World
Journal
of Microbiology
Technical
and Biotechnology,
9, 390-392
Report
Detection of Vibrio c/w/erae 01 in the aquatic environment in Brazil employing direct immunofluorescence microscopy M.T. Martins, P.S. Sanchez, M.I.Z. Sato, P.R. Brayton and RR. Colwell* Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae 01 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. choferae 01 by FA but none was positive by culture, although strains of V. cholerae other than 01 strains were readily isolated. Evidence for V. cholerae 01 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment. Key words: Bacteria, cholera, environment,
immunofluorescence,
The seventh cholera pandemic presented a recrudescence in 1991, when the disease broke out in epidemic form in Latin America. The Latin American epidemic of cholera began in Peru in January, 1991, and spread to neighbouring coastal countries of Latin America. By the end of 1991, at least 14 countries were involved and 342,25 1 cases had been reported, with 3602 deaths (Anon. 1991). Among the measures utilized in cholera surveillance, monitoring of water and sewage is important, not only for epidemiological purposes but also for assessment of risk of the disease (Gangarosa & Mosley 1974; Isaacson et aI. 1974; Colwell et a/. 1981; Bourke et al. 1986; McFarland ef al. 1986; Brayton ef al. 1’987). In the early 1970s, cholera spread to two countries in Europe and nine in Africa. The forecast at that time was the possibility of cholera occurring, not only in areas of Africa and Europe, but also in territories across the Atlantic (Kamal 1974; McFarland et al. 1986). Programmes for cholera surveillance were established in Sao Paulo State, Brazil, in South America, including monitoring of sewage water samples to assess the occurrence and distribution of V. cbolerae 01 in the community (Martins ef al. 1991). M.T. Martins is with the Department of Microbiology, I.C.B. II, University of Sao Paulo, Sao Paulo, S.P. CEP 05508, Brazil. P.S. Sanchez and M.I.Z. Sato are with the State Agency for Environmental Control-CETESB, Sao Paula, S.P. CEP 05459, Brazil. P.R. Brayton and RR. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA; fax: 301-454-8123. RR. Colwell is also with the Center of Marine Biotechnology, Maryland Biotechnology Institute, 600 East Lombard St, Baltimore, MD 21202, USA. *Corresponding author.
@ 1993 Rapid Communications
of Oxford
Ltd
Vibrio, water. Isolation of V. cholerae 01 from the aquatic environment is difficult and, in general, attempts at isolation of the organism from environmental samples brought to the laboratory are not successful (Colwell ef al. 1981; Huq ef ul. 1983, 1990). However, failure ‘to detect Vibrio cholerae 01 in environmental water samples can be explained, at least in part, by the capability of the microorganism to undergo an environmental adaptation (‘dormancy’) which makes its recovery, by methods dependent on culturing, difficult (Xu et al. 1983; Roszak & Colwell 1987; Huq ef ul. 1990). During the programme of research reported here, the World Health Organization requested the assistance of one of us (RRC) in a study, beginning in 1982, to determine whether V. cbolerae 01 could indeed be detected in environmental samples collected in Brazil. During the course of that study, a comparison of culture methods with the fluorescent antibody staining procedure to confirm the potential of V. cbolerae 01 occurrence in Brazil was undertaken, the results of which are reported here.
Materials
and Methods
The initial sample collection was done in September 1982 Sampling stations were located in the southeast of Sao Paula State, Brazil, at about 46.S”W and 23.5’S, on and near the Atlantic Coast. Freshwater samples were obtained from five sites in Billings Reservoir, a source of raw water for a conventional water treatment plant in the city of Sao Bernard0 do Campo, about 40 km from
Vibrio Sao Paul0 City. Seawater and plankton were collected from the beach of Sao Lourenco in Bertioga, at 46.3’W and 24’S, At the time of sampling, this beach was classified as unpolluted, with faecal coliform levels measured by the MF test at 30 c.f.u,/lOO ml, Samples of sewage were collected in the network system of Santos City, a coastal city located 80 km from the city of Sao Paulo, at 46.4’W and 23.9’S For sewage samples, eight sites, four on Jurubatuba Street, Sao Paula City, and four, including an inspection well, inside the Botanical Garden, Santos (Orquidario), were selected. The sites in the Botanical Garden were selected because we had successfully isolated V. &&me 01 from sewage samples
collected at this site in earlier studies (Martins ef ~1. 1991). Sampling stations for sewage and seawater were chosen because V. &&rue
01 had been reported
to occur
in sewage
waters,
hence
examination of such samples was included in this study (Martins et ul.
