sign in sign up
<<
Home , Cholera, Vibrio cholerae

JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1985, p. 572-575 Vol. 22, No. 4 0095-1137/85/100572-04$02.00/0 Copyright © 1985, American Society for Microbiology Isolation of Non-O1 cholerae Associated with Enteric Disease of Herbivores in Western Colorado JOHN B. RHODES,' DARREL SCHWEITZER,' AND JAMES E. OGG2* We, 'rn Slope Animal Diagnostic Laborator, Colorado State Universitv, Grand Jiunctioni, Colorltado 81501,1 andb Dep ment of Microbiology and Environmental Health, Color-ado State University, Fort Collinis, ColoradXo 805232 Received 9 May 1985/Accepted 15 July 1985

Non-OI was isolated from a horse (Equus caballus), a lamb (genus Ovis), and two American buffalo (Bison bison) suffering from enteric disease in the western part of Colorado. In 1981, a foal died of apparent respiratory failure. Necropsy findings included heart failure and . V. cholerae serovar 347 (Smith) was isolated from the colon of this animal. V. cholerae serovar 27 (Smith) was isolated in 1983 from the intestine of a feedlot lamb suffering from and severe watery . In 1984, an enteric disease occurred in a herd of American bison. The sick animals were depressed and separated from the herd, dying in about 3 days. Of approximately 100 adult bison, 7 died. Necropsy of one animal revealed that gross lesions were limited to the . V. cholerae serovar 27 (Smith) was isolated from the abomasum, duodenum, and colon of this animal. A swab specimen from the intestine of another dead bison also yielded V. cholerae serovar 27 (Smith).

Non-O1 Vibrio cholerae is biochemically indistinguishable However, in one of the lambs necropsied (but not represen- from 01 V. cholerae, the causative agent of tative of the herd problem), severe fluid diarrhea and pneu- in humans, but does not agglutinate in 0 group 1 monia were observed. Pasteiuirella halemolytici was isolated antiserum. Some strains of non-O1 V. cholerae produce an from the lungs of this lamb, and V. cholerae was isolated identical to that of 01 V. cholerae (28). Those from the intestine. No similar cases were presented. environmental and clinical isolates that do not produce Bison. In September 1984, a bison raiser noticed an can possess other factors which outbreak of disease in which 7 of approximately 100 adult make them pathogenic (3, 21). Human gastroenteritis caused bison died. The sick bison were depressed and separated by non-O1 V. cholerae is well documented and has been from the herd, dying in about 3 days. Symptoms reported by reported from widespread geographical areas (3, 9, 19, 20). the owner were diarrhea, , serous nasal discharge Human are most often associated with with green frothy ingesta running from the nose, lacrimation, consumption, saltwater exposure, or foreign travel (7, 9, 20). and reddened conjunctiva. Specimens of the intestinal con- In the United States, non-O1 V. cholerae has been found tents, abomasum, duodenum, and colon of the bison yielded along the coasts of the Gulf of Mexico (20), the Chesapeake V. cholerae upon culturing. All of the sick bison were from Bay (6, 11), and the Pacific Ocean (13, 14) and has caused a herd purchased about a month earlier and pastured with his some human infections in inland regions (9; S. M. Gelbart existing herd. The bison had been on a new pasture for about and M. Prabhudesai, Abstr. Annu. Meet. Am. Soc. Micro- 1 week. Examination of the pasture revealed no toxic plants. biol. 1985, C120, p. 230). After completion of necropsy and microbiologic examina- Although non-O1 V. cholerae was isolated from a variety tion, the owner was advised to move the bison out of the new of animals in and (23, 24), no clinical pasture and treat them with , one of the antimi- symptoms of disease were reported in animals under natural crobial agents that inhibits V. choler(w. The owner chose conditions. We report here the isolation of non-O1 V. because of the ease of administration and cholerae from herbivores with enteritis. reported no further problems after initiating this therapeutic (This paper was presented in part at the 85th Annual regimen. Meeting of the American Society for Microbiology, Las Vegas, Nev. [J. B. Rhodes and J. E. Ogg, Abstr. Annu. MATERIALS AND METHODS Meet. Am. Soc. Microbiol. 