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Presence of Aeromonas hydrophila in slaughtered animals
ELSEVIER
International Journal of Food Microbiology 23 (1994) 221-225
International Journal of Food Microbiology
Short Communication
Presence of Aeromonas hydrophila in slaughtered animals S. B u n c i c a,., j. P a n i n b a Meat Industry Research Institute of New Zealand, P.O. Box 617, Hamilton, New Zealand b Yugoslav Institute of Meat Technology, Kacanskog 13, Belgrade, Yugoslacia Received 15 September 1993; revision received 28 February 1994; accepted 21 March 1994)
Abstract
The fate of Aeromonas hydrophila after a bacteriaemia in four healthy pigs intravenously inoculated with 109 and 1015 cells of the bacterium was investigated. In two pigs slaughtered 30 min after inoculation the bacteria were found in blood (8 × 102/ml and 4.2 x 102/ml), meat and fatty tissues (1-10/g), but not in liver, spleen and superficial inguinal lymph nodes. A. hydrophila was not found in blood, meat, fatty tissue, liver, spleen, kidneys and superficial inguinal lymph nodes of other two pigs slaughtered 5.5 h postinoculation, with exception of one superficial inguinal lymph node of one pig.
Keywords: Aeromonas hydrophila; Slaughtered animals; Meat
1. Introduction
Aeromonas hydrophila is a widely spread microorganism (Hazen et al., 1978; Siedler et al., 1980; Burke et al., 1984a,b Gray, 1984). The bacteria is a well-known pathogen of water animals, amphibia and reptiles (Gray, 1984), especially frogs (Stanier, 1943) and fish (Rahim et al., 1984). A. hydrophila have also been found in the faeces of healthy animals - cattle, pigs, sheep, goats, horses and poultry (Annapurna et al., 1977; Gray, 1984; Morgan et al., 1985). Even though A. hydrophila is often present in the faeces of healthy humans, there are few reports of gastroenteritis caused by Aeromonas spp. (Echevarria et al., 1981; Gracey et al., 1982; Burke et al., 1983; Agger et al., 1985). Pitarangsi et al. (1982) and Morgan et al. (1985) showed that peroral ingestion of toxigenic strains of A. hydrophila by resus monkeys and volunteers did not cause diarrhoea. Therefore, the significance * Corresponding author. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 8 - 1 6 0 5 ( 9 4 ) 0 0 0 3 2 - 2
222
S. Buncic, J. Panin / International Journal of Food Microbiology 23 (1994) 221-225
of A. hydrophila as a foodborne pathogen is still unclear. However, it should be kept in mind that this microorganism is an opportunistic pathogen, capable of producing at least 3 different toxins: two haemolysins and an enterotoxin (Ljungh et al., 1981). There are few data in the literature related to the prevalence and fate of A. hydrophila in meat and meat products. The bacteria have been found in minced meat, poultry meat, vacuum- or CO2-packaged meat and raw sausages (Nagel et al., 1960; Molin and Ternstrom, 1979; Myers et al., 1982; Blickstad et al., 1983; Simard et al., 1984; Palumbo et al., 1985), but more detailed investigations concerning routes of contamination of meat with A. hydrophila are lacking. Healthy animals harbouring A. hydrophila are of particular interest from a meat hygiene point of view, as a possible source of intravital (endogenous) or postmortem (secondary) contamination of the meat. There are no published data on the presence of A. hydrophila in deep parts of organs and tissues of slaughtered animals, neither from normal slaughtering nor after artificial inoculation with the bacteria. Therefore, we initiated investigations in Yugoslavia to reveal the fate of the bacteria in animal after an eventual bacteriaemia.
Materials and methods
2.1. Preparation of inoculum of A. hydrophila The A. hydrophila strain used in this experiment is from the collection of Institute for Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen, Denmark), obtained from Prof. Dr. Niels Skovgaard.. This strain (designated as A5) was previously isolated from pork and at Veterinary faculty in Belgrade proved to be hemolytic and enterotoxigenic in suckling mouse assay. Therefore, as considered potentially pathogenic, the strain was selected for the experimental inoculation of pigs. The strain was grown in conical flasks containing 500 ml BHI (Brain Hearth Infusion, Merck) broth, incubated at 30°C for 24 h. The bacteria were centrifuged (2000 g; 10 min; 4°C) and then resuspended in 50 ml of physiological saline. The suspension was then diluted to concentrations of 10S/ml (suspension A) and 1014/ml (suspension B). Concentrations were estimated by surface plating on SA agar as described below.