1991).
Also,
seawater
samples
were collected for study during 1982 from water Beach and verification of this
because V. cho/erae 01 had been isolated samoles collected at Sao Lourenco isolation was needed. The range of sample volumes depending on the type of sample, water. 1
varied from 1 to 1000 ml, i.e. whether sewage or natural
Andysis o~enz~irovmental sumples~or defecfim of Vibrio cholerae 01 Water (1 to 10 ml volumes) and concentrated plankton samples were inoculated directly into alkaline peptone water/bile/tellurite enrichment broth (APWBT) of the following composition: 10 g peptoneil; 5 g ox-bile/l; 0.0005% (w/v) potassium tellurite; pH 8.6. Larger samples, 100 to 1000 ml, were filtered through a 0.2 pm pore filter and the filters (MF) were placed into APWBT enrichment broth. Cultures were maintained at room temperature for 2 h, after which they were incubated at 37’C overnight. The following day, loopfuls of the top I cm of enrichment media were transferred to TCBS agar (Difco) and incubated for 24 h at 37’C. A second loopful was transferred to a marked area on a microscope slide, allowed to dry, fixed with 1 drop of 95% (w/v) ethanol, again air dried, and subsequently examined by immunofluorescence microscopy at the University of Maryland (Brayton & Colwell 1987). All yellow colonies on TCBS agar showing a smooth convex edge with halo were purified and the pure cultures characterized by a set of biochemical tests, according to West & Colwell (1984). Slide agglutination of V. chderne 01 isolates was performed using commercial polyclonal antisera (Difco Laboratories, Detroit, MI), The procedures followed for fluorescent antibody staining were those developed by Brayton & Colwell (1987) and Huq et a[. (1990).
Results
cholerae ia Brazil
by FA, whereas attempts to isolate V, cholerue 01 and non-01 by culturing were unsuccessful. Vibrio cholerue 01 was also detected in plankton samples by FA. These results were provided in a report to the World Health Organization (Colwell 1983). Of 19 samples analysed, 17 (90%) yielded V. cholerue 01 by fluorescent antibody staining, whereas all attempts to culture V. cholerue 01 were unsuccessful. These results were similar to those reported elsewhere (Brayton ef ul. 1987; Huq et ul. 1990). Th us, the superiority of FA for presumptive detection of V. cholerae in environmental water samples is corroborated by these findings and other data reported elsewhere (Xu et ul. 1983, 1984). Martins ef ul. (1991) carried out monitoring of sewage for V, chokrue from 1974 to 1983, examining a total of 12,867 samples of sewage water. Only four of this large number of samples were positive for V. cholerue 01 by culture methods. We conclude that V. cholerue 01 is frequently present in the aquatic environment either in a viable but non-culturable state or in numbers too small to detect by routine culture methods. Under conditions favourable for growth, such V. cholerue 01 may increase in number sufficient to pose an epidemic threat, as sugg.ested by human volunteer experiments (Colwell et ul. 1990). Findings similar to those reported here for V. cholerue 01 in environmental samples have been observed for other pathogenic bacteria, including Cumpylobder jejmi (Rollins & Colwell 1986) and .Suhnonek euferidifis (Roszak ef ul. 1984). We conclude from the results of this study that the FA technique, a sensitive, rapid and reliable method for detecting V. cholerue 01 and other pathogenic bacteria in the aquatic environment, can be useful in monitoring for V. cholerue in the environment. More importantly, from the results of this study, it is concluded that V. cholerae was present in the aquatic environment of Sao Paula, Brazil, prior to the present outbreak of cholera in that country. It is hypothesized that V. cholerue 01 is autochthonous to estuarine and brackish waters of Brazil. Further studies, which are in progress, will provide additional data to confirm this hypothesis.
