coast. Epidemiol. Infect. 112:1994;285.
14. Hlady W.G., Klontz K.C. The epidemiology of Vibrio infections in Florida, 1981-1993. J. Infect. Dis. 173:1996;1176.
15. Kaspar C.W., Tamplin M.L. Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish. Appl. Environ. Microbiol. 59:1993;2425.
16. Kaysner C.A., Abeyta C. Jr, Wekell M.M., De Paola A. Jr, Stott R.F., Leitch J.M. Incidence of Vibrio cholerae from estuaries of the United States West Coast. Appl. Environ. Microbiol. 53:1987;1344.
17. Kaysner C.A., Abeyta C. Jr, Wekell M.M., De Paola A. Jr, Stott R.F., Leitch J.M. Virulent strains of Vibrio vulnificus isolated from estuaries of the United States West Coast. Appl. Environ. Microbiol. 53:1987;1349.
18. O'Neill K.R., Jones S.H., Grimes D.J. Seasonal incidence of Vibrio vulnificus in the Great Bay estuary of New Hampshire and Maine. Appl. Environ. Microbiol. 58:1992;3257.
19. Motes M.L., De Paola A., Cook D.W., Veazey J.E., Hunsucker J.C., Garthright W.E., Blodgett R.J., Chirtel S.J. Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica). Appl. Environ. Microbiol. 64:1998;1459.
20. Motes M.L., De Paola A. Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica). Appl. Environ. Microbiol. 62:1996;3875.
21. Oliver J.D., Warner R.A., Cleland D.R. Distribution and ecology of Vibrio vulnificus and other lactose- fermenting marine Vibrios in coastal waters of the southeastern United States. Appl. Environ. Microbiol. 44:1982;1404.
22. Oliver J.D., Hite F., McDougald D., Andon N.L., Simpson L.M. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl. Environ. Microbiol. 61:1995;2624.
23. Linder K., Oliver J.D. Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. Appl. Environ. Microbiol. 55:1989;2837.
24. Weichart D., Kjelleberg S. Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus. Microbiology. 142:1996;845.
25. Weichart D., McDougald D., Jacobs D., Kjelleberg S. In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus. Appl. Environ. Microbiol. 63:1997;2754.
26. Oliver J.D., Bockian R. In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl. Environ. Microbiol. 61:1995;2620.
27. De Paola A., Capers G.M., Alexander D. Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast. Appl. Environ. Microbiol. 60:1994;984.
28. Jones S.H., Howell T.L., O'Neill K.R. Differential elimination of indicator bacteria and pathogenic Vibrio spp. from Eastern oysters (Crassostrea virginica Gmelin, 1791) in a commercial controlled purification facility in Maine. J. Shellfish Res. 10:1991;105.
29. Tamplin M.L., Capers G.M. Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light. Appl. Environ. Microbiol. 58:1992;1506.
30. Tison D.L., Nishibuchi M., Greenwood J.D., Seidler R.J. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl. Environ. Microbiol. 44:1982;640.
31. Austin B., Austin D.A. Bacterial fish pathogens : disease of farmed and wild fish. 3rd rev :1999;New York: Springer.
32. Amaro C., Biosca E.G., Fouz B., Toranzo A.E., Garay E. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect. Immun. 62:1994;759.
33. Amaro C., Biosca E.G. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl. Environ. Microbiol. 62:1996;1454.
34. Veenstra J., Rietra P.J., Goudswaard J., Kaan J.A., Van-Keulen P.H., Stoutenbeek C.P. [Extra-intestinal infections caused by Vibrio spp. in The Netherlands]. Ned. Tijdschr. Geneeskd. 137:1993;654.
35. Klontz K.C., Lieb S., Schreiber M., Janowski H.T., Baldy L.M., Gunn R.A. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981-1987. Ann. Intern. M. 109:1988;318.
36. Tacket C.O., Brenner F., Blake P.A. Clinical features and an epidemiological study of Vibrio vulnificus infections. J. Infect. Dis. 149:1984;558.
37. Koenig K.L., Mueller J., Rose T. Vibrio vulnificus. Hazard on the half shell. West. J. M. 155:1991;400.
38. Jackson J.K., Murphree R.L., Tamplin M.L. Evidence that mortality from Vibrio vulnificus infection results from single strains among heterogeneous populations in shellfish. J. Clin. Microbiol. 35:1997;2098.
