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Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Toxigenic Vibrio cholerae non‐O:1 isolated from a goat in the Netherlands a
b
a
I. J. R. Visser , E. A. ter Laak , N. W. van Dijk & W. Wouda
a
a
Animal Health Service in Noord‐Nederland , P.O. Box 361, Drachten, 9200 AJ, The Netherlands b
Department of Bacteriology , Central Veterinary Institute , Edelhertweg 15, Lelystad, 8219 PH, The Netherlands Published online: 01 Nov 2011.
To cite this article: I. J. R. Visser , E. A. ter Laak , N. W. van Dijk & W. Wouda (1991) Toxigenic Vibrio cholerae non‐O:1 isolated from a goat in the Netherlands, Veterinary Quarterly, 13:2, 114-118, DOI: 10.1080/01652176.1991.9694293 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694293
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
Downloaded by [Stony Brook University] at 22:47 20 October 2014
forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
84. Voogt HJ de. De hormonale behandeling van prostaatkanker. Ned Tijdschr Geneeskd 1980; 124: 876-83.
85. Vreeburg JTM, Samaun K, Verkade HJ, Verhoef P, Ooms MP, and Weber RFA. Effects of corticosterone on the negative feedback action of testosterone, 5a-dihydrotestosterone and estradiol in the adult male rat. J Steroid Biochem 1988; 29: 93-8. 86. Vreeburg JTM, Greef WJ de, Ooms MP, Wouw P van, and Weber RFA. Effects of adrenocorticotropin and corticosterone on the negative feedback action of testosterone in the adult male rat. Endocrinology 1984; 115: 977-83. 87. Ward IL and Weisz J. Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers. Endocrinology 1984; 114: 1635-44.
88. Wass JAH. The control of gonadotropin secretion. In: JAH Wass and MF Scanlon (Eds) Neuroendocrine Perspectives Vol 6. New York: Springer-Verlag, 1989.
Downloaded by [Stony Brook University] at 22:47 20 October 2014
89. Welsh TH and Johnson BH. Stress-induced alterations in secretion of corticosteroids, progesterone, luteinizing hormone, and testosterone in bulls. Endocrinology 1981; 109: 185-90. 90. Welsh TH, Randel RD, and Johnson BH. Interrelationships of serum corticosteroids, LH and testosterone in male bovine. Arch Andrology 1981; 6: 141-50. 91. Wiepkema PR. Over gedragsstoringen bij dieren in de veehouderij. Tijdschr Diergeneeskd 1985; 110: 12-20. 92.
Yelvington DB, Weiss GK, and Ratner A. Habituation of the prolactin response in rats to psychological stress. Psychoneuroendocrinology 1985; 10: 95-102.
SHORT COMMUNICATIONS
Toxigenic Vibrio cholerae non-0:1 isolated from a goat in the Netherlands I. J. R. Visserl, E. A. ter Laak2, N. W. van Dijki, and W. Woudai SUMMARY. A ease of enterotoxicosis in a goat at necropsy is described. The animal had died without clinical signs. Toxigenic Vibrio cholerae non-0: I was isolated from the intestines.
This species has not been reported earlier from healthy or diseased farm animals, such as goats, in the Netherlands.
INTRODUCTION
Vibrio cholerae serogroup 0:1 causes cholera, a human disease that has been endemic in India and Southeast Asia since ancient times. Periodically the disease spreads to the Middle East and Africa in epidemics and pandemics. Epidemic strains of serogroup 0:1 produce cholera toxin, whose action on the mucosal cells of the small intestine causes the diarrhoea that is characteristic of cholera (10). There are at least 72 other serogroups of V. cholerae that are called non-0:1 (7). Formerly these strains were called non-cholera vibrios (NCV) or non-agglutinable vibrios (NAG). Although the majority of the non-0:1 strains of V. cholerae isolated from the environment does not appear to be enteropathogenic (4), many strains of V cholerae non-0:1 produce a toxin identical or very similar to cholera toxin
(2, 4, 9, 23). Other strains, which do not produce cholera toxin, are enteropaI
2
Animal Health Service in Noord-Nederland, P.O. Box 361, 9200 AJ Drachten, The Netherlands. Central Veterinary Institute, Department of Bacteriology, Edelhertweg 15, 8219 PH Lelystad, The Netherlands.