and Discussion
Four of the five samples of freshwater from the Billings Reservoir presented positive results for V. cholerue 01 by FA. No strains of V. chokrue, 01 or non-01, were detected by culture in these samples. All samples of sewage water collected from the sites on Jurubatuba Street and in the Orquidario of Santos yielded V. cholerue non-01 by culture, confirmed by subsequent bacteriological analysis at the University of Maryland. Vibrio chderue 01 was detected by immunofluorescence microscopy in all of these samples, however. Three of four samples collected at Sao Lourenco Beach in Bertioga yielded low numbers of V, cholerue 01
Acknowledgements Travel support for R.R. Colwell to Brazil in September 1982, was provided by the World Health Organization, Geneva, making this collaboration possible. Assistance was provided by P. West in the confirmation of V. cholerue 01 and non-01, which is gratefully acknowledged. The research work was funded, in part, by National Science Foundation Grant No. BSR 84-03197 and Co-operative Agreement No. CR-81-2246-01-O between the Environmental Protection Agency and the University of Maryland,
World Jmmal of Microbmfogy and Bioteckmhgy,
Vol !? ?993
391
M.T. Mar-fins et al.
References Anon. 1991 CDC Update: Cholera-Western Hemisphere 1991. &forbidify and Morfalify World Reports 40, 860. Bourke, A.T.C., Cossins, Y.N., Gray, B.R.W., Hunney, T.J., Rostron, N.A., Holmes, R.V., Grigges, E.R., Larsen, D.J. & Kelk, V.R. 1986 Investigation of cholera acquired from the riverine environment in Queensland. Medical Journal of Australia 144, 229-234. Brayton, P.R. & Colwell, R.R. 1987 Fluorescent antibody staining method for enumeration of viable environmental Vibrio cholerae 01. ]ournal of Microbiological Mefhods, 6, 309-314. Brayton, P.R., Tamplin, M.L., Huq, A. & Colwell, R.R. 1987 Enumeration of V. cholerae 01 in Bangladesh waters by fluorescent-antibody direct viable count. Applied and Environmental Microbiology 53, 2862-2865. Colwell, R.R. 1983 Report to the World Health Organization, February 28, 1983. Geneva: World Health Organization. Colwell, R.R., Seidler, R.J., Kaper, J., Joseph, S.W., Garges, S., Lockman, H., Maneval, D., Bradford, H., Roberts, N., Remmers, E., Huq, I. & Huq, A. 1981 Occurrence of Vibrio cholerae serotype 01 in Maryland and Louisiana estuaries. Applied and Environmental Microbiology 41, 555-558. Colwell, R.R., Tamplin, M.L., Brayton, P.R., Gauzens, A.L., Tall, B.D., Herrington, D., Levine, M.M., Hall, S., Huq, A. & Sack, D.A. 1990 Environmental aspects of Vibrio cho!erae in transmission of cholera. Advances in Research on Cholera and Related Diarrheas 7, 327-343. Gangarosa, E.J. & Mosley, W.H. 1974 Epidemiology and surveillance of cholera. In Cholera, eds. Barua, D. & Burrows, W. pp. 381-403. London: W.B. Saunders. Huq, A., Colwell, R.R., Rahman, R., Ali, A., Chowdhury, M.A.R., Parveen, S., Sack, D.A. & Russek-Cohen, E. 1990 Detection of Vibrio cholerae 01 in the aquatic environment by fluorescentmonoclonal antibody and culture methods. Applied and Environmenfal Microbiology 56, 2370-2373. Huq, A., Small, E.B., West, P.A., Huq, M.I., Rahman, R. & Colwell, R.R. 1983 Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Applied and Environmental Microbiology 4.5, 275-283.