2.2. Pigs inoculated with A. hydrophila Four pigs (age 7 months; 100 kg live weight) were used in this experiment. The pigs originated from a Yugoslav farm registered as free of illnesses and were observed during 3 days prior to experiment with respect to normal intake of feed and water and the absence of any abnormal clinical symptoms. The pigs were considered healthy, with normal immunological status. A ten ml volume of suspension A corresponding to 10 9 cells of A. hydrophila was inoculated into the ear vein
S. Buncic, J. Panin/ InternationalJournalof FoodMicrobiology23 (1994)221-225
223
of pigs No. 1 and 2. A ten ml volume of suspension B corresponding to 1015 bacteria was inoculated in same manner into pigs No. 3 and 4. Pigs No. 1 and 3 were slaughtered after 30 min, and pigs No. 2 and 4 after 5.5 h. The number of A. hydrophila in the meat, fatty tissue, liver, spleen, kidneys and superficial inguinal lymph nodes was determined after sampling and homogenization by the direct surface spread plate on SA agar as described below. The presence of A. hydrophila was examined simultaneously in 1 g of meat (M. gracilis and M. longissimus dorsii) and in 1 g of fatty tissue (from double chin) using the enrichment method as described below.
2.3. Enumeration of A. hydrophila Determination of colony forming units. The surface of the meat samples and organs was burned, samples of 10 g were aseptically taken from the centre, homogenised with 90 ml of physiological saline for 2 min in a Stomacher R (Seward Medical, London, UK) and decimal dilutions prepared. The number of A. hydrophila in the homogenate was determined on Starch Ampicillin (SA) agar (Palumbo et al., 1985). From appropriate dilutions 1 ml (10 -1) or 0.1 ml (from higher decimal dilutions) were plated onto SA agar plates. The 1 ml inocula were distributed on two plates. Enrichment. Ten ml of nutrient broth (Torlak, Belgrade, Yugoslavia) with added ampicillin (10/zg/ml) was inoculated with 1 g of sample, incubated at 30°C for 24 h, and 0.1 ml subcultured onto SA agar plates. Confirmation ofA. hydrophila. SA agar plates were incubated at 30°C for 24 h and then carefully overflowed with Lugol solution to determine typical amylase-positive colonies. Two to three typical colonies from each plate were selected, streaked onto Blood Agar plates (containing 5% of defibrinated veal blood, Torlak, Belgrade, Yugoslavia) to obtain pure cultures and observe for haemolysis, and the following confirmatory tests for A. hydrophUa were performed: Gram-stain, catalase, oxidase, Voges-Proskauer reaction, aesculin hydrolysis, fermentation of glucose, resistance to vibriostatic agent 0/129, and reaction on API 20NE strips (Bio Merieux, Marcy-I, Etoile, France).