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thogenic to animals experimentally (4, 5). These strains do not cause cholera epidemics, but do cause individual cases and outbreaks of cholera-like diarrhoea in humans. These had been only rarely reported from places all over the world since 1960 (21) but have been more frequently reported since 1972 (12). An additional virulence factor such as intestinal adhesion of the bacteria is also important in producing disease (4, 6). Other toxins produced by non-0:1 strains have also been described: for example, enterotoxins related to cholera toxin (23) and a toxin similar to the Escherichia coli heat stable enterotoxin (2). In contrast to V. cholerae 0:1, non-0:1 strains may also cause bacteraemia (22). The ecology of V cholerae was poorly understood until recently. Since 1977 V cholerae has been detected in water and mud samples from aquatic areas where cholera epidemics have never occurred, such as in England and the USA (3, 12). It has also been isolated from aquatic animals, such as shellfish, fishes, frogs and ducks (5, 12, 21), but these animal species act only as carriers for V cholerae. Most of the strains that have been found in brackish and fresh water areas worldwide are non-0:1 strains. The few 0:1 strains are generally non-toxigenic and have been found mostly in brackish water (4, 7). It is now believed that an aquatic environment is the natural reservoir for V. cholerae (1, 4, 7, 10). Although it has been reported that farm animals may also carry the organisms, references are very scarce (8, 20). After an outbreak of cholera, Sanyal et al. (18) isolated V cholerae from the faeces of cattle, goats, dogs and chickens in Varanasi, India, where cholera
has not been endemic. Some of the strains that were isolated from cattle and chickens were of the 0:1 type. Although cows appeared to be infected for 8 months at most, V cholerae was isolated only occasionally from goats. Clinical signs of disease were not observed in any of these animals. Under experimental conditions,
however, toxigenic strains of both 0:1 and non-0:1 type are pathogenic for a variety of laboratory animals (18). Only one report, however, exists on V cholerae in relation to enteric disease in animals. Rhodes et al. isolated V cholerae non-
0:1 from a foal, a lamb and two bisons with enteric disease in Colorado (17). This report describes the isolation of a toxigenic V cholerae non-0:1 strain from the small intestines of a goat raised in the Netherlands. CASE REPORT
In the spring of 1989 a goat was found dead and was destroyed without being examined. In July of 1989 two goats with no clinical signs of disease died within two days on the same farm. The second dead goat was sent to the Animal Health
Service for necropsy. The goat was found to be in a thriving condition. The intestines were extended and flabby and contained copious watery fluid. Other pathological lesions were not observed. The goat originated from a farm with 74 milk goats and 22 kids, all of Saanen and Toggenburger breed. The owner started goat husbandry in 1986 and all goats were bought in the Netherlands. Most kids were kept for breeding to enlarge the goat population on the farm. Except for the sudden deaths of the three goats noted above, the herd had had no disease problems. Numerous wild waterfowl and migrating birds lived in the environment of the farm. Laboratory examination Samples collected from the intestines contained no worm eggs or coccidia. For bacteriological examination, intestinal samples were inoculated onto two blood agar (BA) plates containing blood agar base no. 2 (Oxoid CM 271, Haarlem, The
Netherlands) and 7% sheep erythrocytes and onto a brilliant green agar plate (Oxoid CM 329). One BA plate was incubated anaerobically (GasPak anaerobic system; BBL, Becton Dickinson); the other media were incubated aerobically at THE VETERINARY QUARTERLY. VOL. 13, No. 2, APRIL 1991
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37° C. After 24 h many flat, gray-brown, B-haemolytic colonies were detected on
the aerobically incubated BA in nearly pure culture. They contained Gramnegative, small, curved rods. Strain no. 16847 was isolated and further investigated. The oxidase test was positive. The API 20 E system (Api-Biomerieux, 's-
Hertogenbosch, The Netherlands) was used to identify the strain as Vibrio cholerae.
On the anaerobically incubated BA, many colonies with a double B-haemolytic
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zone were detected. These contained large, Gram-positive rods that were tentatively identified as Clostridium perfringens. No other microorganisms were isolated. A smear from the intestinal wall was examined microscopically and contained many Gram-positive, large, rod-like clostridia as well as Gram-negative rods. Because of the numerous clostridia detected in the smear and in the anaerobically cultured BA enterotoxaemia caused by Clostridium perfringens was the presumptive diagnosis. At that time, 24 h after the necropsy, the identification of the Gramnegative strain was started. The isolated strain of V cholerae was then sent to the Central Veterinary Institute for further examination and confirmation of the diagnosis. It was found that gray to cream coloured colonies with a brownish aspect grew rapidly on sheep blood agar. The diameter of colonies after 24 h incubation was 2 mm. Colonies were surrounded by a 13-haemolytic zone and contained Gram-negative curved rods.