Isaacson, M., Clarke, K.R., Ellacombe, G.H., Smit, W.A., Smit, P., Koomhof, H.J., Smith, L.S. & Kriel, L.J. 1974 The recent outbreak in the South African gold mining industry: a preliminary report. South Africa Medical Journal 48, 2557-2560. Kamal, A.M. 1974 The seventh pandemic of cholera. In Cholera, eds. Barua, D. & Burrows, W. p. 9. London: W.B. Saunders. Martins, M.T., Pessoa, G.V.A., Sanchez, P.S., Sato, M.I.Z., Coimbrao, C.A., Monteiro, C.K. & Marques, E. 1991 Occurrence of V. cholerae 01 non-toxigenic in wastewaters from Sao Paula, Brazil. Water Science Technology 24, 363-366. McFarland, L., Bradford, H.B. & Matheson, J. 1986 Cholera in Louisiana update. Journal of the American Medical Association 256,
3080. Rollins, D.M. & Colwell, R.R. 1986 Viable but not nonculturable stage of Campylobacterjejuni and its role in survival in the aquatic environment. Applied and Environmental Microbiology 52,
531-538. Roszak, D.B. & Colwell, R.R. 1987 Survival strategies of bacteria in the environment. Microbiological Reviews 5 1, 465-479. Roszak, D.B., Grimes, D.J. & Colwell, R.R. 1984 Viable but non-recoverable stage of Salmonella enteritidis in aquatic systems. Canadian journal of Microbiology 30, 334-338. West, P.A. & Colwell, R.R. 1984 Identification and classification of Vibrionaceaean overview. In Vibrios in the Environmenf, ed. Colwell, R.R. pp. 285-363. New York: John Wiley & Sons. Xu, HS., Roberts, N.C., Adams, L.B., West, P.A., Siebling, R.J., Huq, A., Huq, M.I., Rahman, R. & Colwell, R.R. 1984 An indirect fluorescent antibody staining procedure for detection of Vibrio cholerae serovar 01 in aquatic environmental samples. Journal of Microbiological Mefhoak 2, 221-231. Xu, H.S., Roberts, N., Singleton, F.L., Attwell, R.W., Grimes, D.J. & Colwell, R.R. 1983 Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microbial Ecology 8, 213-223.
(Received 18 November 1992; accepfed 16 December 1992)
Journal
of Microbiology
Technical
and Biotechnology,
9, 390-392
Report
Detection of Vibrio c/w/erae 01 in the aquatic environment in Brazil employing direct immunofluorescence microscopy M.T. Martins, P.S. Sanchez, M.I.Z. Sato, P.R. Brayton and RR. Colwell* Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae 01 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. choferae 01 by FA but none was positive by culture, although strains of V. cholerae other than 01 strains were readily isolated. Evidence for V. cholerae 01 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment. Key words: Bacteria, cholera, environment,
immunofluorescence,
The seventh cholera pandemic presented a recrudescence in 1991, when the disease broke out in epidemic form in Latin America. The Latin American epidemic of cholera began in Peru in January, 1991, and spread to neighbouring coastal countries of Latin America. By the end of 1991, at least 14 countries were involved and 342,25 1 cases had been reported, with 3602 deaths (Anon. 1991). Among the measures utilized in cholera surveillance, monitoring of water and sewage is important, not only for epidemiological purposes but also for assessment of risk of the disease (Gangarosa & Mosley 1974; Isaacson et aI. 1974; Colwell et a/. 1981; Bourke et al. 1986; McFarland ef al. 1986; Brayton ef al. 1’987). In the early 1970s, cholera spread to two countries in Europe and nine in Africa. The forecast at that time was the possibility of cholera occurring, not only in areas of Africa and Europe, but also in territories across the Atlantic (Kamal 1974; McFarland et al. 1986). Programmes for cholera surveillance were established in Sao Paulo State, Brazil, in South America, including monitoring of sewage water samples to assess the occurrence and distribution of V. cbolerae 01 in the community (Martins ef al. 1991). M.T. Martins is with the Department of Microbiology, I.C.B. II, University of Sao Paulo, Sao Paulo, S.P. CEP 05508, Brazil. P.S. Sanchez and M.I.Z. Sato are with the State Agency for Environmental Control-CETESB, Sao Paula, S.P. CEP 05459, Brazil. P.R. Brayton and RR. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD 20742, USA; fax: 301-454-8123. RR. Colwell is also with the Center of Marine Biotechnology, Maryland Biotechnology Institute, 600 East Lombard St, Baltimore, MD 21202, USA. *Corresponding author.
