Studies on escherichia coli serotypes in animals

Media

Part of Acta Medica Philippina

Title: Studies on escherichia coli serotypes in animals
Creator: Jacalne, Antonio V.; Arago, Potenciano R.
Language: English
Source: Volume XV (1) July-September 1958
Year: 1958
Subject: Escherichia coli
Rights
Fulltext: STUDIES ON ESCHERICHIA COLI SEROTYPES IN ANIMALS¥ ANTO~IO V. JACALNE, D.V.ll. POTE1'"CIANO R. ARAGON, M.D., l'tl.P.H. Department of Medical Microbiology, Institute of Hygiene University of the Philippines The coli group of organisms has long been recognized as normal inhabitants of the intestinal tract of man and animals and their presence in food and water has been universally used as index of fecal contamination. During the past few years, a number of European innstigators (1, 2, 3) reported the association of Esclierichia coli serotypes with outbreaks of infant diarrhea and gastro-enteritis (infantile coli-enteritis) in young children. Essentially similar epidemiologic and serologic findings were likewise reported by various American investigators (4, 5, 6, 7, 8, 9) since the discovery of the first E.coli serotype by Bray (10) in 1945 ancl the second serotype by Giles, et. al. (11) in 1947. In confirmation of these reports, feeding experiments on adult human \'Olunteers and infants have been described by Ferguson, (12) Neter, (13) and June (14). The transmissibility and reproducibility of the disease in animals have also been successfully demonstrated and confirmed by Namioka (15) and Dunne, d. al. (16). As a result of these pioneering experiments in man and animals, it is now universally recognized that certain "enteropathogenic E. coli cause epidemic gastro-coli-enteritis in infants and young children. With the development of serologic technic by Kauffman (17) and the improvement of methods of isolation and identification by Ewing (18), more E. coli serotypes have been reported in recent years not only in young children and animals but a)so from sources like soil and ,·egetables. While marked progress has been made abroad in the study of Eschel'ichia coli serotypes, very limited information has been • Supported b~' a rese111'd1 grnnt from tlw l'nin•rsity o( tlw Philip11i1ws. 44 ACTA MEDICA PHILIPPl~A reported 1ocally. The first report on the existence of enteror.athogenie E.coli (026:B6, 055:B5, Olll:B4 and 0127:B8) associated with diarrhea-enteritis in infants in the Philippines were made by Aragon, et. al. (19) and Guerrero, et. al. (20) in separate investigations carried out in the Philippine General Hospital and San Lazaro Hospital, respectively. In addition, Guerrero points out that A.P. de Roda has isolated eleven E. rnli serotypes in the City of Manila. The occurrence, however, of enteropathogenic E. coli in animals has never been reported locally. The isolation of pathogenic E. coli from cases of "calf scours" or calf diarrhea was previously reported by Smith (~~1) in 1927 and by Lovell (22) in 1937. These reports were later confirmed by Orskov (23) in 1951 and in 1955, Glanta (24) successfully isolated E. coli serotype 026:B6 from calves with "white scours." The present study was carried out to determine whether four of the most common enteropathogenic Escheri.chi.a roli serotypes (026:B6, 055 :B5, 0111 :B4 and 0127 :BS) are present in the feces of animals closely associated with man. MATERIALS AND METHODS The present study was conducted in the Department of Medical Microbiology, Institute of Hygiene, University of the Philippines, during the period from August 1957 to February 1959, Animals examined: The 856 animals included in this study consisted of 368 rats, 232 dogs and 256 pigs. The rats (R. ratt:us, R. non1egicus and M-us mw~c-ulus) were obtained from the Bureau of Quarantine and the Section of Insect and Vermin Control of the Manila Health Department. A number of rats caught in the neighboring provinces of Batangas, Bulacan