This is to certify that the thesis entitled ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA IN VETERINARY CLINICAL SPECIMENS presented by Mary Fraser Sit has been accepted towards fulfillment of the requirements for Master's degree in Microbiology (9 fié‘mflg‘flaM/V/Zéé . Major professor Date MaY 19. 1978 04639 LIBRARY Michigan Stan University THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM VETERINARY CLINICAL SPECIMENS By Mary Fraser Sit A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1978 ABSTRACT THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM VETERINARY CLINICAL SPECIMENS By Mary Fraser Sit Little is known about the role of anaerobic bacteria in diseases of domestic animals at the present time. Therefore, the present investigation was undertaken to isolate and identify anaerobic bacteria in veterinary clinical specimens. Specimens were collected aseptically in anaerobic transport tubes and processed using Hungate anaerobic techniques (65) and an anaerobic glove chamber. Most isolates were identified to the species level according to the procedures described in the VP1 Anaerobe Laboratory Manual (63). A total of seventy-one clinical cases, primarily from dogs, cattle, horses, sheep, and swine, were examined for anaerobic organisms. Of these, forty-five cases (64%) were positive for one or more anaerobic species. Seventy-five strains of anaerobic bacteria were isolated, of which 50.7% were 9, perfringens; l7.3% other Clostidium sp.; 9.3% Gram-negative, non-sporulating rods; 9.3% Gram-positive anaerobic cocci; 6.7% Gram-positive, non-sporulating rods; and 6.7% Actinomyces sp. While some of these organisms have previously been associated with various disease conditions in animals, many are being reported here for the first time. Mary Fraser Sit The clostridial species isolated in this study included: g, perfringens, Q, sphenoides, g, sordellii, Q, carnis, Q, barati, g, butyricum, g, glycolicum, Q, tertium, Q, botulinum non-proteolytic BEF, Q, bifermentans, Q, ramosum and g, acetobutylicum. The clinical conditions from which these organisms were isolated included: eye, ear and skin infections, allergic sinusitis, a thoracic effusion, mastitis, enterotoxemias malignant edema, diarrhea, and infertility. The non-sporulating anaerobes isolated included: Bacteroides sp., g, clostridiiformis subspecies clostridiiformis, Fusobacterium varium, f, necrogenes, E. necrophorum, Eubacterium sp., g, cylindroides, Propionibacterium sp., Actinomyces sp., A, bovis and Lactobacillus sp. These organisms were isolated from such disease conditions as: eye, skin and joint infections, fistulous wounds, mastitis, allergic sinusitis, lymphadenitis and an abscess. The results showed conclusively that various types of sporula- ting and non-sporulating anaerobes occur much more commonly in a wide variety of animal infections than has previously been reported. ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. C. A. Reddy for his guidance, advice and support in developing this study. I would also like to thank Dr. Gordon R. Carter fOr his assistance in obtaining specimens and for his advice and encouragement. I am particularly indebted to the members of the staff of the Clinical Microbiology Laboratory, Mrs. Dorothy Boettger, Mrs. Betty Wei, Mr. Harold McAllister, Dr. Wayne Roberts, and Dr. M. M. Chengappa. Without their often-sought technical assistance, their aid in securing supplies and much more, this study would not have been possible. My thanks also to Mr. Peter Cornell and Dr. Martha Harris for their assistance in getting me started. And finally, to Siu Po, who not only made the impossible, possible, but made it worthwhile as well. Thank you. ii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. LEGEND OF SYMBOLS FOR TABLES, FIGURES I. INTRODUCTION. II. LITERATURE REVIEN . A. Anaerobiosis. Molecular Oxygen Organic Peroxides . Catalase . . . Oxidation- Reduction Potential . Oxidation of Intracellular Components Superoxide Dismutase . . . . mmwa—I o o o o o o B. Anaerobic Methodology. 