39. Buchrieser C., Gangar V.V., Murphree R.L., Tamplin M.L., Kaspar C.W. Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis. Appl. Environ. Microbiol. 61:1995;1163.
40. Arias C.R., Pujalte M.J., Garay E., Aznar R. Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR. Appl. Environ. Microbiol. 64:1998;3403.
41. Hoi L., Dalsgaard A., Larsen J.L., Warner J.M., Oliver J.D. Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus. Appl. Environ. Microbiol. 63:1997;1674.
42. Warner J.M., Oliver J.D. Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other Vibrio species. Appl. Environ. Microbiol. 65:1999;1141.
43. Stelma G.N. Jr, Reyes A.L., Peeler J.T., Johnson C.H., Spaulding P.L. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl. Environ. Microbiol. 58:1992;2776.
44. Tison D.L., Kelly M.T. Virulence of Vibrio vulnificus strains from marine environments. Appl. Environ. Microbiol. 51:1986;1004.
45. Krovacek K., Baloda S.B., Dumontet S., Mansson I. Detection of potential virulence markers of Vibrio vulnificus strains isolated from fish in Sweden, Comp. Immunol. Microbiol. Infect. Dis. 17:1994;63.
46. Moreno M.L., Landgraf M. Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood. J. Appl. Microbiol. 84:1998;747.
47. Morris J.G.J., Wright A.C., Simpson L.M., Wood P.K., Johnson D.E., Oliver J.D. Virulence of Vibrio vulnificus: association with utilization of transferrin-bound iron, and lack of correlation with levels of cytotoxin or protease production. FEMS Microbiol. Lett. 40:1987;55.
48. Rodrigues D.P., Ribeiro R.V., Hofer E. Analysis of some virulence factors of Vibrio vulnificus isolated from Rio de Janeiro, Brazil. Epidemiol. Infect. 108:1992;463.
49. Wright A.C., Simpson L.M., Oliver J.D., Morris J.G. Jr.,. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect. Immun. 58:1990;1769.
50. Yoshida S., Ogawa M., Mizuguchi Y. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect. Immun. 47:1985;446.
51. Tamplin M.L. The ecology of Vibrio vulnificus. Proceedings of the 1994Vibrio vulnificusWorkshop. 1995;75 U. S. Food and Drug Administration, Washington, DC.
52. Zuppardo A.B., Siebeling R.J. An epimerase gene essential for capsule synthesis in Vibrio vulnificus. Infect. Immun. 66:1998;2601.
53. Zuppardo A.B., Siebeling R.J. Identification of capsule genes of Vibrio vulnificus, B/D-324. American Society for Microbiology 99th General Meeting. 1999;93 Chicago, IL, ASM.
54. Wright A.C., Powell J.L., Whitehead L.A., Kaper J.B., Morris J.G. The wza gene of Vibrio vulnificus is required for capsular polysaccharide outer membrane transport and virulence, B/D-328. American Society for Microbiology 99th General Meeting. 1999;94 Chicago, IL, ASM.
55. Johnson D.E., Calia F.M., Musher D.M., Goree A. Resistance of Vibrio vulnificus to serum bactericidal and opsonizing factors: relation to virulence in suckling mice and humans. J. Infect. Dis. 150:1984;413.
56. Tamplin M.L., Specter S., Rodrick G.E., Friedman H. Differential complement activation and susceptibility to human serum bactericidal action by Vibrio species. Infect. Immun. 42:1983;1187.
57. Tamplin M.L., Specter S., Rodrick G.E., Friedman H. Vibrio vulnificus resists phagocytosis in the absence of serum opsonins. Infect. Immun. 49:1985;715.
58. Powell J.L., Wright A.C., Wasserman S.S., Hone D.M., Morris J.G. Jr.,. Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models. Infect. Immun. 65:1997;3713.
59. Devi S.J., Hayat U., Powell J.L., Morris J.G. Jr.,. Preclinical immunoprophylactic and immunotherapeutic efficacy of antisera to capsular polysaccharide-tetanus toxoid conjugate vaccines of Vibrio vulnificus. Infect. Immun. 64:1996;2220.