1985, C121, p. 320].) Necropsy and histopathology. Necropsies of the foal, lamb, CASE REPORTS and one bison and histopathologic examination of tissues were Horse. In July 1981, a foal (about 2.5 months old) died from the foal and bison performed. after treated for 1 week for apparent viral pneumonia. Microbiology. Samples from the colon of the foal, the being and The foal had respiratory difficulty and a temperature of intestine of the lamb, and the abomasum, duodenum, bison were cultured on the 39.5°C and had lost 50 pounds in 3 days. No diarrhea was colon of the necropsied following reported; however, lesions were found in the colon at media: Trypticase (BBL Microbiology Systems, Cockeys- blood and Hektoen necropsy. V. cholerae and were ville, Md.) soy agar with 5% sheep (BA) isolated from these lesions. enteric agar (HE) (BBL). Selenite-F Enrichment (BBL) was same to test for Lamb. In August 1983, a lamb feedlot operator noticed inoculated with the samples sudden deaths of lambs approximately 6 months old. The species. Swab specimens obtained from the intestines of two and mailed to the Western Animal of the outbreak was found to be Haemophills somnlus. other dead bison Slope Diagnostic Laboratory were inoculated directly onto thiosul- fate-citrate- -sucrose (TCBS) agar. The cultures * Corresponding author. were incubated at 37°C for 18 to 24 h. HE plates were 572 VOL. 22, 1985 V. CHOLERAE ASSOCIATED WITH ENTERITIS IN HERBIVORES 573 streaked with Selenite-F cultures, incubated for 24 h, and identified these two colony types as V. cholerae and A. examined for lactose-nonfermenting colonies. hydrophila. Only yellow-orange colonies were present on Eleven specimens of from the bison herd (after the HE plate; they were assumed at the time to be Esche- oxytetracycline treatment had been initiated) and two richia coli. samples from the pasture pond and ditch were incubated (ii) Lamb. Necropsy of the lamb revealed that the ventral overnight at 37°C in alkaline peptone water (1% peptone, 1% portions of the lungs were consolidated and covered with NaCl [pH 8.4]). The alkaline peptone water enrichments fibrin. Fluid and a mucopurulent exudate filled the lower were streaked onto TCBS agar, and the plates were incu- airways. The intestinal wall was thin and moderately red- bated for up to 48 h and examined for growth typical of V. dened. The lumen was dilated and contained excess watery cholerae. Isolated colonies appearing on the agar media green feces containing shreds of mucosa. were subcultured onto BA to establish stock cultures. BA plates streaked with intestinal contents from the lamb were identified by methods outlined in the Man- yielded mainly smooth, hemolytic, oxidase-positive colonies ual of Clinical Microbiology (17), supplemented when nec- that imparted a greenish color to the hemolyzed areas of the essary by reference to Bergey's manuals (5, 15) and by use of medium. HE plates yielded predominantly yellow-orange the API 20E system (Analytab Products, Plainview, N.Y.). colonies assumed to be E. coli. Further testing of subcul- The fermentation reactions of bacteria were determined with tures of the hemolytic colonies showed them to be V. the aid of the DMS Rapid CH system (Analytab Products). cholerae. No Salmonella species were isolated from The directions of the manufacturer were followed when Selenite-F Enrichment inoculated with specimens of the using the API 20E system and the DMS Rapid CH system. intestinal contents. Isolates giving biochemical reactions typical of V. cholerae (iii) Bison. Gross lesions in the bison were limited to the were tested for agglutination in 01 V. cholerae polyvalent gastrointestinal tract. The mucosa of the rumen was red- antisera obtained from Harry L. Smith, Jr. (Vibrio Refer- dened, and the villi adhered to each other. The abomasal ence Laboratory, Jefferson Medical College, Philadelphia, wall was thickened by edema, and the mucosa was dark Pa.). red-purple. Shallow, coalescing erosions and ulcerations Serotyping of the non-O1 V. cholerae isolated from were seen in the and were most severe in the the foal was performed by Harry L. Smith, Jr. Biochemical