1. Surface Culture. . 2. Roll Tube Methods . . . 3. Anaerobic Chamber Techniques C. Role of Anaerobes in Health and Disease. l. Indigenous Microflora. . . 2. Anaerobes Associated with Disease. III. METHODS AND MATERIALS. Source of Specimens. Type of Specimens . . Specimen Collection and Transport . Page vii Liquid . Swab. Tissue . Preparation of Media Blood Agar Plates (BAP) . Chopped Meat Glucose (CMG) Peptone-Yeast Extract . Glove Chamber. Isolation Procedures Identification IV. RESULTS Clostridial Isolates C. perfringens . Other Clostridia. Non-Sporulating Isolates . Gram-Negative Non-Sporing Bacteria. Gram-Positive Non-Sporing Bacteria. Anaerobic Cocci . V. DISCUSSION BIBLIOGRAPHY. iv Page 49 49 50 50 El 52 53 55 58 58 58 65 83 83 103 108 l21 Table 0150)“) ll. l2. l3. I4. 15. LIST OF TABLES The O. R. Potential at pH 7.0 of Some Common Indicators . Evolution of Anaerobic Culture Methods. Incidence of Anaerobes as Normal Flora in Humans Conditions Pre-Disposing to Infection with Anaerobes . Relative Frequency of Bacterial Species of the Normal Fecal Flora of 20 Japanese-Hawaiians. The Number of Viable Bacteria Found in the Feces of Ten Adult Animals of Each of Eleven Species. . . Clinical Hints Suggesting Infection with Anaerobes. Human Infections Commonly Involving Anaerobic Bacteria Neurotropic Intoxications Caused by Clostridia . Enterotoxemias and Infections of Hepatic Origin Caused by Clostridia (often associated with liver fluke infestation). Gas Gangrene and Round Infections Caused by Clostridia Isolation of Clostridial Species from Various Clinical Conditions in Canines. . . . . Isolation of Clostridia from Different Clinical Conditions in Various Species of Domestic Animals . . Cellular and Colonial Morphology of Clostridial Isolates. Biochemical Characteristics and Acid Metabolic Products of g, perfringens Isolates . Page ll l8 l9 25 29 36 36 40 41 42 60 62 64 67 Table l6. I7. 18. 19. 20. 2l. Biochemical Characteristics and Metabolic Products of Clostridial Species Other than 9, perfringens . Isolation of Non-Spore Forming Bacteria from Clinical Conditions in Various Domestic Animals Cellular and Colonial Morphology of Non-Spore Forming Bacteria . . . . . . . . . . . . . . Biochemical Characteristics and Acid Metabolic Products of Gram-Negative, Non-Sporing Bacteria . . Biochemical Characteristics and Acid Metabolic Products of Gram-Positive, Non-Sporing Rod-Shaped Bacteria Biochemical Characteristics and Acid Metabolic Products of Gram-Positive, Anaerobic Cocci . . . . vi Page 69 84 87 93 96 105 LIST OF FIGURES Figure l. to 10. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of a typical strain of g, perfringens . . . . . . . . . . A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of C. sphenoides(#l0b). A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of a typical strain of C. sordellii (#ZOc) . . . . . . . . A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of Q, carnis (#25c). A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of Q, barati (#26c). A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, butyricum (#l4b). . . . . . . . . . . . . . . A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, glycolicum #l7c . . . . . . . . . . . . A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of C, tertium #44b . . . . . . . . . A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of C. botulinum non-prot. BEF (#44c). . . . . A gas chromatogram showing the volatile (left) and non- volat31e (right) acid end products of C. bifermentans #44d . . . . . . . . . vii Page 66 70 71 72 73 74 75 76 77 78 Figure 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. gas chromatogram volatile (right) (#34). . gas chromatogram volatile (right) (#37b) . gas chromatogram volatile (right) (#29) . . gas chromatogram volatile (right) (#6) .. . gas chromatogram volatile (right) 55 clostridiiformis (#23a). showing the volatile (left) and non- acid end