60. Hayat U., Reddy G.P., Bush C.A., Johnson J.A., Wright A.C., Morris J.G. Jr.,. Capsular types of Vibrio vulnificus: an analysis of strains from clinical and environmental sources. J. Infect. Dis. 168:1993;758.
61. Bush C.A., Patel P., Gunawardena S., Powell J., Joseph A., Johnson J.A., Morris J.G. Classification of Vibrio vulnificus strains by the carbohydrate composition of their capsular polysaccharides. Anal. Biochem. 250:1997;186.
62. Wright A.C., Powell J.L., Tanner M.K., Ensor L.A., Karpas A.B., Morris J.G. Jr., Sztein M.B. Differential expression of Vibrio vulnificus capsular polysaccharide. Infect. Immun. 67:1999;2250.
63. McPherson V.L., Watts J.A., Simpson L.M., Oliver J.D. Physiological effects of the lipopolysaccharide of Vibrio vulnificus on mice and rats. Microbios. 67:1991;141.
64. Espat N.J., Auffenberg T., Abouhamze A., Baumhofer J., Moldawer L.L., Howard R.J. A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction. Ann. Surg. 223:1996;428.
65. Strauss K.A., Trigiani E.R., Morris J.G. Jr., Powell J.L. Purification and biological activity of Vibrio vulnificus and Vibrio cholerae O139 lipopolysaccharide extracted using the PCP method, B/D-49. American Society for Microbiology 99th General Meeting. 1999;38 Chicago, IL, ASM.
66. Amaro C., Fouz B., Biosca E.G., Marco-Noales E., Collado R. The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels. Infect. Immun. 65:1997;2475.
67. Strom M.S., Lory S. Structure-function and biogenesis of the type IV pili. Annu. Rev. Microbiol. 47:1993;565.
68. Gander R.M., Larocco M.T. Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus. J. Clin. Microbiol. 27:1989;1015.
69. Paranjpye R.N., Lara J.C., Pepe J.C., Pepe C.M., Strom M.S. The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice. Infect. Immun. 66:1998;5659.
70. Pepe C.M., Eklund M.W., Strom M.S. Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion. Mol. Microbiol. 19:1996;857.
71. Fussenegger M., Rudel T., Barten R., Ryll R., Meyer T.F. Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae - a review. Gene. 192:1997;125.
72. Kallstrom H., Islam M.S., Berggren P.O., Jonsson A.B. Cell signaling by the type IV pili of pathogenic Neisseria. J. Biol. Chem. 273:1998;21777.
73. Mattick J.S., Whitchurch C.B., Alm R.A. The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa - a review. Gene. 179:1996;147.
74. Tonjum T., Koomey M. The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships - a review. Gene. 192:1997;155.
75. Waldor M.K., Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 272:1996;1910.
76. Lory S., Strom M.S. Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa - a review. Gene. 192:1997;117.
77. Gray L.D., Kreger A.S. Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect. Immun. 48:1985;62.
78. Gray L.D., Kreger A.S. Mouse skin damage caused by cytolysin from Vibrio vulnificus and by V. vulnificus infection. J. Infect. Dis. 155:1987;236.
79. Kook H., Lee S.E., Baik Y.H., Chung S.S., Rhee J.H. Vibrio vulnificus hemolysin dilates rat thoracic aorta by activating guanylate cyclase. Life Sci. 59:1996;L41.
80. Kook H., Rhee J.H., Lee S.E., Kang S.Y., Chung S.S., Cho K.W., Baik Y.H. Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin. Eur. J. Pharmacol. 365:1999;267.
81. Yamamoto K., Wright A.C., Kaper J.B., Morris J.G. Jr.,. The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene. Infect. Immun. 58:1990;2706.
82. Gray L.D., Kreger A.S. Detection of anti-Vibrio vulnificus cytolysin antibodies in sera from mice and a human surviving V. vulnificus disease. Infect. Immun. 51:1986;964.
83. Wright A.C., Morris J.G. Jr,. The extracellular cytolysin of Vibrio vulnificus: inactivation and relationship to virulence in mice. Infect. Immun. 59:1991;192.
84. Chang T.M., Chuang Y.C., Su J.H., Chang M.C. Cloning and sequence analysis of a novel hemolysin gene (vllY) from Vibrio vulnificus. Appl. Environ. Microbiol. 63:1997;3851.