colon and cecum. verification, measurements of cholera toxin production, and Histopathologic examination of tissues from the bison serotyping of the lamb and bison isolates with Smith antisera revealed that the most significant lesions were limited to the against non-O1 V. cholerae (25) were performed at the gastrointestinal tract and consisted of nonsuppurative in- Enteric Bacteriology Section, Centers for Disease Control, flammation. The abomasal mucosa was focally necrotic, and Atlanta, Ga. Additional biochemical verification of V. chol- large numbers of bacteria were located deep within these erae isolates was done at the National Veterinary Services necrotic foci. In some segments of the intestine changes Laboratory, Ames, Iowa (for the lamb isolates) and at the were even more severe and the mucosa was nearly de- Colorado Department of Health, Denver (for the bison stroyed. isolates). All BA plate cultures prepared from the specimens taken Samples from the intestine of the necropsied bison were from the abomasum, duodenum, and colon of the bison examined for enteric parasites by direct microscopic exam- yielded a predominance of large, smooth, hemolytic colonies ination and for parasite ova and coccidial oocysts after that imparted a greenish color to the hemolyzed areas of the concentration by sulfate flotation. The direct micro- medium. HE plates inoculated at the same time as BA plates scopic examination of intestinal contents was used for the yielded only yellow-orange colonies. No Salmonella species detection and presumptive identification of Campylobacter were isolated after incubation of specimens of intestinal species. Wet-mount preparations were observed with the aid contents in Selenite-F Enrichment. Smooth, yellow colonies of dark-field microscopy for bacteria of characteristic size appeared on one of the plates of TCBS agar streaked with and morphology and exhibiting the corkscrew, darting type "mailed-in" swabs of intestinal contents from other dead of motility of campylobacters (12, 15). No attempt was made bison. The hemolytic colonies on BA and the smooth, yellow to detect and culture enteric and chlamydiae in colonies on TCBS agar were oxidase positive and identified specimens collected during necropsy of the animals. as V. cholerae. No enteric parasites or Campylobacter species were ob- RESULTS served microscopically in specimens from the bison intes- Histopathologic and microbiologic findings. (i) Horse. The tine. principal necropsy findings in the foal were chronic passive No colonies of V. cholerae appeared on the TCBS agar congestion resulting from heart failure due to a large patent plates streaked with alkaline peptone water enrichment ductus arteriosus. Thrombosis of the anterior mesenteric cultures prepared from the 11 specimens of feces collected artery due to verminous arteritis caused poor circulation to from the bison herd after the initiation of oxytetracycline the intestine. Additional findings were an inflamed and therapy. Similarly, alkaline peptone water enrichment cul- edematous gastrointestinal tract. tures of the two water samples taken from the water sources Microscopic lesions in the foal consisted of erosion and serving the bison herd did not yield growth typical of V. focal ulceration of the gastric mucosa, with chronic nonsup- cholerae when plated on TCBS agar. purative inflammation and bacterial colonization. The intes- Characteristics of V. cholerae isolates. Excellent identifica- tine was hyperemic and edematous, and both mononuclear tion of V. cholerae was obtained with the API 20E system. and polymorphonuclear leukocytes were found in increased All the animal isolates failed to agglutinate in antiserum numbers in the mucosa. against V. cholerae 0 group 1. The non-O1 V. cholerae The BA plate inoculated with a sample from the colon of strain from the horse was Smith serovar 347; the strains from the foal had two types of hemolytic colonies in approxi- the lamb and bison were Smith serovar 27. Cholera toxin mately equal numbers, and the confluent areas of hemolysis production by non-O1 V. cholerae strains from the lamb and were greenish in color. Using the API 20E system, we bison was not demonstrated by either