products of C. beijerinckii showing the volatile (left) and non- acid end products of Q, ramosum showing the volatile (left) and non- acid end products of C, acetobutylicum showing the volatile (left) and non- acid end products of Bacteroides sp. showing the volatile (left) and non- acid end products of B. clostridiiformis gas chromatogram volatile (right) gas chromatogram volatile (right) (#23c). . gas chromatogram volatile (right). (#27). gas chromatogram volatile (right) (#186). . . gas chromatogram volatile (right) gas chromatogram volatile (right) gas chromatogram volatile (right) (#28c). . gas chromatogram volatile (right) gas chromatogram volatile (right) showing the volatile (left) and non- acid end products of F varium (#23b) . showing the volatile (left) and non- acid end products of F. necrogenes showing the volatile (left) and non- acid end products of f, necrophorum showing the volatile (left) and non- acid end products of E, cylindroides showing the volatile (left) and non- acid end products of A, bovis (#19) showing the volatile (left) and non- acid end products of E, lentum (#28b)- showing the volatile (left) and non- acid end products of L, fermentum showing the volatile (left) and non- acid end products of E. limosum (#36) showing the volatile (left) and non- acid end products of P. acnes (#32) viii Page 79 80 81 88 89 9O 91 92 97 98 99 . 100 . 101 . 102 Figure Page 25. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, intermedius (#23d) 106 26. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, anaerobius (#30b) . . . . . . . . . . . . . . . . 107 ix man ic iv py LEGEND OF SYMBOLS FOR TABLES, FIGURES positive reaction negative reaction strong acid, pH 5.5 or below .umupzoeu can uvpvgnocuoocuvs .uvnocmco monspucv u + access: once. a, “acumen appeam: n u unumucn ap—uama a — Lo_:oocc_ n.H .Amnv .ceg seen. was; so acaow no: a c exocxcn n = m—chou ace xzxcuznoco .xczcacnomac .momummun puma: movapucp u e _ _ P .u _ o N = N o .u ee.u.> a .N .N P _ o .u a o o .u .Leeeea o P o _ _ = o = e e o e._.e.eee puccouxw _ N .H . 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All strains produced acetic and butyric acids as their major fermentation end-products, with variable amounts of lactic acid. Nhen present, pyruvic and succinic acids were found in trace amounts only. A typical gas chromatogram for g, perfringens is shown in Figure l, with the range of variation in butyric. Pyruvic and lactic acid peaks included. The biochemical characteristics and metabolic products for each of the isolated are presented in Table 12. Other Clostridia In addition to Q, perfringens, thirteen other Clostridium species were isolated from eleven different clinical cases (Tables 12 and I3). Many of these species had never been reported from clinical conditions in animals before. 9, sphenoides, Q, sordellii, g, carnis and g, barati were isolated from canines. all in association with at least one strain of g, perfringens. Q, tertium, Q, botulinum (non- prot. BEF) and g, bifermentans were isolated from a single case of bovine enterotexmia. In addition, 9, butyricum and g, glycolicum were isolated from an aborted bovine fetus and from a case of bovine mastitis, respectively. In all three of the above bovine cases, the organisms were isolated in conjunction with g: perfringens. Two organisms most resembling g, beijerinckii and Q, botulinum (non-prot. BEF) were isolated in pure culture from separate cases of equine infertility. g, ramosum was isolated, in addition to Q, perfringens, from a swine hockjoint and Q, acetobutylicum was isolated in pure culture from a case of blindness in a goat. 66 Im— P LIL—b B s __.L Fig. 1. A gas chromatogram showing the voiatile (left) and non- volatile (right) acid end products of a typicai strain of g, perfringens. The extent of variation in acid production between strains is indicated by the dotted lines. For expianation of symbols, refer to p. x. 67' Biochemica] Characteristics and Acid Metaboiic Products of