@ 1993 Rapid Communications
of Oxford
Ltd
Vibrio, water. Isolation of V. cholerae 01 from the aquatic environment is difficult and, in general, attempts at isolation of the organism from environmental samples brought to the laboratory are not successful (Colwell ef al. 1981; Huq ef ul. 1983, 1990). However, failure ‘to detect Vibrio cholerae 01 in environmental water samples can be explained, at least in part, by the capability of the microorganism to undergo an environmental adaptation (‘dormancy’) which makes its recovery, by methods dependent on culturing, difficult (Xu et al. 1983; Roszak & Colwell 1987; Huq ef ul. 1990). During the programme of research reported here, the World Health Organization requested the assistance of one of us (RRC) in a study, beginning in 1982, to determine whether V. cbolerae 01 could indeed be detected in environmental samples collected in Brazil. During the course of that study, a comparison of culture methods with the fluorescent antibody staining procedure to confirm the potential of V. cbolerae 01 occurrence in Brazil was undertaken, the results of which are reported here.
Materials
and Methods
The initial sample collection was done in September 1982 Sampling stations were located in the southeast of Sao Paula State, Brazil, at about 46.S”W and 23.5’S, on and near the Atlantic Coast. Freshwater samples were obtained from five sites in Billings Reservoir, a source of raw water for a conventional water treatment plant in the city of Sao Bernard0 do Campo, about 40 km from
Vibrio Sao Paul0 City. Seawater and plankton were collected from the beach of Sao Lourenco in Bertioga, at 46.3’W and 24’S, At the time of sampling, this beach was classified as unpolluted, with faecal coliform levels measured by the MF test at 30 c.f.u,/lOO ml, Samples of sewage were collected in the network system of Santos City, a coastal city located 80 km from the city of Sao Paulo, at 46.4’W and 23.9’S For sewage samples, eight sites, four on Jurubatuba Street, Sao Paula City, and four, including an inspection well, inside the Botanical Garden, Santos (Orquidario), were selected. The sites in the Botanical Garden were selected because we had successfully isolated V. &&me 01 from sewage samples
collected at this site in earlier studies (Martins ef ~1. 1991). Sampling stations for sewage and seawater were chosen because V. &&rue
01 had been reported
to occur
in sewage
waters,
hence
examination of such samples was included in this study (Martins et ul.
1991).
Also,
seawater
samples
were collected for study during 1982 from water Beach and verification of this
because V. cho/erae 01 had been isolated samoles collected at Sao Lourenco isolation was needed. The range of sample volumes depending on the type of sample, water. 1
varied from 1 to 1000 ml, i.e. whether sewage or natural
Andysis o~enz~irovmental sumples~or defecfim of Vibrio cholerae 01 Water (1 to 10 ml volumes) and concentrated plankton samples were inoculated directly into alkaline peptone water/bile/tellurite enrichment broth (APWBT) of the following composition: 10 g peptoneil; 5 g ox-bile/l; 0.0005% (w/v) potassium tellurite; pH 8.6. Larger samples, 100 to 1000 ml, were filtered through a 0.2 pm pore filter and the filters (MF) were placed into APWBT enrichment broth. Cultures were maintained at room temperature for 2 h, after which they were incubated at 37’C overnight. The following day, loopfuls of the top I cm of enrichment media were transferred to TCBS agar (Difco) and incubated for 24 h at 37’C. A second loopful was transferred to a marked area on a microscope slide, allowed to dry, fixed with 1 drop of 95% (w/v) ethanol, again air dried, and subsequently examined by immunofluorescence microscopy at the University of Maryland (Brayton & Colwell 1987). All yellow colonies on TCBS agar showing a smooth convex edge with halo were purified and the pure cultures characterized by a set of biochemical tests, according to West & Colwell (1984). Slide agglutination of V. chderne 01 isolates was performed using commercial polyclonal antisera (Difco Laboratories, Detroit, MI), The procedures followed for fluorescent antibody staining were those developed by Brayton & Colwell (1987) and Huq et a[. (1990).