Results and discussion
Our investigation of healthy animals slaughtered in meat plants in Yugoslavia revealed that A. hydrophila can be found in deep parts of liver in 1.6% of pigs and 6.6% of cattle (unpublished data). This phenomenon raises the question what is their fate in organism of healthy animals, in case of bacteriaemia. Therefore, four pigs were intravenously inoculated with broth culture of A. hydrophila. None of the animals showed any symptoms prior to slaughter or any patho-morphological changes at the post-mortem meat inspection. The bacteria were present 30 min
224
S. Buncic, J. Panin / International Journal of Food MicrobiologLv 23 (1994) 221-225
Table 1 Findings of Aeromonas hydrophila in organs and tissues of pigs after intravenous inoculation Samples
Blood Liver Spleen Kidney Muscles Fatty tissue Lymph node
Count of A. hydrophila ( c f u / g or ml) Pigs slaughtered 30 min after inoculation
Pigs slaughtered 5.5 h after inoculation
(A)
(B)
(A)
(B)
8 x 10 2 () (/ 3 x 101 +* +* (/
4.2?< 10 2 0 (I
() 0 0
0 0 (I
0
0
(I
+* +* 0
0 0 0
0 0 2 × 10 3
+* Aeromonas hydrophila detected only with enrichment (A) Pigs inoculated with 109 cells of A. hydrophila (B) Pigs inoculated with 1015 ceils of A. hydrophila
after inoculation in the blood, but not in the liver, spleen and inguinal lymph nodes (Table 1). A. hydrophila was found in the meat and fatty tissue of the same pigs using only the enrichment method, but not direct spread plate method. The kidneys from one pig slaughtered 30 rain after inoculation with 10 9 cells harboured A. hydrophila, probably due to the great volume of blood passing through these organs. In two other pigs which were slaughtered 5.5 h after inoculation A. hydrophila was eliminated from the blood, organs, meat and fatty tissue. The bacteria was detected only in one inguinal lymph node of one pig. Our results, although obtained with a small number of inoculated animals, may indicate that endogenous contamination of meat with A. hydrophUa will not occur in pigs that are healthy, non-stressed and rested before slaughter. These results might, however, be influenced by specific immunological status of the pigs with regard to Aeromonas spp. At present, it is not known whether the normal intake of A. hydrophila via feed and water could increase the resistance of pigs to this microorganism. The behaviour of A. hydrophila in immunocompromised, stressed or otherwise exhausted animals remains to be investigated.
References Agger, W.A., McCormick, J.D. and Gurwith, M.J. (1985) Clinical and microbiological features of Aeromonas hydrophila - associated diarrhoea. J. Clin. Microbiol. 21,909-913. A n n a p u r n a , E. and Sanyal, S.C. (1977) Enterotoxicity of Aeromonas hydrophila. J. Med. Microbiot. 10, 317-323. Blickstad, E. and Molin, G. (1983) Carbon dioxide as a controller of the spoilage flora of pork, with special reference to temperature and sodium chloride. J. Food Prot. 46, 756-763. Burke, V., Gracey, M., Robinson, J., Peck, D., Beaman, J. and Bundell, C. (1983) The microbiology of childhood gastroenteritis: Aeromonas species and other infective agents. J. Infect. Dis. 148, 68-74.
S. Buncic, J. Panin / International Journal of Food Microbiology 23 (1994) 221 225
225