and Rizal and which were used in a separate investigation were also included in this study. All the live rats were killed with ether and were immediately autopsied in the laboratory. The intestines of each rat were isolated with forceps and were examined for any gastrointestinal disorder prior to the collection of the specimen. The consistency of the intestinal contents was examined and a small portion was streaked on a differential plating media with a sterile inoculating loop. Dead rats were similarly examined. ESCHERICHIA COLI SEROTYPES 45 The dogs used in this study included those found free of diarrhea and which were confined in the Pasay and Manila city pounds and SPCA (Society for the Prevention of Cruelty to Animals) compound, Dogs brought to the Veterinary Clinic of Dr. Nicanor Carlos in the district of Malate for rabies vaccination were also included. Clinical materials from these animals were collected with the use of sterile rectal swabs moistened with sterile normal saline and placed in individual sterile test tubes. The swabs were brought immediately to the laboratory and inoculated in the plating media. All the rectal swabs from pigs were collected at the Manila city abattoir before the pigs were butchered. E. coli typing sera: The anti "OB" and anti "O" specific typing scra employed in this study were prepared by immunizing healthy rabbits with pathogenic E. coli serotypes 055 :B5, 0111 :B4, 0127 :BS and 026:B6 obtained from the Communicable Disease Center, United States Public Health Service. The technic used in the preparation of the typing sera was based on the method described by Ewing (25). Method of isolation and identification: The method employed in the isolation and identification of E. coli serotypes from the animals was based on the techni(]ue prescribed by Ewing and Edwards (18, 26) with some modifications. All fecal samples and rectal swabs from the different animals were inoculated into eosin methylene blue (EMB) agar, MacConkey agar and occasionally blood agar plates. This was done by surface streaking and swabbing. After 16-20 hours incubation at 37°C. The inoculated plates were examined for the presence of Escherichia coli colonies. Typical E. coli colonies on EMB agar are smooth, round and low convex with entire edge 1-2 mm. in diameter and with characteristic -greenish metallic lustre. They are red in color in MacConkey and grayish white in blood agar, generally producing no hemolysis. With the l'Se of a sterile inoculating needle, several typical E. coli colonies ~bout 10-15 in number were picked from the different parts of ihe plating media and each was transferred into nutrient agar slants. The slants were incubated at 37°C ancl after ~O hours. 46 ACTA MEDICA PHILIPPINA a small portion of the surface growth from each slant was obtained with a sterile inoculating loop and tested by slide agglutination for preliminary screening. The test was accomplished by using pooled anti "OB" typing sera (Anti "OB" sera against 026:86, 055 :B5, 0127:88 and 0111 :B4). If agglutination Occurs, th~ test was repeated by using single anti "OB" specific typing sera. Again, if agglutination is observed, with any one of the four single anti "OB" typing sera, the growth was harvested with normal saline and heated at 100°C in a water bath for a period of one hour, to destroy the "B" component of the antigen. The heat killed antigen was then centrifuged for 30 minutes at 2,500 rpm and washed with normal saline. A thick suspension of the heated antigen was then prepared and retested by slide agglutination using single anti "OB" specific typing serum as above. A positive slide agglutination test with the individual anti "OB" typing sera is indicative of the presence of any one of the four E. coli serotypes. Confirmation as to the presence of the "0" component of the antigen was done by using single anti "O" specific typing sera