g, perfringens Isolates.a TABLE 15. muuauoca ecu u.u< “.mapoEuz «cm: x..: omogusm omovn._az mmoccm: amoupa: vacuum; mucus—u 332cm m,m»_ogv»z cv—aumm cones: gowns—om~ Animai Species AprLs AB d 28 Cg cg Canine 28 10A 12 16 18a 20a AB] Apr15 A8 28 C9 28 28 28 B Aprls AB cgd cg C9 C9 20b 21 Apris Ab 28 24a AB 24b 25a AB 28 C9 C9 28 ABLs A85 A8 d 25b 266 28 8 C9 ct cd C9 C9 26b 28a 30a 41 A815 AB 28 AB 28 28 A85 A81 45a 45b ABL 28 C9 C9 C9 C9 C9 C9 3a Bovine A315 28 28 ABLs ABLs A8 28 d 28 28 11a ABLs AB 11b 14a 17a Ab 28 28 C9 cd C9 A8 17b 44a A815 AB 28 Equine AB 28 28 C9 C9 13 AB 15a 22 A8 A85 A8 28 C9 37a 38 Swine AB Rabbit AB 28 Chicken Cat AB 28 C9 35 For expianation of symbols refer to p. ix. 68 Except for g, ramosum, the cellular morphologies of these clostridial isolates were comparable to the Q, perfringens isolates. However, considerable differences in motility and sporulation patterns were noted amongst the isolates. The cellular and colonial morphologies, motility and sporulation patterns of these organisms are given in Table l4. Spores were not observed in the strains of g, sordellii, Q, botulinum non-prot. BEF, or g, beijerinckii isolated. However these organisms survived a heat test (63) which indicated that they were sporulating clostridia. Each of the unusual clostridial isolates was identified on the basis of their biochemical characteristics (Table l6) and acid metabolic end products (Figures 2 through l3). The results showed that each of the clostridial species isolated in this study was very similar to previous descriptions (l8, 63) with the exception of some minor vari- ations. Some of the salient features of these species are noted below. 9, sphenoides was a large, Gram-positive, motile rod with swollen, subterminal spores that typically hydrolyzed gelatin and produced indol but was negative for lecithinase and lipase. A major amount of acetate and a minor amount lactate were produced from glucose. 9, sordellii was similar to previously described strains of this organism; e.g., a large, Gram-positive motile rod that produced lecithinase and urease, but not lipase. However, meat was not digested. Acid end products included major amounts of acetate and formate, with minor amounts of propionate, isovalerate and lactate. Consistent with previous reports, 9, garnj§_was able to initiate growth on conventional aerobic media but produced spores only under anaerobic conditions. The organism appeared as a large, Gram-positive, (59 TABLE l6. Biochemical Characteristics and Metabolic Products of Clostridial Species Other than g. perfringens.a " A A ’3 I: .51 a e l: A V A 5 V N A A v- v .D an - 6— u m ‘~' é: ‘8 '~’ IE :: E: 5 I; "" I: m - 8 2 s a v Em u 0 gm >1 :2 :: u .2 g em = s e c g 2 z .2 ."3 'z '5 .. :8 E b :3 3L 8 O U C «I U +3 3&- 0) 3&- +3 .c L L L a L no. u- - an. S m u .9. 3 3 3 "- 3 22;; z 3 3;», a 3,. o o o o o 0. 02¢ a: a: .28 I: 08 LN col col co: cal cal c). caFvsv' cekw EJF' cokw~v «aha cahw Canine Bovine Equine Swine Caprine Esculin hydrolysis + + + + + + + - + + - + + Fructose A H A A A A A A H A A A A Glucose A H A A A A A A A A A A A Lactose A - A A A - A - - A - A A Maltose H H H A A A A A H A A A A Mannitol H - - - - - H - - - - - A Mannose A H A A A - A A - A A A A Melibiose - - - - A - A - - A - A - Starch pH - - - - A - A A - H A - A Sucrose H - A A A - A A - A A A H Xylose - - - - A A A - - w H H - Gelatin - + - - - - - + + - + - H Milk c cd - - c - - - d - - c cd Meat - - - - — 1 - - d - - - - Indol + + - - - - - - + - - - - Lecithinase - + - + - - - - + - - Lipase — - - - - - - + - - - Hemolysis - - a - - - - B B - - - 8 Motility + + - - + + + + + + + - + Urease + - cné’ + Acid products Al AFpivl BAFL BALs BAFls ApibAF BAls AFpib BALS BAFl La BALS ivl bls bivic Alcohol . products 15(2) 16 (2,3, 2(3) (2.3.15) (2.3) 15) aFor explanation of symbols refer to p. ix. bGlucose-Hinimal Salts-Biotin medium. cParentheses indicate minor alcohol production. 70 4 H) Fig. 2. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g. sphenoides (#lOb). For explanation of symbols, refer to p. x. 71 __—U iv f-_;—_p Fig. 3. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, sordellii (#20c). For explanation of symbols, refer to p. x. 72 F L 4 l "‘—l Fig. 4. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, carnis (#25c). For explanation of symbols, refer to p. x. 73 L , B s -—l Til Fig. 5. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, barati (#26c). For explanation of symbols, refer to p. x. 74 —l c) s Fig. 6. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, butyricum (#l4b). For explanation of symbols, refer to p. x. 75 4 F—l Fig. 7. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of Q. glycolicum (#l7C). For explanation of symbols, refer to p. x. 76 —l *4 Fig. 8. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, tertium (#44b). For explanation of symbols, refer to p. x. 77 —u Lfl Fig. 9. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, botulinum non-prot. BEF (#44c). For explanation of symbols, refer to p. x. 78 l cl Fig. l0. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of Q, bifermentans (#44d). For explanation of symbols, refer to p. x. 79 —l L-¥/-—l 8‘ Fig. ll. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of Q, beijerinckii (#34). For explanation of symbols, refer to p. x. 80 -i *1) Fig. 12. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of g, ramosum (#37b). For explanation of symbols, refer to p. x. 81 Lac—:4 B s. Fig. 13. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of C. acetobutylicum (#29). For explanation of symbols, refer to p. x. 82 non-motile rod with swollen, subterminal spores. Acetate, formate, butyrate and lactate were produced in major amounts. 9, barati was a large, Gram-positive, motile rod with swollen, subterminal spores that was positive for lecithinase and negative for lipase and gelatin hydrolysis. Atypically, the organism produced no change in milk. Major amounts of acetate, butyrate and lactate with a trace amount of succinate were produced from glucose. 9, butyricum was a large, Gram- positive, motile rod with swollen, subterminal spores that closely resembled the classical description of this species. Lecithinase, lipase, indol and gelatin hydrolysis were all negative, while growth in glucose-minimal salts-biotin (GMB) medium was positive (distinguishing this species from g, beijerinckii). Q, glycolicum appeared as a large, Gram-positive, motile rod with subterminal spores. Gelatin hydrolysis, indol, lecithinase and lipase were all negative, with major amounts of acetate and minor amounts of propionate, isobutyrate, isovalerate and lactate produced from glucose. 9, tertium also grew aerobically but produced spores only under anaerobic conditions. The organism was a large, Gram-positive, motile rod with swollen, terminal spores. Atypically, milk was not coagulated. Acid end products included major amounts of acetate and formate with trace amounts of butyrate, lactate and succinate. The two strains of Q, botulinum non-prot. BEF (#36, #44c) were large, Gram-positive rods that produced major amounts of acetic and butyric acids with trace amounts of formic and/or lactic acids. Strain #36 was lost prior to completion of testing, however the isolate was tentatively identified as g, botulinum non-prot. BEF on the basis of gelatin hydrolysis, fermentation of glucose and negative indol and lactose results. Strain #44 typically produced lipase and 83 lacked lecithinase. Both strains failed to coagulate milk unlike the majority of strains previously described. 