The bacteria were highly motile, like rockets. The strain was able to grow anaerobically and produced acid but no gas from D-glucose, sucrose, maltose and mannitol. It produced catalase, oxidase, indole, lysine decarboxylase, ornithine decarboxylase and acetylmethylcarbinol. The strain reduced nitrate, hydrolysed gelatin and utilised citrate (Simmons medium) and was positive in the string test (19). The strain produced no acid from lactose, did not hydrolyse urea, produced
no H2S, was not susceptible to polymyxin (disc = 50 pm) and did not haemagglutinate chicken erythrocytes. At this stage the culture was typed as V cholerae, similar to biotype eltor, but negative in haemagglutination test. An API 20 E test produced number 5147124: Vibrio cholerae, with a 99.7% chance of correct identification. An API 20 NE test produced number 7074747: Vibrio cholerae, with a 98.5% chance of correct identification. The identification of the strain was confirmed by Dr. P. A. M. Guinée, National Institute for Public Health and Environmental Protection, Bilthoven, the Nether-
lands, who obtained additional results. Because antiserum raised against V cholerae 0:1 did not agglutinate the strain, it was concluded that the strain belonged to the NCV-group (V cholerae non-0:1). Furthermore, because the strain produced a verotoxin, it may have caused the pathologic signs of disease. Investigation into the origin of the infection
In September 1989, six water samples were collected from ditches around the grasslands where the goates grazed. Faeces samples were collected rectally from five goats chosen at random. These samples were investigated for the presence of V. cholerae by culturing them on BA, Plate Count Agar (Oxoid CM 325) and a selective medium according to Monsur (13). Vibrio cholerae was not detected in these samples. DISCUSSION
A high number of V. cholerae non-0:1 was isolated in nearly pure culture on a non-selective medium, incubated aerobically, from the small intestines of a goat. The only signs of disease in the goat were extended and flabby intestines containing 116
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copious watery fluid. We suggest the term enterotoxicosis to describe the pathological findings. If the V. cholerae non-0:1 was isolated from a human being, the strain would be considered the cause of the intestinal disorder. Whether V cholerae non-0:1 alone
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caused enterotoxicosis in the goat is unclear, because many C. perfringens organisms were also isolated from the same samples after anaerobic incubation. Clostridium perfringens is generally regarded as a cause of enterotoxaemia in various animal species, including goats (11, 16). Clostridium perfringens may also cause haemorrhagic or necrotic enteritis, but a severe watery diarrhoea is not characteristic in goats (11). Hence the isolated V cholerae strain might have been the cause of the disease, because toxin was also detected. Whether the two other goats that died earlier were infected with V cholerae non-0:1 is not known. The organism was not isolated from faeces samples from healthy goats of the farm, nor from water samples collected from ditches around the farm, although it may have been present in low undetectable numbers (1, 10). The origin and significance
of this infection is unclear. Vibrio cholerae is probably common in aquatic environments in the Netherlands. Some investigators have reported that the organism is more prevalent in summer, when temperatures are higher (3, 14, 15),
but others do not report a correlation between season and incidence (10, 12). Migrating ducks also contribute to periodically higher numbers of V cholerae in the environment (5). It seems worthwhile to look for the prevalence and pathogenicity of this organism in animals with intestinal disorders by using selective culture media (4, 13) to establish a possible association of cholera-like diseases in farm animals and V
cholerae. It would be of special interest to examine animals from areas with a rich waterfowl population. ACKNOWLEDGEMENTS
We thank C. M. F. Wagenaars (Lelystad, The Netherlands) for technical assistance and the late Dr. P. A. M. Guinée, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands, for confirmation of the diagnosis and supply of the Monsur agar plates. REFERENCES 1.
2. 3.
Anonymous. Aquatic niche is haven for Vibrio cholerae. ASM News 1989; 55: 642. Arita M, Takeda T, Honda T, and Miwatani T. Purification and characterization of Vibrio cholerae non-0:1 heat-stable enterotoxin. Infect Immun 1986; 52: 45-9. Bashford DJ, Donovan TJ, Furniss AL, and Lee JV. Vibrio cholerae in Kent. Lancet 1979; 37: 436-7.
4. Baumann P and Schubert RHW. Family II. Vibrionaceae Veron 1965, 5245 AL. In: Bergey's manual of systematic bacteriology I, p. 516-38, 1984. Ed: NR Krieg and JG Holt. Williams & 5. 6.
7.
Wilkins, Baltimore. Bisgaard M, Sakazaki R, and Shimada T. Prevalence of Non-Cholera Vibrios in cavum nasi and pharynx of ducks. Acta Pathol Microbiol Scand Sect B 1978; 86: 261-6.