against any of the four serotypes. Control tests were also made for each specimen to rule out false positive reaction. Other E. coli serotypes were not determined due to the lack of specific typing sera. No attempt was made to isolate other enteric pathogens and saprophytic bacteria. The physiological and morphological characteristics of the isolates were also studied to confirm the serologic findings. RESULTS The Escherichia coli serotypes isolated from diarrhea-free animals are shown in the table below. Of the 856 animals examined, 11.8 percent yielded pathogenic E. coli serotypes. From the table, it can be seen that Escherichia coli serotypes 0127 :BS is the predominating type in rats and dogs, while in swine, 055 :B5 is the most frequently isolated serotype. Of the three species of animals examined, rats yielded the highest percentage of E. coli serotypes. From the results obtained, it is evident that pathogenic E. coli serotypes isolated locally from children with diarrhea are also found in the feces of diarrhea-free animals. These results confirm the findings of Sakazaki and Namioka (27) in Japan ESCHERICHIA COLI SEROTYPES 47 PRIVALENCE OF PATHOGENIC ESCHERICHIA COLI SEROTYPES IN ANIMALS, MANILA, Aug. 1957 - Feb. 1959 s~~~;~e ·~~::~d~~~-!!::a~~:\'in~· ! ~~e:~ 026:B6 368 11 3.01 256 4 1.6 232 5 2.2 055:B5 368 12 :t:i: 256 IO 3.9 232 6 2.6 Olll:B4 0127:B8 368 368 !) 2.4! 256 IG 4.3, 256 8 3.1 232 7 2.7 232 3 1.3 10 4.3 who isolated pathogenic E. coli serotype 025 from dogs, swine and cattle, serotypes 055, 0126, and 0112 from dogs and cattle, and serotype 0124 from dogs and horses, except that they failed to isolated 0127 :BB and 0111 :B4 in the animals they examined. It is a well known fact that animals transmit a number of bacterial, viral and parasitic infections to man. According to Hull (28) there are forty diseases that are transmitted from animals to man, eight of which are primarily of human origin. In the Philippines, records show that rats, dogs and pigs share with man in the transmission of a number of bacterial, viral and parasitic infections. Since pathogenic E. coli serotypes has been established in infants in the Philippines, and since these organisms could be isolated from the feces of normal rats, dogs and pigs, it is not improbable that animals acquire the organisms from man or vice versa and therefore may serve as carriers and possibly reservoirs of pathogenic E. coli serotypes. It is not to be inferred, however, that animals are the chief sources of infection. U.S. Army laboratory personnel stationed in Japan (29) found that pathogenic E. coli were isolated in 8.'3 per cent of 3,620 soil samples and 3.9 per cent of 4,077 vegetables examined. Although a high percentage of pathogenic E. coli was isolated from these animals, it is not justifiable to draw definite conclusions as to whether animals are actually carriers of pa thogenic E.coli. Further studies on the association of pathogenic E. coli serotypes in animals and man will probably be fruitful in clarifying the role of animals as pathogenic E. coli carriers. 48 ACTA MEDICA PBILIPPINA SUMMARY A total of 856 animals were examined for the presence of enteropathogenic E. coli serotypes. About 11.8 per cent of the animals yielded pathogenic E. coli of the following serntypes: 055:B6, 026:B6, 0127:B8 and Olll:B4. ACKNOWLEDGMENTS We would like to express our gratitude to Dr. Nicanor Carlos of the City Pound; Dr. Dominador Rubio of the Manila City Abattoir; Dr. Atanacio Montaire of the SPCA; Dr. Jesus Azurin of the Bureau of Quarantine; and Mr. Irineo Montemajor of the Section of Insect & Vermin Control, Manila Health Deaprtment, for their kind cooperation in providing the animals used in this study. Our thanks are also due to Dr. Victor Valenzuela, Head Department of Biostatistics, Institute of Hygiene, U.P., for his invaluable suggestions in the preparation of the manuscript. REFERENCES 1. SMITH, J., GALLOWAY, W.H. and SPEIRS, A.L.: Infantile gastroenteritis with Speeial Reference to the Specific Serologirnl Type 055:B5 H6 (Beta type) of Bacterium coli. Jour. Hyg., 48:472, 1950. 