Burke, V., Robinson, J., Gracey, M., Paterson, D. and Partridge K. (1984) Isolation of Aeromonas hydrophila from a metropolitan water supply: Seasonal correlation with clinical isolates. Appl. Environ. Microbiol. 48, 361-366. Burke, V., Robinson, J., Gracey, M., Peterson, D., Meyer, N. and Haley, V. (1984) Isolation of Aeromonas spp. from an unchlorinated domestic water supply. Appl. Environ. Microbiol. 48, 367-370. Echeverria, P., Blacklow, N.R., Sanford, L.B. and Cukor, G.G. (1981) Traveler's diarrhoea among american peace corps volunteers in rural Thailand. J. Infect. Dis. 143, 767-771. Gracey, M., Burke, V. and Robinson, J. (1982) Aeromonas-associated gastroenteritis. Lancet 2, 1304-1306. Gray, S.J. (1984) Aeromonas hydrophila in livestock: Incidence, biochemical characteristics and antibiotic susceptibility. J. Hyg. Cambridge 92, 365-375. Ljungh, A., Wretlind, B. and Mollby, R. (1981) Separation and characterization of enterotoxin and two baemolysins from Aeromonas hydrophila. Acta Pathogenica Microbiol. Scand. Sec. B. 89, 387-397. Molin, G. and Ternstrom, A. (1979) Effect of packaging under carbon dioxide, nitrogen or air on the microbial flora of pork stored at 4°C.J. Appl. Bacteriol. 47, 197-208. Morgan, D.R., Johnson, P.C., DuPont, H.L., Satterwhite, T.K. and Wood, L.V. (1985) Lack of correlation between known virulence properties of Aeromonas hydrophila and enteropathogenicity for humans. Infection and Immunity 50, 62-65. Myers, B.R., Morsholl, P.T., Edmonson, J.E. and Stringer, W. (1982) Isolation of pectinolytic Aeromonas hydrophila and Yersinia enterocoliticfa from vacuum packaging pork. J. Food Prot. 45, 33-37. Nagel, Ch.W., Simpson K.L., Ng, H., Waughn, R.H. and Stewart, G.F. (1960) Microorganisms associated with spoilage of refrigerated poultry. Food Technol. 21, 21-23. Palumbo, S.A., Maxino, F., Williams, A.C., Buchannan, R.L. and Thayer, D.W. (1985) Starch-Ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl. Environ. Microbiol. 50, 1027-1030. Pitarangsi, C., Echevarria, P., Whitmire, R., Tirapat, C., Formal, S., Dammin, G.J. and Tingtalapong, M. (1982) Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: Prevalence among individuals with and without diarrhoea in Thailand. Infection and Immunity 35, 666-673. Popoff, M. (1984) Aeromonas. In: Bergey's Manual of Systematic Bacteriology. William & Wilkins, Baltimore/London, Vol. 1, pp. 545-548. Rahim, Z., Sanyal, S.C., Aziz, K.M.S., Huq, M.I. and Chowdhury, A.A. (1984) Isolation of enterotoxigenic, hemolytic and antibiotic-resistant Aeromonas hydroiphila strains from infected fish in Bangladesh. Appl. Environ. Microbiol. 48, 865-867. Seidler, J.R., Allen, D.A., Lockman, H., Colwell, R.R., Joseph, S.W. and Daily O.P. (1980) Isolation, enumeration and characterization of Aeromonas from polluted waters encountered in diving operations. Appl. Environ. Microbiol. 39, 1010-1018. Simard, R.E., Zee, J. and L'Hevreux, L. (1984) Microbial growth in carcasses and boxed beef during storage. J. Food Prot. 47, 773-777. Stanier, R.Y. (1943) A note on the taxonomy of Proteus hydrophilus. J. Bacteriol. 46, 213-214.
International Journal of Food Microbiology 23 (1994) 221-225
International Journal of Food Microbiology
Short Communication
Presence of Aeromonas hydrophila in slaughtered animals S. B u n c i c a,., j. P a n i n b a Meat Industry Research Institute of New Zealand, P.O. Box 617, Hamilton, New Zealand b Yugoslav Institute of Meat Technology, Kacanskog 13, Belgrade, Yugoslacia Received 15 September 1993; revision received 28 February 1994; accepted 21 March 1994)
Abstract
The fate of Aeromonas hydrophila after a bacteriaemia in four healthy pigs intravenously inoculated with 109 and 1015 cells of the bacterium was investigated. In two pigs slaughtered 30 min after inoculation the bacteria were found in blood (8 × 102/ml and 4.2 x 102/ml), meat and fatty tissues (1-10/g), but not in liver, spleen and superficial inguinal lymph nodes. A. hydrophila was not found in blood, meat, fatty tissue, liver, spleen, kidneys and superficial inguinal lymph nodes of other two pigs slaughtered 5.5 h postinoculation, with exception of one superficial inguinal lymph node of one pig.