9, bifermentans was a large, Gram-positive, motile rod with subterminal spores that was positive for lecithinase and negative for lipase and urease. A major amount of acetate and formate, with minor amounts of propionate, isobutyrate, butyrate. isovalerate and isocaproate were produced from glucose. Strain #34 was tentatively identified as g, beijerinckii on the basis of the acid end products produced, weak acid production in starch and xylose, and negative results for gelatin hydrolysis and indol. Q, ramosum appeared as a medium-sized, thin Gram-positive rod with swollen, terminal spores. The organism was negative for indol, lecithinase and lipase, and produced a major amount of lactate with a trace of acetate. No formate was detected, contrary to what was expected. _C_. acetobutylicum was a large, Gram-positive, motile rod with swollen, subterminal spores. The organism was very typical of this species with the exception that gelatin was weakly hydrolyzed and milk was both coagulated and digested. Non-Sporulating Isolates Gram-Negative Non-Sporing Bacteria Seven strains of Gram-negative NSF bacteria were isolated from five clinical cases (see Table l7). A Bacteroides sp. was isolated from a case of canine conjunctivities. A fistulous tract infection in a dog yielded g, clostridiiformis ss. clostridiiformis, Fuso- bacterium varium and f, necrogenes, in addition to E, intermedius and several facultative organisms. A post-surgical follow-up yielded an organism most resembling E, necrogenes. 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'uoou o>.u.moo-5oco Ase o..ov mo.:opou noeoo p—uEm mouuouoummuumogummm xaopogogoz Lops—.mu zoo—ozoco: po.:o—ou smpcomco EIE' 'I I..-|‘h"”flrn N4 I“-IIDEE,rhh.nlu.h.EbE~g’.'-‘*i~ i .owcouoom oopsgou accom-ooz Lo zuopogogoz .owco—ou nee Lopoppou .m. mom>£+ AS >812. :- z- z: x: + apivls P 8H ApBLs 31:4- III-.- P 8H apBls apBls asz a + 'U asz aFor explanation of symbols refer to p. ix. 94 small, non-motile, Gram-negative rod that produced acetate, butyrate and lactate in major amounts with minor amounts of propionate and succinate. Indol was positive and propionate was produced from threonine, but not from lactate. The two strains of E, necrogenes (#23c, #33) were long, slender, Gram-negative rods with tapered ends that produced large amounts of butyrate with trace amounts of acetate, propionate, lactate and succinate. Propionate was produced from threonine but not from lactate and indol was negative. The two strains of E, necrophorum (#27, #33) both appeared as long, thin, Gram-negative rods that became increasingly pleomorphic with age. Butyrate was the sole major acid produced, along with minor amounts of acetate, propionate and succinate. Strain #27 was lost prior to completion of testing but was tentatively identified as E, necrophorum on the basis of a positive indol test; negative results for esculin hydrolysis, lactose and mannose; and coagulation in milk. Strain #43b characteristically produced propionate from lactate and threonine, and was positive for lipase. Gram-Positive Non-Sporing Bacteria Eight clinical cases were positive for Gram-positive NSF bacteria, yielding a total of lo strains; (5) Actinomyces , (3) Eg; bacterium, (l) Lactobacillus, and (l) Propionibacterium (Table l7). Only one strain of Actinomyces, isolated from a canine eye disorder, fitted the classical description of A, bovis. 0n the basis of starch hydrolysis, a second strain (isolated from ovine lympha- denitis) was also identified as A, bovis, although the biochemical test results were skewed due to apparent utilization of nutrients in 95 the basal medium. Three other strains of Actinomyces, from sheep intestine (l) and swine abscess (2), did not key to any known species. They were designated Actinomyces on the basis of the characteristic acid end products produced and on the distinctive cellular morphology observed. E, cylindroides and an organism resembling E, limosum were isolated from cases of canine pyoderma and bovine mastitis, respectively, both in conjunction with strains of E, perfringens. Organisms very similar to E, lgflEgm_and E, fermentum were isolated from a case of conjunctivitis in a dog. A strain of Propionibacterium, most similar to E, ggngg, was isolated from a case of fistulous withers in a horse, in addition to several facultative organisms. The cellular morphology of Actinomyces, as noted previously, was very