Datta-Roy K, Dasgupta C, and Ghose AC. Haemagglutination and intestinal adherence properties of clinical and environmental isolates of non-0:1 Vibrio cholerae. Appl Environ Microbiol 1989; 55: 2403-6. Farmer III JJ, Hickman-Brenner FW, and Kelly MT. Vibrio. In: Manual of Clinical Microbiology,
Ed: Lenette EH, Valows A, Hausler Jr WJ, and Shadomy HJ. Fourth ed. Am Soc for Microbiology, Washington DC 1985; 282-301. 8. Hamza PA and Chatterjee BD. Vibrios of public health importance - occurrence in animal faeces. Indian Vet J 1982; 59: 1-4.
9. Honda T, Lertpocasombat K, Hata A, Miwatani T, and Finkelstein RA. Purification and characterization of a protease produced by Vibrio cholerae non-0:1 and comparison with a protease of V cholerae 0:1. Infect Immun 1989; 57: 2799-803. 10. Janda M, Powers C, Bryant RG, and Abbott SL. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin Microbiol Rev 1988; 1: 245-67. THE VETERINARY QUARTERLY. VOL 13,
No. 2, APRIL 1991
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11. Jubb KVE, Kennedy PC, and Palmer N. Pathology of domestic animals. Third ed. Academic Press, Inc New York 1985; 149-55. 12. Kaper J, Lockman H, Colwel RR, and Joseph SW. Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay. Appl and Environ Microbiol 1979; 37: 91-103. 13. Monsur KA. Bacteriological diagnosis of cholera under field conditions. Bull WHO 1963; 28: 387-9.
Muller HE, Bockemiihl J, Burkhardt F und Graevenitz A von. Isolierung und Identifizierung von Vibrionaceae. Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1980; 248: 148-61. 15. Nacescu N and Ciufecu C. Serotypes of Nag Vibrios isolated from clinical and environmental sources. Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1978; 240: 334-8. 16. Oxer DT. Enteroxaemia in goats. Aust Vet J 1956; 32: 62-6.
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14.
17. Rhodes JB, Schweitzer D, and Ogg JE. Isolation of non-0:1 Vibrio cholerae associated with enteric disease of herbivores in Western Colorado. J Clin Microbiol 1985; 22: 572-5. 18. Sanyal SC, Singh SJ, Tiwari IC, Sen PC, Marwah SM, Hazarika UR, Singh H, Shimada T, and Sakazaki R. Role of household animals in maintenance of cholera infection in a community. J Infect Dis 1974; 130: 575-9. 19. Smith Jr HL. A presumptive test for vibrios: the 'string' test. Bull WHO 1970; 42: 817-8. 20. Shewan JM and Véron M. Genus I. Vibrio Pacini 1854, 411. In: Bergey's manual of determinative
bacteriology, p 340-45. Eight ed. 1974. Ed: RE Buchanan and NE Gibbons. The Williams & Wilkins Company, Baltimore.
21. Szeness L, Sey L und Szeness A. Bakteriologische Untersuchungen des Darminhalts von Wasservogeln, Fischen und Fröschen mit besonderer Beriicksichtigung des Vorkommens von Nicht-Cholera-Vibrionen (NCV). Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1979; 245: 8995.
22. Wistrom J. A case of non-0:1 Vibrio cholerae bacteremia from Northern Europe. J Infect Dis 1989; 160: 732.
23. Yamamoto K, Takeda Y, Miwatani T, and Graig JP. Evidence that a non-0:1 Vibrio cholerae produces enterotoxin that is similar but not identical to cholera enterotoxin. Infect Immun 1983; 41: 896-901.
Prevalence of Leptospira interrogans serovar hardjo antibodies in milk in Belgian dairy herds P. P. Dom1, F. Haesebrouckl, R. Vandermeersch2, J. Descamps3, and K. Van Ommeslaeghe4 SUMMARY. Pooled milk samples were collected from 2000 Belgian dairy herds in the autumn
of 1989. Antibodies against Leptospira interrogans serovar hardjo were detected in 9.2% of the herds, with the incidence being higher in the southern part of the country. INTRODUCTION
Leptospira interrogans serovar hardjo (L. hardjo) is the most common causative agent of bovine leptospirosis in countries throughout the world. In all the countries
bordering on Belgium (the Netherlands, Germany and France), L. hardjo I
2 3
4
Laboratory of Veterinary Bacteriology, Faculty of Veterinary Medicine, University of Ghent, Casinoplein 24, B-9000 Ghent, Belgium. Regional Veterinary Investigation Centre, Industrielaan 15, B-8820 Torhout, Belgium. SmithKline Beecham Animal Health Products, Rue de l'Institut 89, B-1330 Rixensart, Belgium. SmithKline Beecham Animal Health Products, Place de l'Université 16, B-1348 Louvain-la Neuve, Belgium.