the Y-1 adrenal cell 574 RHODES ET AL. J. CLIN. MICROBIOL. assay or the enzyme-linked immunosorbent assay; the V. because (i) it was the predominant organism isolated from all cholerae strain from the horse was not tested for toxin the cultured tissues; (ii) it was isolated from two dead bison; production. The characteristics of the non-01 V. cholerae (iii) no other enteropathogenic bacteria were isolated from isolates are shown in Table 1. the intestinal contents; (iv) some tissue damage of the intestinal wall observed in the dead bison appeared to be DISCUSSION similar to that described in infant rabbits infected experi- mentally with cholera toxin-negative strains of non-01 V. The non-01 V. cholerae strain isolated from the foal may cholerae (18); (v) no enteric parasites were observed in have contributed to the gastroenteritis, but the A. hydrophila specimens of intestinal contents or in the histopathologic strain also could have been involved. Perhaps both of these preparations of tissues from the dead bison; (vi) organisms were opportunistic invaders that multiplied be- treatment to control V. cholerae appeared to resolve the cause of impaired intestinal circulation due to heart failure disease outbreak in the herd; and (vii) non-01 V. cholerae is and verminous arteritis. an enteric and the herd of bison had an enteric The pathogenic role of V. cholerae in the intestinal infec- disease. tion in the lamb cannot be determined from the one case Malignant catarrhal fever and bovine diarrhea occur presented in this report; however, the lamb did have a in bison, but gross and microscopic examination of appro- voluminous "rice-water" stool, a condition reported for priate tissues revealed no lesions characteristic of these some non-01 V. cholerae infections in humans (1, 8, 19). diseases. Although other viruses and chlamydiae cannot be Non-01 V. cholerae serovar 27 most likely contributed to entirely eliminated as potential agents in this outbreak of the outbreak of the gastrointestinal disease in the bison acute diarrheal disease in the bison, we are not aware of reports of these organisms causing enteritis in adult animals. Campylobacter species are common inhabitants of the TABLE 1. Characteristics of non-O1 V. cholerae from horse, intestinal tracts of animals, and some species have been lamb, and bison associated with intestinal diseases of cattle (2, 10). Direct dark-field microscopic examination of specimens from the Test or substrate Reactiona intestines of the bison did not reveal bacteria with the Indophenol oxidase. + characteristic morphology and motility of campylobacters. Arginine dihydrolase . Although this procedure may not be as sensitive as selective Lysine decarboxylase. + culture methods for the detection of Campylobacter species, Ornithine decarboxylase. + it was an indication that this pathogen, if present, was not Indole. + present in large numbers in the gastrointestinal tract of the Urease . dead bison examined. Simmons citrate. + The source(s) of the non-01 V. cholerae in these cases of Kligler agar...... Alk/A gastroenteritis in herbivores in western Colorado remains H2S (in Kligler iron agar). is more widespread in inland o-Nitrophenyl-l(-D-galactopyranoside ...... + unknown. Perhaps V. cholerae Voges-Proskauer (37C) ...... + areas in this country than previously thought. Non-01 and Motility ...... + 01 V. cholerae strains are usually found in brackish water Gelatinase ...... + along coastal areas (6, 11), although they have been isolated Nitrate reductase ...... + in inland areas. In several surveys conducted in Kent, MacConkey agar growth ...... + , V. cholerae was found to occur sporadically in Hemolysis (sheep blood) ...... + small numbers in but regularly in large numbers Growth in 0, 3, or 5% NaCl ...... + in brackish water during the summer months (16, 26). Growth in 7% NaCl. Brackish water can be found in nonmountainous areas of Growth at 25, 35, or 40°C ...... + + western Colorado, especially in mid to late summer, when all "String test" (17) ...... these Gas produced from . of these outbreaks occurred. The surface water in Acid produced from: areas has a relatively high concentration of salts, particularly Glucose ...... + in