85. Kothary M.H., Kreger A.S. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J. Gen. Microbiol. 133:1987;1783.
86. Miyoshi N., Shimizu C., Miyoshi S., Shinoda S. Purification and characterization of Vibrio vulnificus protease. Microbiol. Immunol. 31:1987;13.
87. Smith G.C., Merkel J.R. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect. Immun. 35:1982;1155.
88. Miyoshi S., Nakazawa H., Kawata K., Tomochika K., Tobe K., Shinoda S. Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family. Infect. Immun. 66:1998;4851.
89. Chuang Y.C., Chang T.M., Chang M.C. Cloning and characterization of the gene (empV ) encoding extracellular metalloprotease from Vibrio vulnificus. Gene. 189:1997;163.
90. Cheng J.C., Shao C.P., Hor L.I. Cloning and nucleotide sequencing of the protease gene of Vibrio vulnificus. Gene. 183:1996;255.
91. Miyoshi S., Wakae H., Tomochika K., Shinoda S. Functional domains of a zinc metalloprotease from Vibrio vulnificus. J. Bacteriol. 179:1997;7606.
92. Shao C.P., Hor L.I. Characterization of metalloprotease-deficient Vibrio vulnificus mutant generated by allelic exchange technique, B/D-314. American Society for Microbiology 99th General Meeting. 1999;91 Chicago, IL, ASM.
93. Stelma G.N., Spaulding P.L., Reyes A.L., Johnson C.H. Production of enterotoxin by Vibrio vulnificus. J. Food Protect. 51:1988;192.
94. Somerville C.C., Colwell R.R. Sequence analysis of the beta-N-acetylhexosaminidase gene of Vibrio vulnificus: evidence for a common evolutionary origin of hexosaminidases. Proc. Natl. Acad. Sci. USA. 90:1993;6751.
95. Wortman A.T., Somerville C.C., Colwell R.R. Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe. Appl. Environ. Microbiol. 52:1986;142.
96. Testa J., Daniel L.W., Kreger A.S. Extracellular phospholipase A2 and lysophospholipase produced by Vibrio vulnificus. Infect. Immun. 45:1984;458.
97. Martinez J.L., Delgado-Iribarren A., Baquero F. Mechanisms of iron acquisition and bacterial virulence, FEMS. Microbiol. Rev. 6:1990;45.
98. Otto B.R., Verweij-Van Vught A.M., Maclaren D.M. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit. Rev. Microbiol. 18:1992;217.
99. Wright A.C., Simpson L.M., Oliver J.D. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect. Immun. 34:1981;503.
100. Simpson L.M., Oliver J.D. Ability of Vibrio vulnificus to obtain iron from transferrin and other iron-binding compounds. Curr. Microbiol. 15:1987;155.
101. Helms S.D., Oliver J.D., Travis J.C. Role of heme compounds and haptoglobin in Vibrio vulnificus pathogenicity. Infect. Immun. 45:1984;345.
102. Simpson L.M., Oliver J.D. Siderophore production by Vibrio vulnificus. Infect. Immun. 41:1983;644.
103. Litwin C.M., Rayback T.W., Skinner J. Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect. Immun. 64:1996;2834.
104. Okujo N., Akiyama T., Miyoshi S., Shinoda S., Yamamoto S. Involvement of vulnibactin and exocellular protease in utilization of transferrin- and lactoferrin-bound iron by Vibrio vulnificus. Microbiol. Immunol. 40:1996;595.
105. Litwin C.M., Calderwood S.B. Role of iron in regulation of virulence genes. Clin. Microbiol. Rev. 6:1993;137.
106. Litwin C.M., Calderwood S.B. Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J. Bacteriol. 175:1993;706.
107. Litwin C.M., Byrne B.L. Cloning and characterization of an outer membrane protein of Vibrio vulnificus required for heme utilization: regulation of expression and determination of the gene sequence. Infect. Immun. 66:1998;3134.
108. De Paola A., McLeroy S., McManus G. Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats. Appl. Environ. Microbiol. 63:1997;2464.
109. De Paola A., Motes M.L., Chan A.M., Suttle C.A. Phages infecting Vibrio vulnificus are abundant and diverse in oysters (Crassostrea virginica) collected from the Gulf of Mexico. Appl. Environ. Microbiol. 64:1998;346.