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Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Toxigenic Vibrio cholerae non‐O:1 isolated from a goat in the Netherlands a
b
a
I. J. R. Visser , E. A. ter Laak , N. W. van Dijk & W. Wouda
a
a
Animal Health Service in Noord‐Nederland , P.O. Box 361, Drachten, 9200 AJ, The Netherlands b
Department of Bacteriology , Central Veterinary Institute , Edelhertweg 15, Lelystad, 8219 PH, The Netherlands Published online: 01 Nov 2011.
To cite this article: I. J. R. Visser , E. A. ter Laak , N. W. van Dijk & W. Wouda (1991) Toxigenic Vibrio cholerae non‐O:1 isolated from a goat in the Netherlands, Veterinary Quarterly, 13:2, 114-118, DOI: 10.1080/01652176.1991.9694293 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694293
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
Downloaded by [Stony Brook University] at 22:47 20 October 2014
forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
84. Voogt HJ de. De hormonale behandeling van prostaatkanker. Ned Tijdschr Geneeskd 1980; 124: 876-83.
85. Vreeburg JTM, Samaun K, Verkade HJ, Verhoef P, Ooms MP, and Weber RFA. Effects of corticosterone on the negative feedback action of testosterone, 5a-dihydrotestosterone and estradiol in the adult male rat. J Steroid Biochem 1988; 29: 93-8. 86. Vreeburg JTM, Greef WJ de, Ooms MP, Wouw P van, and Weber RFA. Effects of adrenocorticotropin and corticosterone on the negative feedback action of testosterone in the adult male rat. Endocrinology 1984; 115: 977-83. 87. Ward IL and Weisz J. Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers. Endocrinology 1984; 114: 1635-44.
88. Wass JAH. The control of gonadotropin secretion. In: JAH Wass and MF Scanlon (Eds) Neuroendocrine Perspectives Vol 6. New York: Springer-Verlag, 1989.
Downloaded by [Stony Brook University] at 22:47 20 October 2014
89. Welsh TH and Johnson BH. Stress-induced alterations in secretion of corticosteroids, progesterone, luteinizing hormone, and testosterone in bulls. Endocrinology 1981; 109: 185-90. 90. Welsh TH, Randel RD, and Johnson BH. Interrelationships of serum corticosteroids, LH and testosterone in male bovine. Arch Andrology 1981; 6: 141-50. 91. Wiepkema PR. Over gedragsstoringen bij dieren in de veehouderij. Tijdschr Diergeneeskd 1985; 110: 12-20. 92.
Yelvington DB, Weiss GK, and Ratner A. Habituation of the prolactin response in rats to psychological stress. Psychoneuroendocrinology 1985; 10: 95-102.
SHORT COMMUNICATIONS
Toxigenic Vibrio cholerae non-0:1 isolated from a goat in the Netherlands I. J. R. Visserl, E. A. ter Laak2, N. W. van Dijki, and W. Woudai SUMMARY. A ease of enterotoxicosis in a goat at necropsy is described. The animal had died without clinical signs. Toxigenic Vibrio cholerae non-0: I was isolated from the intestines.
This species has not been reported earlier from healthy or diseased farm animals, such as goats, in the Netherlands.
INTRODUCTION
Vibrio cholerae serogroup 0:1 causes cholera, a human disease that has been endemic in India and Southeast Asia since ancient times. Periodically the disease spreads to the Middle East and Africa in epidemics and pandemics. Epidemic strains of serogroup 0:1 produce cholera toxin, whose action on the mucosal cells of the small intestine causes the diarrhoea that is characteristic of cholera (10). There are at least 72 other serogroups of V. cholerae that are called non-0:1 (7). Formerly these strains were called non-cholera vibrios (NCV) or non-agglutinable vibrios (NAG). Although the majority of the non-0:1 strains of V. cholerae isolated from the environment does not appear to be enteropathogenic (4), many strains of V cholerae non-0:1 produce a toxin identical or very similar to cholera toxin
(2, 4, 9, 23). Other strains, which do not produce cholera toxin, are enteropaI
2
Animal Health Service in Noord-Nederland, P.O. Box 361, 9200 AJ Drachten, The Netherlands. Central Veterinary Institute, Department of Bacteriology, Edelhertweg 15, 8219 PH Lelystad, The Netherlands.