Keywords: Aeromonas hydrophila; Slaughtered animals; Meat
1. Introduction
Aeromonas hydrophila is a widely spread microorganism (Hazen et al., 1978; Siedler et al., 1980; Burke et al., 1984a,b Gray, 1984). The bacteria is a well-known pathogen of water animals, amphibia and reptiles (Gray, 1984), especially frogs (Stanier, 1943) and fish (Rahim et al., 1984). A. hydrophila have also been found in the faeces of healthy animals - cattle, pigs, sheep, goats, horses and poultry (Annapurna et al., 1977; Gray, 1984; Morgan et al., 1985). Even though A. hydrophila is often present in the faeces of healthy humans, there are few reports of gastroenteritis caused by Aeromonas spp. (Echevarria et al., 1981; Gracey et al., 1982; Burke et al., 1983; Agger et al., 1985). Pitarangsi et al. (1982) and Morgan et al. (1985) showed that peroral ingestion of toxigenic strains of A. hydrophila by resus monkeys and volunteers did not cause diarrhoea. Therefore, the significance * Corresponding author. 0168-1605/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 8 - 1 6 0 5 ( 9 4 ) 0 0 0 3 2 - 2
222
S. Buncic, J. Panin / International Journal of Food Microbiology 23 (1994) 221-225
of A. hydrophila as a foodborne pathogen is still unclear. However, it should be kept in mind that this microorganism is an opportunistic pathogen, capable of producing at least 3 different toxins: two haemolysins and an enterotoxin (Ljungh et al., 1981). There are few data in the literature related to the prevalence and fate of A. hydrophila in meat and meat products. The bacteria have been found in minced meat, poultry meat, vacuum- or CO2-packaged meat and raw sausages (Nagel et al., 1960; Molin and Ternstrom, 1979; Myers et al., 1982; Blickstad et al., 1983; Simard et al., 1984; Palumbo et al., 1985), but more detailed investigations concerning routes of contamination of meat with A. hydrophila are lacking. Healthy animals harbouring A. hydrophila are of particular interest from a meat hygiene point of view, as a possible source of intravital (endogenous) or postmortem (secondary) contamination of the meat. There are no published data on the presence of A. hydrophila in deep parts of organs and tissues of slaughtered animals, neither from normal slaughtering nor after artificial inoculation with the bacteria. Therefore, we initiated investigations in Yugoslavia to reveal the fate of the bacteria in animal after an eventual bacteriaemia.
Materials and methods
2.1. Preparation of inoculum of A. hydrophila The A. hydrophila strain used in this experiment is from the collection of Institute for Veterinary Microbiology, Royal Veterinary and Agricultural University, Copenhagen, Denmark), obtained from Prof. Dr. Niels Skovgaard.. This strain (designated as A5) was previously isolated from pork and at Veterinary faculty in Belgrade proved to be hemolytic and enterotoxigenic in suckling mouse assay. Therefore, as considered potentially pathogenic, the strain was selected for the experimental inoculation of pigs. The strain was grown in conical flasks containing 500 ml BHI (Brain Hearth Infusion, Merck) broth, incubated at 30°C for 24 h. The bacteria were centrifuged (2000 g; 10 min; 4°C) and then resuspended in 50 ml of physiological saline. The suspension was then diluted to concentrations of 10S/ml (suspension A) and 1014/ml (suspension B). Concentrations were estimated by surface plating on SA agar as described below.
2.2. Pigs inoculated with A. hydrophila Four pigs (age 7 months; 100 kg live weight) were used in this experiment. The pigs originated from a Yugoslav farm registered as free of illnesses and were observed during 3 days prior to experiment with respect to normal intake of feed and water and the absence of any abnormal clinical symptoms. The pigs were considered healthy, with normal immunological status. A ten ml volume of suspension A corresponding to 10 9 cells of A. hydrophila was inoculated into the ear vein
S. Buncic, J. Panin/ InternationalJournalof FoodMicrobiology23 (1994)221-225
223
of pigs No. 1 and 2. A ten ml volume of suspension B corresponding to 1015 bacteria was inoculated in same manner into pigs No. 3 and 4. Pigs No. 1 and 3 were slaughtered after 30 min, and pigs No. 2 and 4 after 5.5 h. The number of A. hydrophila in the meat, fatty tissue, liver, spleen, kidneys and superficial inguinal lymph nodes was determined after sampling and homogenization by the direct surface spread plate on SA agar as described below. The presence of A. hydrophila was examined simultaneously in 1 g of meat (M. gracilis and M. longissimus dorsii) and in 1 g of fatty tissue (from double chin) using the enrichment method as described below.