2. KAUFFMAN, F. and DUPONT, A.: Esche1·ichia strains from Infantile Epidemic Castro-enteritis: Acta Path. Micro. Sca11cl., 27:5;)2, 1950. 3. TAYLOR, J. and CHARTER, R.E.: Isolation of Serological tnws of Bacterium coli in Two Residential Nu1"Se1·ies and Their Relation to Infantile Castro-enteritis. Jour. Path. and HaC"t., 1;4:7U:., 1952. 4. NETER, E., WEBB, C.R., SHUMWAY, C.N. MURDOCK, !\.I.R.: Study on Etiology, Epidemiology and Antibiotic Therapy of Infantile Diarrhea with Partit'ular Reference to Certain Serotyp(S of Es~heri chia coli. Am. Jour. Public Health, 41 :1490, ( Decembe1·), 1951. 5. MODICA, R.l., FERGUSON, W. W. DUCEY, E. F.: Epidemi · Infantile Diarrhea Associated with EsC"herichia coli lll:B4, Jour. Lab. Clin. Med. 39:122, 1952. 6. STOCK, A.H., and SHUMAN, M.E.: Epidemiologic Studies on Enteropathogenic Escherichia coli Diarrhea. Ann. of N.Y. Acacl. of Sci. 1;1;:108, (August), 1956. 7. STULBERG, C.S., ZUELZER, W.W. and PAGE, R.H.: Escherichia coli 0127:B8 A Pathogenic strain Causing Infantile Diarrhea. Ill-The ESCHERICHIA COLI SEROTYPES 49 Antibody Response of Infants. J. Immunology. ;1;:281, (April), 1956. 8. NETER, E., ZALEWSKI and Jo'ERGUSON, W.W.: Escherichia coli Hemagglutinin Response of Adult Volunteers to Ingested E. coli 055:B5. Pl'oc. Soc. Exptl. Biol. Med. 82:215, (FebJ"uuy), 1953. 9. NETER, E., WESTPHAL, 0., LUDERJTZ, 0. GINO, R.r.I. aml GOR· ZYNSKI, E.A.: D~monstration of antibodies Against Ente1·opathogenic Escherichia coli in Sera of Childl'en of Val'ious A!:'es. Pedia· tries, 16:801, 1955. 10. BRAY, J., Isolation of Antigenically Homogenous Strains of Ha("tel'ium coli Neopolitanum From Summer Diarrheas of Infants. Jo11r. Path. & Bact • .57:239, (April), 1945. 11. GILES, C., SANGSTER, G. and Sl\IITH, J.: E1iidcmic Gash·o-<'ntcritis of Infants in Aberdeen During 1947. Arch. Disease ChiMhoorl. f-':45, (March), 1949. 12. FERGUSON, W.W., and JUNE, R. C.: Experiments on Feeding Adult Volunteers with Escherichia coli lll:B4, A coliform organism Associated with I11fant Diarrhea. A111. Jou1·. Hyg . .5.i:l55, 1952. 13. NETER, E., KORNS, R. F., TRUSSELL, R. E.: Association of Escherichia coli scro·irroup 0111 with Two Hospital Outbreaks of E1iidemic Diarrhea of the Newborn Infant in New Ym·k State During 1947. Pediatrics, 12:377 (October), 1953. 14. JUNE, R. C., FERGUSON, W.W., & WORFEL, :\I. T.: Expe1·imcnts in Feeding Adult Volunteers with Esche1·ichia coli 55:B5 a coliform organism Associated with Infant Diarrhea. Am. J. Hyg . .i7:222 (March), 1953. 15. NAMIOKA, S., URUSHIDO, i\I., SAKAZAKI, R.: Eschel'ichia coli Isolated from Transmissibie Gastro-enteritis in Pigs. Jap. Jou1·. Med. Sci. and Biol. 11 :Ml (June), 1958. 16. DUNNE, H. W., GLANTZ, P. J., HOKANSON, J. F. & BOitTHEE, A. L.: Escheri<'hia coli as a Cause of Diarrhea in Calves. Ann. of N. Y. Acad. of Sd. 1;1;:129 (August), 1956. 17. KAUFFMANN, F.: Serology of the coli group. J. Jmmu11ology, !ii: 71, 1947. 18. EWING, W. H., EDWARDS, P.A.: Isolation and P1·cli111i11a1·y Identification of Eschel'ichia coli Sc!'otypes Associated with Cas<'S of Diarrhea of the Newborn. The Publk Health Lab. J2:'i5, 1954. 19. ARAGON, P.R., STRANSK)', E., SANTOS-OCAMPO, P.: Escherichia coli 3el'otypes Associated with Dia1Theal Conditions in Manila. Jour. Phil. Med. Asso .. ~.i :20, 1959. 20. GUERRERO, R. Ma., DE RODA, A. P., BELMONTE, C.R. & REYES, F.: Neomycin (Non· USP) In the Trl'atment of Infectious Diarrheas in Children. Jour. Phil. i\led. Asso .. /,5 :25, 1959. ~1. 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G.: Diseases T1·ansmitted Ft'Om Animals to Man. 4th edition 1955. Charles C. Thomas, Springfield, Illinois, U.S.A.
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