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thogenic to animals experimentally (4, 5). These strains do not cause cholera epidemics, but do cause individual cases and outbreaks of cholera-like diarrhoea in humans. These had been only rarely reported from places all over the world since 1960 (21) but have been more frequently reported since 1972 (12). An additional virulence factor such as intestinal adhesion of the bacteria is also important in producing disease (4, 6). Other toxins produced by non-0:1 strains have also been described: for example, enterotoxins related to cholera toxin (23) and a toxin similar to the Escherichia coli heat stable enterotoxin (2). In contrast to V. cholerae 0:1, non-0:1 strains may also cause bacteraemia (22). The ecology of V cholerae was poorly understood until recently. Since 1977 V cholerae has been detected in water and mud samples from aquatic areas where cholera epidemics have never occurred, such as in England and the USA (3, 12). It has also been isolated from aquatic animals, such as shellfish, fishes, frogs and ducks (5, 12, 21), but these animal species act only as carriers for V cholerae. Most of the strains that have been found in brackish and fresh water areas worldwide are non-0:1 strains. The few 0:1 strains are generally non-toxigenic and have been found mostly in brackish water (4, 7). It is now believed that an aquatic environment is the natural reservoir for V. cholerae (1, 4, 7, 10). Although it has been reported that farm animals may also carry the organisms, references are very scarce (8, 20). After an outbreak of cholera, Sanyal et al. (18) isolated V cholerae from the faeces of cattle, goats, dogs and chickens in Varanasi, India, where cholera
has not been endemic. Some of the strains that were isolated from cattle and chickens were of the 0:1 type. Although cows appeared to be infected for 8 months at most, V cholerae was isolated only occasionally from goats. Clinical signs of disease were not observed in any of these animals. Under experimental conditions,
however, toxigenic strains of both 0:1 and non-0:1 type are pathogenic for a variety of laboratory animals (18). Only one report, however, exists on V cholerae in relation to enteric disease in animals. Rhodes et al. isolated V cholerae non-
0:1 from a foal, a lamb and two bisons with enteric disease in Colorado (17). This report describes the isolation of a toxigenic V cholerae non-0:1 strain from the small intestines of a goat raised in the Netherlands. CASE REPORT
In the spring of 1989 a goat was found dead and was destroyed without being examined. In July of 1989 two goats with no clinical signs of disease died within two days on the same farm. The second dead goat was sent to the Animal Health
Service for necropsy. The goat was found to be in a thriving condition. The intestines were extended and flabby and contained copious watery fluid. Other pathological lesions were not observed. The goat originated from a farm with 74 milk goats and 22 kids, all of Saanen and Toggenburger breed. The owner started goat husbandry in 1986 and all goats were bought in the Netherlands. Most kids were kept for breeding to enlarge the goat population on the farm. Except for the sudden deaths of the three goats noted above, the herd had had no disease problems. Numerous wild waterfowl and migrating birds lived in the environment of the farm. Laboratory examination Samples collected from the intestines contained no worm eggs or coccidia. For bacteriological examination, intestinal samples were inoculated onto two blood agar (BA) plates containing blood agar base no. 2 (Oxoid CM 271, Haarlem, The
Netherlands) and 7% sheep erythrocytes and onto a brilliant green agar plate (Oxoid CM 329). One BA plate was incubated anaerobically (GasPak anaerobic system; BBL, Becton Dickinson); the other media were incubated aerobically at THE VETERINARY QUARTERLY. VOL. 13, No. 2, APRIL 1991
115
37° C. After 24 h many flat, gray-brown, B-haemolytic colonies were detected on
the aerobically incubated BA in nearly pure culture. They contained Gramnegative, small, curved rods. Strain no. 16847 was isolated and further investigated. The oxidase test was positive. The API 20 E system (Api-Biomerieux, 's-
Hertogenbosch, The Netherlands) was used to identify the strain as Vibrio cholerae.
On the anaerobically incubated BA, many colonies with a double B-haemolytic
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zone were detected. These contained large, Gram-positive rods that were tentatively identified as Clostridium perfringens. No other microorganisms were isolated. A smear from the intestinal wall was examined microscopically and contained many Gram-positive, large, rod-like clostridia as well as Gram-negative rods. Because of the numerous clostridia detected in the smear and in the anaerobically cultured BA enterotoxaemia caused by Clostridium perfringens was the presumptive diagnosis. At that time, 24 h after the necropsy, the identification of the Gramnegative strain was started. The isolated strain of V cholerae was then sent to the Central Veterinary Institute for further examination and confirmation of the diagnosis. It was found that gray to cream coloured colonies with a brownish aspect grew rapidly on sheep blood agar. The diameter of colonies after 24 h incubation was 2 mm. Colonies were surrounded by a 13-haemolytic zone and contained Gram-negative curved rods.