Results
cholerae ia Brazil
by FA, whereas attempts to isolate V, cholerue 01 and non-01 by culturing were unsuccessful. Vibrio cholerue 01 was also detected in plankton samples by FA. These results were provided in a report to the World Health Organization (Colwell 1983). Of 19 samples analysed, 17 (90%) yielded V. cholerue 01 by fluorescent antibody staining, whereas all attempts to culture V. cholerue 01 were unsuccessful. These results were similar to those reported elsewhere (Brayton ef ul. 1987; Huq et ul. 1990). Th us, the superiority of FA for presumptive detection of V. cholerae in environmental water samples is corroborated by these findings and other data reported elsewhere (Xu et ul. 1983, 1984). Martins ef ul. (1991) carried out monitoring of sewage for V, chokrue from 1974 to 1983, examining a total of 12,867 samples of sewage water. Only four of this large number of samples were positive for V. cholerue 01 by culture methods. We conclude that V. cholerue 01 is frequently present in the aquatic environment either in a viable but non-culturable state or in numbers too small to detect by routine culture methods. Under conditions favourable for growth, such V. cholerue 01 may increase in number sufficient to pose an epidemic threat, as sugg.ested by human volunteer experiments (Colwell et ul. 1990). Findings similar to those reported here for V. cholerue 01 in environmental samples have been observed for other pathogenic bacteria, including Cumpylobder jejmi (Rollins & Colwell 1986) and .Suhnonek euferidifis (Roszak ef ul. 1984). We conclude from the results of this study that the FA technique, a sensitive, rapid and reliable method for detecting V. cholerue 01 and other pathogenic bacteria in the aquatic environment, can be useful in monitoring for V. cholerue in the environment. More importantly, from the results of this study, it is concluded that V. cholerae was present in the aquatic environment of Sao Paula, Brazil, prior to the present outbreak of cholera in that country. It is hypothesized that V. cholerue 01 is autochthonous to estuarine and brackish waters of Brazil. Further studies, which are in progress, will provide additional data to confirm this hypothesis.
and Discussion
Four of the five samples of freshwater from the Billings Reservoir presented positive results for V. cholerue 01 by FA. No strains of V. chokrue, 01 or non-01, were detected by culture in these samples. All samples of sewage water collected from the sites on Jurubatuba Street and in the Orquidario of Santos yielded V. cholerue non-01 by culture, confirmed by subsequent bacteriological analysis at the University of Maryland. Vibrio chderue 01 was detected by immunofluorescence microscopy in all of these samples, however. Three of four samples collected at Sao Lourenco Beach in Bertioga yielded low numbers of V, cholerue 01
Acknowledgements Travel support for R.R. Colwell to Brazil in September 1982, was provided by the World Health Organization, Geneva, making this collaboration possible. Assistance was provided by P. West in the confirmation of V. cholerue 01 and non-01, which is gratefully acknowledged. The research work was funded, in part, by National Science Foundation Grant No. BSR 84-03197 and Co-operative Agreement No. CR-81-2246-01-O between the Environmental Protection Agency and the University of Maryland,
World Jmmal of Microbmfogy and Bioteckmhgy,
Vol !? ?993
391
M.T. Mar-fins et al.