2.3. Enumeration of A. hydrophila Determination of colony forming units. The surface of the meat samples and organs was burned, samples of 10 g were aseptically taken from the centre, homogenised with 90 ml of physiological saline for 2 min in a Stomacher R (Seward Medical, London, UK) and decimal dilutions prepared. The number of A. hydrophila in the homogenate was determined on Starch Ampicillin (SA) agar (Palumbo et al., 1985). From appropriate dilutions 1 ml (10 -1) or 0.1 ml (from higher decimal dilutions) were plated onto SA agar plates. The 1 ml inocula were distributed on two plates. Enrichment. Ten ml of nutrient broth (Torlak, Belgrade, Yugoslavia) with added ampicillin (10/zg/ml) was inoculated with 1 g of sample, incubated at 30°C for 24 h, and 0.1 ml subcultured onto SA agar plates. Confirmation ofA. hydrophila. SA agar plates were incubated at 30°C for 24 h and then carefully overflowed with Lugol solution to determine typical amylase-positive colonies. Two to three typical colonies from each plate were selected, streaked onto Blood Agar plates (containing 5% of defibrinated veal blood, Torlak, Belgrade, Yugoslavia) to obtain pure cultures and observe for haemolysis, and the following confirmatory tests for A. hydrophUa were performed: Gram-stain, catalase, oxidase, Voges-Proskauer reaction, aesculin hydrolysis, fermentation of glucose, resistance to vibriostatic agent 0/129, and reaction on API 20NE strips (Bio Merieux, Marcy-I, Etoile, France).
Results and discussion
Our investigation of healthy animals slaughtered in meat plants in Yugoslavia revealed that A. hydrophila can be found in deep parts of liver in 1.6% of pigs and 6.6% of cattle (unpublished data). This phenomenon raises the question what is their fate in organism of healthy animals, in case of bacteriaemia. Therefore, four pigs were intravenously inoculated with broth culture of A. hydrophila. None of the animals showed any symptoms prior to slaughter or any patho-morphological changes at the post-mortem meat inspection. The bacteria were present 30 min
224
S. Buncic, J. Panin / International Journal of Food MicrobiologLv 23 (1994) 221-225
Table 1 Findings of Aeromonas hydrophila in organs and tissues of pigs after intravenous inoculation Samples
Blood Liver Spleen Kidney Muscles Fatty tissue Lymph node
Count of A. hydrophila ( c f u / g or ml) Pigs slaughtered 30 min after inoculation
Pigs slaughtered 5.5 h after inoculation
(A)
(B)
(A)
(B)
8 x 10 2 () (/ 3 x 101 +* +* (/
4.2?< 10 2 0 (I
() 0 0
0 0 (I
0
0
(I
+* +* 0
0 0 0
0 0 2 × 10 3
+* Aeromonas hydrophila detected only with enrichment (A) Pigs inoculated with 109 cells of A. hydrophila (B) Pigs inoculated with 1015 ceils of A. hydrophila
after inoculation in the blood, but not in the liver, spleen and inguinal lymph nodes (Table 1). A. hydrophila was found in the meat and fatty tissue of the same pigs using only the enrichment method, but not direct spread plate method. The kidneys from one pig slaughtered 30 rain after inoculation with 10 9 cells harboured A. hydrophila, probably due to the great volume of blood passing through these organs. In two other pigs which were slaughtered 5.5 h after inoculation A. hydrophila was eliminated from the blood, organs, meat and fatty tissue. The bacteria was detected only in one inguinal lymph node of one pig. Our results, although obtained with a small number of inoculated animals, may indicate that endogenous contamination of meat with A. hydrophUa will not occur in pigs that are healthy, non-stressed and rested before slaughter. These results might, however, be influenced by specific immunological status of the pigs with regard to Aeromonas spp. At present, it is not known whether the normal intake of A. hydrophila via feed and water could increase the resistance of pigs to this microorganism. The behaviour of A. hydrophila in immunocompromised, stressed or otherwise exhausted animals remains to be investigated.
References Agger, W.A., McCormick, J.D. and Gurwith, M.J. (1985) Clinical and microbiological features of Aeromonas hydrophila - associated diarrhoea. J. Clin. Microbiol. 21,909-913. A n n a p u r n a , E. and Sanyal, S.C. (1977) Enterotoxicity of Aeromonas hydrophila. J. Med. Microbiot. 10, 317-323. Blickstad, E. and Molin, G. (1983) Carbon dioxide as a controller of the spoilage flora of pork, with special reference to temperature and sodium chloride. J. Food Prot. 46, 756-763. Burke, V., Gracey, M., Robinson, J., Peck, D., Beaman, J. and Bundell, C. (1983) The microbiology of childhood gastroenteritis: Aeromonas species and other infective agents. J. Infect. Dis. 148, 68-74.