The bacteria were highly motile, like rockets. The strain was able to grow anaerobically and produced acid but no gas from D-glucose, sucrose, maltose and mannitol. It produced catalase, oxidase, indole, lysine decarboxylase, ornithine decarboxylase and acetylmethylcarbinol. The strain reduced nitrate, hydrolysed gelatin and utilised citrate (Simmons medium) and was positive in the string test (19). The strain produced no acid from lactose, did not hydrolyse urea, produced
no H2S, was not susceptible to polymyxin (disc = 50 pm) and did not haemagglutinate chicken erythrocytes. At this stage the culture was typed as V cholerae, similar to biotype eltor, but negative in haemagglutination test. An API 20 E test produced number 5147124: Vibrio cholerae, with a 99.7% chance of correct identification. An API 20 NE test produced number 7074747: Vibrio cholerae, with a 98.5% chance of correct identification. The identification of the strain was confirmed by Dr. P. A. M. Guinée, National Institute for Public Health and Environmental Protection, Bilthoven, the Nether-
lands, who obtained additional results. Because antiserum raised against V cholerae 0:1 did not agglutinate the strain, it was concluded that the strain belonged to the NCV-group (V cholerae non-0:1). Furthermore, because the strain produced a verotoxin, it may have caused the pathologic signs of disease. Investigation into the origin of the infection
In September 1989, six water samples were collected from ditches around the grasslands where the goates grazed. Faeces samples were collected rectally from five goats chosen at random. These samples were investigated for the presence of V. cholerae by culturing them on BA, Plate Count Agar (Oxoid CM 325) and a selective medium according to Monsur (13). Vibrio cholerae was not detected in these samples. DISCUSSION
A high number of V. cholerae non-0:1 was isolated in nearly pure culture on a non-selective medium, incubated aerobically, from the small intestines of a goat. The only signs of disease in the goat were extended and flabby intestines containing 116
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copious watery fluid. We suggest the term enterotoxicosis to describe the pathological findings. If the V. cholerae non-0:1 was isolated from a human being, the strain would be considered the cause of the intestinal disorder. Whether V cholerae non-0:1 alone
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caused enterotoxicosis in the goat is unclear, because many C. perfringens organisms were also isolated from the same samples after anaerobic incubation. Clostridium perfringens is generally regarded as a cause of enterotoxaemia in various animal species, including goats (11, 16). Clostridium perfringens may also cause haemorrhagic or necrotic enteritis, but a severe watery diarrhoea is not characteristic in goats (11). Hence the isolated V cholerae strain might have been the cause of the disease, because toxin was also detected. Whether the two other goats that died earlier were infected with V cholerae non-0:1 is not known. The organism was not isolated from faeces samples from healthy goats of the farm, nor from water samples collected from ditches around the farm, although it may have been present in low undetectable numbers (1, 10). The origin and significance
of this infection is unclear. Vibrio cholerae is probably common in aquatic environments in the Netherlands. Some investigators have reported that the organism is more prevalent in summer, when temperatures are higher (3, 14, 15),
but others do not report a correlation between season and incidence (10, 12). Migrating ducks also contribute to periodically higher numbers of V cholerae in the environment (5). It seems worthwhile to look for the prevalence and pathogenicity of this organism in animals with intestinal disorders by using selective culture media (4, 13) to establish a possible association of cholera-like diseases in farm animals and V
cholerae. It would be of special interest to examine animals from areas with a rich waterfowl population. ACKNOWLEDGEMENTS
We thank C. M. F. Wagenaars (Lelystad, The Netherlands) for technical assistance and the late Dr. P. A. M. Guinée, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands, for confirmation of the diagnosis and supply of the Monsur agar plates. REFERENCES 1.
2. 3.
Anonymous. Aquatic niche is haven for Vibrio cholerae. ASM News 1989; 55: 642. Arita M, Takeda T, Honda T, and Miwatani T. Purification and characterization of Vibrio cholerae non-0:1 heat-stable enterotoxin. Infect Immun 1986; 52: 45-9. Bashford DJ, Donovan TJ, Furniss AL, and Lee JV. Vibrio cholerae in Kent. Lancet 1979; 37: 436-7.
4. Baumann P and Schubert RHW. Family II. Vibrionaceae Veron 1965, 5245 AL. In: Bergey's manual of systematic bacteriology I, p. 516-38, 1984. Ed: NR Krieg and JG Holt. Williams & 5. 6.
7.
Wilkins, Baltimore. Bisgaard M, Sakazaki R, and Shimada T. Prevalence of Non-Cholera Vibrios in cavum nasi and pharynx of ducks. Acta Pathol Microbiol Scand Sect B 1978; 86: 261-6.