References Anon. 1991 CDC Update: Cholera-Western Hemisphere 1991. &forbidify and Morfalify World Reports 40, 860. Bourke, A.T.C., Cossins, Y.N., Gray, B.R.W., Hunney, T.J., Rostron, N.A., Holmes, R.V., Grigges, E.R., Larsen, D.J. & Kelk, V.R. 1986 Investigation of cholera acquired from the riverine environment in Queensland. Medical Journal of Australia 144, 229-234. Brayton, P.R. & Colwell, R.R. 1987 Fluorescent antibody staining method for enumeration of viable environmental Vibrio cholerae 01. ]ournal of Microbiological Mefhods, 6, 309-314. Brayton, P.R., Tamplin, M.L., Huq, A. & Colwell, R.R. 1987 Enumeration of V. cholerae 01 in Bangladesh waters by fluorescent-antibody direct viable count. Applied and Environmental Microbiology 53, 2862-2865. Colwell, R.R. 1983 Report to the World Health Organization, February 28, 1983. Geneva: World Health Organization. Colwell, R.R., Seidler, R.J., Kaper, J., Joseph, S.W., Garges, S., Lockman, H., Maneval, D., Bradford, H., Roberts, N., Remmers, E., Huq, I. & Huq, A. 1981 Occurrence of Vibrio cholerae serotype 01 in Maryland and Louisiana estuaries. Applied and Environmental Microbiology 41, 555-558. Colwell, R.R., Tamplin, M.L., Brayton, P.R., Gauzens, A.L., Tall, B.D., Herrington, D., Levine, M.M., Hall, S., Huq, A. & Sack, D.A. 1990 Environmental aspects of Vibrio cho!erae in transmission of cholera. Advances in Research on Cholera and Related Diarrheas 7, 327-343. Gangarosa, E.J. & Mosley, W.H. 1974 Epidemiology and surveillance of cholera. In Cholera, eds. Barua, D. & Burrows, W. pp. 381-403. London: W.B. Saunders. Huq, A., Colwell, R.R., Rahman, R., Ali, A., Chowdhury, M.A.R., Parveen, S., Sack, D.A. & Russek-Cohen, E. 1990 Detection of Vibrio cholerae 01 in the aquatic environment by fluorescentmonoclonal antibody and culture methods. Applied and Environmenfal Microbiology 56, 2370-2373. Huq, A., Small, E.B., West, P.A., Huq, M.I., Rahman, R. & Colwell, R.R. 1983 Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Applied and Environmental Microbiology 4.5, 275-283.
Isaacson, M., Clarke, K.R., Ellacombe, G.H., Smit, W.A., Smit, P., Koomhof, H.J., Smith, L.S. & Kriel, L.J. 1974 The recent outbreak in the South African gold mining industry: a preliminary report. South Africa Medical Journal 48, 2557-2560. Kamal, A.M. 1974 The seventh pandemic of cholera. In Cholera, eds. Barua, D. & Burrows, W. p. 9. London: W.B. Saunders. Martins, M.T., Pessoa, G.V.A., Sanchez, P.S., Sato, M.I.Z., Coimbrao, C.A., Monteiro, C.K. & Marques, E. 1991 Occurrence of V. cholerae 01 non-toxigenic in wastewaters from Sao Paula, Brazil. Water Science Technology 24, 363-366. McFarland, L., Bradford, H.B. & Matheson, J. 1986 Cholera in Louisiana update. Journal of the American Medical Association 256,
3080. Rollins, D.M. & Colwell, R.R. 1986 Viable but not nonculturable stage of Campylobacterjejuni and its role in survival in the aquatic environment. Applied and Environmental Microbiology 52,
531-538. Roszak, D.B. & Colwell, R.R. 1987 Survival strategies of bacteria in the environment. Microbiological Reviews 5 1, 465-479. Roszak, D.B., Grimes, D.J. & Colwell, R.R. 1984 Viable but non-recoverable stage of Salmonella enteritidis in aquatic systems. Canadian journal of Microbiology 30, 334-338. West, P.A. & Colwell, R.R. 1984 Identification and classification of Vibrionaceaean overview. In Vibrios in the Environmenf, ed. Colwell, R.R. pp. 285-363. New York: John Wiley & Sons. Xu, HS., Roberts, N.C., Adams, L.B., West, P.A., Siebling, R.J., Huq, A., Huq, M.I., Rahman, R. & Colwell, R.R. 1984 An indirect fluorescent antibody staining procedure for detection of Vibrio cholerae serovar 01 in aquatic environmental samples. Journal of Microbiological Mefhoak 2, 221-231. Xu, H.S., Roberts, N., Singleton, F.L., Attwell, R.W., Grimes, D.J. & Colwell, R.R. 1983 Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microbial Ecology 8, 213-223.
(Received 18 November 1992; accepfed 16 December 1992)