S. Buncic, J. Panin / International Journal of Food Microbiology 23 (1994) 221 225
225
Burke, V., Robinson, J., Gracey, M., Paterson, D. and Partridge K. (1984) Isolation of Aeromonas hydrophila from a metropolitan water supply: Seasonal correlation with clinical isolates. Appl. Environ. Microbiol. 48, 361-366. Burke, V., Robinson, J., Gracey, M., Peterson, D., Meyer, N. and Haley, V. (1984) Isolation of Aeromonas spp. from an unchlorinated domestic water supply. Appl. Environ. Microbiol. 48, 367-370. Echeverria, P., Blacklow, N.R., Sanford, L.B. and Cukor, G.G. (1981) Traveler's diarrhoea among american peace corps volunteers in rural Thailand. J. Infect. Dis. 143, 767-771. Gracey, M., Burke, V. and Robinson, J. (1982) Aeromonas-associated gastroenteritis. Lancet 2, 1304-1306. Gray, S.J. (1984) Aeromonas hydrophila in livestock: Incidence, biochemical characteristics and antibiotic susceptibility. J. Hyg. Cambridge 92, 365-375. Ljungh, A., Wretlind, B. and Mollby, R. (1981) Separation and characterization of enterotoxin and two baemolysins from Aeromonas hydrophila. Acta Pathogenica Microbiol. Scand. Sec. B. 89, 387-397. Molin, G. and Ternstrom, A. (1979) Effect of packaging under carbon dioxide, nitrogen or air on the microbial flora of pork stored at 4°C.J. Appl. Bacteriol. 47, 197-208. Morgan, D.R., Johnson, P.C., DuPont, H.L., Satterwhite, T.K. and Wood, L.V. (1985) Lack of correlation between known virulence properties of Aeromonas hydrophila and enteropathogenicity for humans. Infection and Immunity 50, 62-65. Myers, B.R., Morsholl, P.T., Edmonson, J.E. and Stringer, W. (1982) Isolation of pectinolytic Aeromonas hydrophila and Yersinia enterocoliticfa from vacuum packaging pork. J. Food Prot. 45, 33-37. Nagel, Ch.W., Simpson K.L., Ng, H., Waughn, R.H. and Stewart, G.F. (1960) Microorganisms associated with spoilage of refrigerated poultry. Food Technol. 21, 21-23. Palumbo, S.A., Maxino, F., Williams, A.C., Buchannan, R.L. and Thayer, D.W. (1985) Starch-Ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl. Environ. Microbiol. 50, 1027-1030. Pitarangsi, C., Echevarria, P., Whitmire, R., Tirapat, C., Formal, S., Dammin, G.J. and Tingtalapong, M. (1982) Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: Prevalence among individuals with and without diarrhoea in Thailand. Infection and Immunity 35, 666-673. Popoff, M. (1984) Aeromonas. In: Bergey's Manual of Systematic Bacteriology. William & Wilkins, Baltimore/London, Vol. 1, pp. 545-548. Rahim, Z., Sanyal, S.C., Aziz, K.M.S., Huq, M.I. and Chowdhury, A.A. (1984) Isolation of enterotoxigenic, hemolytic and antibiotic-resistant Aeromonas hydroiphila strains from infected fish in Bangladesh. Appl. Environ. Microbiol. 48, 865-867. Seidler, J.R., Allen, D.A., Lockman, H., Colwell, R.R., Joseph, S.W. and Daily O.P. (1980) Isolation, enumeration and characterization of Aeromonas from polluted waters encountered in diving operations. Appl. Environ. Microbiol. 39, 1010-1018. Simard, R.E., Zee, J. and L'Hevreux, L. (1984) Microbial growth in carcasses and boxed beef during storage. J. Food Prot. 47, 773-777. Stanier, R.Y. (1943) A note on the taxonomy of Proteus hydrophilus. J. Bacteriol. 46, 213-214.