Datta-Roy K, Dasgupta C, and Ghose AC. Haemagglutination and intestinal adherence properties of clinical and environmental isolates of non-0:1 Vibrio cholerae. Appl Environ Microbiol 1989; 55: 2403-6. Farmer III JJ, Hickman-Brenner FW, and Kelly MT. Vibrio. In: Manual of Clinical Microbiology,
Ed: Lenette EH, Valows A, Hausler Jr WJ, and Shadomy HJ. Fourth ed. Am Soc for Microbiology, Washington DC 1985; 282-301. 8. Hamza PA and Chatterjee BD. Vibrios of public health importance - occurrence in animal faeces. Indian Vet J 1982; 59: 1-4.
9. Honda T, Lertpocasombat K, Hata A, Miwatani T, and Finkelstein RA. Purification and characterization of a protease produced by Vibrio cholerae non-0:1 and comparison with a protease of V cholerae 0:1. Infect Immun 1989; 57: 2799-803. 10. Janda M, Powers C, Bryant RG, and Abbott SL. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin Microbiol Rev 1988; 1: 245-67. THE VETERINARY QUARTERLY. VOL 13,
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11. Jubb KVE, Kennedy PC, and Palmer N. Pathology of domestic animals. Third ed. Academic Press, Inc New York 1985; 149-55. 12. Kaper J, Lockman H, Colwel RR, and Joseph SW. Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay. Appl and Environ Microbiol 1979; 37: 91-103. 13. Monsur KA. Bacteriological diagnosis of cholera under field conditions. Bull WHO 1963; 28: 387-9.
Muller HE, Bockemiihl J, Burkhardt F und Graevenitz A von. Isolierung und Identifizierung von Vibrionaceae. Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1980; 248: 148-61. 15. Nacescu N and Ciufecu C. Serotypes of Nag Vibrios isolated from clinical and environmental sources. Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1978; 240: 334-8. 16. Oxer DT. Enteroxaemia in goats. Aust Vet J 1956; 32: 62-6.
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17. Rhodes JB, Schweitzer D, and Ogg JE. Isolation of non-0:1 Vibrio cholerae associated with enteric disease of herbivores in Western Colorado. J Clin Microbiol 1985; 22: 572-5. 18. Sanyal SC, Singh SJ, Tiwari IC, Sen PC, Marwah SM, Hazarika UR, Singh H, Shimada T, and Sakazaki R. Role of household animals in maintenance of cholera infection in a community. J Infect Dis 1974; 130: 575-9. 19. Smith Jr HL. A presumptive test for vibrios: the 'string' test. Bull WHO 1970; 42: 817-8. 20. Shewan JM and Véron M. Genus I. Vibrio Pacini 1854, 411. In: Bergey's manual of determinative
bacteriology, p 340-45. Eight ed. 1974. Ed: RE Buchanan and NE Gibbons. The Williams & Wilkins Company, Baltimore.
21. Szeness L, Sey L und Szeness A. Bakteriologische Untersuchungen des Darminhalts von Wasservogeln, Fischen und Fröschen mit besonderer Beriicksichtigung des Vorkommens von Nicht-Cholera-Vibrionen (NCV). Zentralbl Bakteriol Hyg Abt I Orig Reihe A, 1979; 245: 8995.
22. Wistrom J. A case of non-0:1 Vibrio cholerae bacteremia from Northern Europe. J Infect Dis 1989; 160: 732.
23. Yamamoto K, Takeda Y, Miwatani T, and Graig JP. Evidence that a non-0:1 Vibrio cholerae produces enterotoxin that is similar but not identical to cholera enterotoxin. Infect Immun 1983; 41: 896-901.
Prevalence of Leptospira interrogans serovar hardjo antibodies in milk in Belgian dairy herds P. P. Dom1, F. Haesebrouckl, R. Vandermeersch2, J. Descamps3, and K. Van Ommeslaeghe4 SUMMARY. Pooled milk samples were collected from 2000 Belgian dairy herds in the autumn
of 1989. Antibodies against Leptospira interrogans serovar hardjo were detected in 9.2% of the herds, with the incidence being higher in the southern part of the country. INTRODUCTION
Leptospira interrogans serovar hardjo (L. hardjo) is the most common causative agent of bovine leptospirosis in countries throughout the world. In all the countries
bordering on Belgium (the Netherlands, Germany and France), L. hardjo I
2 3
4
Laboratory of Veterinary Bacteriology, Faculty of Veterinary Medicine, University of Ghent, Casinoplein 24, B-9000 Ghent, Belgium. Regional Veterinary Investigation Centre, Industrielaan 15, B-8820 Torhout, Belgium. SmithKline Beecham Animal Health Products, Rue de l'Institut 89, B-1330 Rixensart, Belgium. SmithKline Beecham Animal Health Products, Place de l'Université 16, B-1348 Louvain-la Neuve, Belgium.
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