areas where the water is stagnant and evaporating. Lactose ...... Our inability to detect V. cholerae in the water samples Sucrose ...... + taken from the pasture where the bison herd grazed may Arabinose. have been due to a number of factors, including the limited Cellobiose. volume of water tested, the incubation temperature (37°C) of Inositol . the enrichment broth, competing microflora, or noncultur- Mannitol ...... + c able but viable cells or all of these. V. cholerae, E. coli, Mannose ...... in Gluconate ...... + Salmonella enteritidis, and Shigella species may exist Glycerol ...... + aquatic environments in a state (4, Trehalose ...... + 22, 27). Salicin . To our knowledge, this is the first report of V. cholerae Galactose ...... + associated with enteric disease in herbivores under natural Maltose ...... + conditions. The strains isolated from the bison and lamb did Vibriostatin 0/129 ...... S not produce cholera toxin; however, it is not unusual for Polymyxin B (50 U) ...... R these types of non-01 V. cholerae to still be pathogenic (18, Cholera toxin...... _d 21). The finding of V. cholerae in these herbivores points to a +, Positive reaction; -, negative reaction; S, susceptible; R, resistant; another possible vector, in addition to aquatic birds (16, 26), Alk/A, alkaline slant, acid butt in 24 h. for this organism in the environment in inland areas of the b Lamb strain negative; horse and bison strains positive in 3 days. States. we cannot rule out the possibility c Lamb strain positive; horse and bison strains negative. United However, d The horse strain was not tested for cholera toxin. that V. cholerae may be indigenous to certain , VOL. 22. 1985 V. CHOLERAE ASSOCIATED WITH ENTERITIS IN HERBIVORES 575 especially brackish water (6, 26), and therefore that herbi- 88:602-606. vores drinking the water may become transient or chronic 10. Jones, F. S., M. Orcutt, and R. B. Little. 1931. (Vibrio carriers of the organism. Also, under the proper conditions, jejuni, N.SP.) associated with intestinal disorders of cows and calves. J. Med. 53:853-864. the contribute to or cause enteritis in Exp. organism may possibly 11. Kaper, J., H. Lockman, R. R. Colwell, and S. W. Joseph. 1979. herbivores. Ecology, serology, and enterotoxin production of Vibrio chol- It is important to point out that the V. clholerae in these ercae in Chesapeake Bay. Appl. Environ. Microbiol. 37:91-103. cases was first detected by the hemolysis and greenish color 12. Kaplan, R. L. 1980. Campylobacter, p. 235-241. In E. H. on the BA plates. The HE plates had only yellow-orange Lennette. A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.). colonies that were mistakenly assumed to be all lactose- Manual of clinical microbiology, 3rd ed. American Society for fermenting coliforms (e.g., E. coli). However, V. cholerae Microbiology, Washington, D.C. does grow on HE plates, forming yellow-orange colonies 13. Kenyon, J. E., D. C. Gillies, D. R. Piexoto, and B. Austin. 1983. through sucrose fermentation. These colonies are easily Vibrici cholerae (non-O1) isolated from California coastal wa- mistaken for E. coli colonies. should be ters. Appl. Environ. Microbiol. 46:1232-1233. Microbiologists 14. Kenyon, J. E., D. R. Piexoto, B. Austin, and D. C. Gillies. 1984. aware of this when culturing for enteric . The Seasonal variation in numbers of Vibrio cholerae (non-O1) hemolysis and greenish color have also been observed with isolated from California coastal waters. Appl. Environ. Micro- A. hydrophila strains. biol. 47:1243-1245. The isolation of V. cholerae is becoming more common in 15. Krieg, N. R., and J. G. Holt (ed.). 1974. Bergey's manual of the United States because clinical microbiologists recognize systematic bacteriology. vol. 1. The Williams & Wilkins Co., its role in human disease. Normally, microbiologists in Baltimore. animal diagnostic laboratories do not attempt to culture V. 16. Lee, J. V., D. J. Bashford, T. J. Donovan, A. L. Furniss, and cholerace from animals with gastroenteritis. We hope that P. A. West. 1982. The incidence of Vibrio cholerae in water, this will make veterinarians and more animals and birds in Kent, England. J. Appl. Bacteriol. report microbiologists 52:281-291. aware of V. cholerae as another possible participant in 17. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and J. P. Truant enteric infections of herbivores. (ed.). 1980. Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. ACKNOWLEDGMENTS 18. Madden, J. M., W. P. Nematollahi, W. E. Hill, B. A. McCardell, We thank Betty J. Ogg for her excellent secretarial assistance, and R. M. Twedt. 1981. Virulence of three clinical isolates of Harry L. Smith, Jr., for serotyping the horse isolate and supplying Vibrio cholerae non-O-1 serogroup in experimental enteric the V. cholerae 0 group 1 antisera, and Archibald F. Alexander infections in rabbits. Infect. Immun. 33:616-619. (Director, Colorado State University Animal Diagnostic Laborato- 19. McIntyre, 0. R., J. C. Feeley, W. B. Greenough III, A. S. ries) for his review of the manuscript and helpful suggestions. We are Benenson, S. I. Hassan, and A. Saad. 1965. Diarrhea caused by grateful to the Enteric Bacteriology Section, Centers for Disease non-cholera vibrios. Am. J. Trop. Med. 14:412-418. Control, for serotyping the lamb and bison isolates and testing them 20. Morris, J. G., R. Wilson, B. R. Davis, I. K. Wachsmuth, C. F. for the production of cholera toxin. Riddle, H. G. Wathen, R. A. Pollard, and P. A. Blake. 1981. Non-O group 1 Vibrio cholerae gastroenteritis in the United LITERATURE CITED States: clinical, epidemiologic, and laboratory characteristics of 1. Aldova, E., K. Laznickova, E. Stepankova, and J. Lietava. 1968. sporadic cases. Ann. Intern. Med. 94:656-658. 21. R. Isolation of nonagglutinable vibrios from an enteritis outbreak in Nishibuchi, M., J. Seidler, D. M. Rollins, and S. W. Joseph. Czechoslovakia. J. Infect. Dis. 118:25-31. 1983. Vibrio factors cause rapid fluid accumulation in suckling 2. Al-Mashat, R. R., and D. J. Taylor. 1980. Canmpvlobacter spp in mice. Infect. Immun. 40:1083-1091. enteric lesions in cattle. Vet. Rec. 107:31-34. 22. Roszak, D. B., D. J. Grimes, and R. R. Colwell. 1984. Viable but 3. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Diseases of nonrecoverable stage of Salmonella enteritidis in aquatic sys- humans (other than cholera) caused by vibrios. Annu. Rev. tems. Can. J. Microbiol. 30:334-338. Microbiol. 34:341-367. 23. Sack, R. B. 1973. A search for canine carriers of Vibrio. J. 4. Brayton, P., D. Roszak, S. A. Huq, L. M. Palmer, D. J. Grimes, Infect. Dis. 127:709-712. and R. R. Colwell. 1984. significance of human 24. Sanyal, S. C., S. J. Singh, I. C. Tiwari, P. C. Sen, S. M. pathogens in the ocean, p. 263-267. In Oceans 84. Marine Marwah, U. R. Hazarika, H. Singh, T. Shimada, and R. Technology Society, Washington, D.C. Sakazaki. 1974. Role of household animals in maintenance of 5. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's cholera in a community. J. Infect. Dis. 130:575-579. manual of determinative bacteriology. 8th ed. The Williams & 25. Smith, H. L., Jr. 1979. Serotyping of non-cholera vibrios. J. Wilkins Co., Baltimore. Clin. Microbiol. 10:85-90. 6. Colwell, R. R., and J. Kaper. 1977. Vibrio cholerae. Vibrio 26. West, P. A., and J. V. Lee. 1982. Ecology of Vibrio species, parahaemol'ticus and other vibrios: occurrence and distribu- including cholerae in natural waters in Kent, England. J. Appl. tion in Chesapeake Bay. Science 198:394-396. Bacteriol. 52:435-448. 7. DePaola, A. 1981. Vibri-i cholerae in marine and environ- 27. Xu, H.-S., N. Roberts, F. L. Singleton, R. W. Attwell, D. J. mental waters: a literature review. J. Sci. 46:66-70. Grimes, and R. R. Colwell. 1982. Survival and viability of 8. El-Shawi, N., and A. J. Thewaini. 1969. Non-agglutinable nonculturable and Vibrio cholerae in the estu- vibrios isolated in the 1966 epidemic of cholera in Iraq. Bull. arine and marine environment. Microb. Ecol. 8:313-323. W.H O. 40:163-166. 28. Yamamoto, K., Y. Takeda, T. Miwatani, and J. P. Craig. 1983. 9. Hughes, J. M., D. G. Hollis, E. J. Gangarosa, and R. E. Weaver. Purification and some properties of a non-O1 Vibrio cholerae 1978. Non-cholera vibrio infections in the United States: clini- enterotoxin that is identical to cholera enterotoxin. Infect. cal, epidemiologic, and laboratory features. Ann. Intern. Med. Immun. 39:1128-1135.