distinctive; appearing as a tangled mass of Gram-positive rods. The colonial morphology was similar for all strains of Actinomyces isolated. E, gylindroides and E, limosum were similar in cellular morphology (thin Gram-positive rods) but were distinguished by very different colonial morphologies (see Table l8). E, lggEgm_was similar to E, limosum in colonial appearance, but was easily distinguished from the latter by its very small cell size. The E, gggg§_isolate was a Gram-positive cocco-bacillus while E, fermentum was a medium-sized, thin, Gram-positive rod. All were non-motile. The biochemical characteristics and metabolic products of the isolates are given in Table 20. Typical gas chromatograms for the different species are presented in Figures l9 through 24. Salient characteristics of these organisms are noted below. The Actinomyces isolates were distinguished on the basis of cellular morphology as well as on their production of acetic, formic, 96 TABLE 20. Biochemical Characteristics and Acid Metabolic Products of Gram-Postive, Non-Sporing, Rod-Shaped. Bacteria.a '3 .A E 22 . sea 3&7 Z: 9’3 st” 393 5’3 ., .. .. 5:3 8 {2 E}? 4% fig 3 3 w §§ .5. 43151 a5 +35! .53: E . 59 2: 1: £5: 13?, 3?] 8. :3. '5. 2d 3d 2 .3 ‘ 3 ' a 3:5? 2% 2% 2... Canine Bovine Equine Ovine Swine P! Growth - - - - - - H - - - Amygdalin - - - - - H H H - ‘Arabinose - - - - - H A H A Cellobiose A - - - H - H - H A Erythritol - - u u A u Esculin pH A - - - w - u u A u hydrolysis + - - - + - - + + - Fructose A A - - A - H A H A Glucose A A - A A H A A A A Glycogen - - A A A A Inositol - - H A A A Lactose - - - A - — A A H H Maltose - H - A - - A A A A Mannitol - - - - A - H A - A Mannose A A - - - - H u w u Melezitose - - - - - H H H H Melibiose - - - - - - H H H H Raffinose - - - A - A H H - Rhamnose - - - - - A - - H Ribose A H H H A A Salicin A - H A A A Sorbitol - - H A A A Starch pH - - - . - H - A A A - hydrolysis - + - - - - + - - - Sucrose - - - - - - u - u u Trehalose - - - - - A - u u Xylose - - - A H - H H A A Gelatin - - - - - - + - - - Milk - C - - - - c c - - Meat - - - - - - - - - - Indol - - - - - - - - - Nitrate + + + - - + - - - - Catalase - - - - - - - - - - Lecithinase - - - - - - Lipase - - - - - - Hemolysis - - - - - - - - - - Motility - - - - - - - - - - Gas 2+ - - - - - - - - - Acid end products. AB AFLS al aLs AbLs aP AFLS AFLS AFLS AFLS aFor explanation of symbols refer to p. ix. 97 —1 c—J. Fig. l9. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, cylindroides (#le). For explanation of symbols, refer to p. x. 98 —l H Fig. 20. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of A, bovis (#l9). For explanation of symbols, refer to p. x. 99 —l c—J. Fig. 2l. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, lentum (#28b). For explanation of symbols, refer to p. x. 100 4 /-+ Fig. 22. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, fermentum (#28c). For explanation of symbols, refer to p. x. 101 4 *4 Fig. 23. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, limosum (#36). For explanation of symbols, refer to p. x. 102 4 *4 Fig. 24. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E. acnes (#32). For explanation of symbols, refer to p. x. 103 lactic and succinic acids. E, cylindroides was a small, readily de- colorized, Gram-positive rod that produced major amounts of acetate and butyrate only. The organism was negative for lactose, maltose, mannitol and indol, and was positive for esculin hydrolysis, fructose and mannose. Strain #28b was tentatively identified as E, lggEEm_on the basis of cellular morphology, the production of trace amounts of aCetate and lactate only, and its failure to ferment fructose. Strain #36 was identified as E, limosum on the basis of the acid end products produced (major amounts of acetate and lactate, minor amounts of butyrate and succinate), the production of acid in mannitol and failure to produce any change in milk. Strain #28c was tentatively identified as E, fermentum on the basis of the acid end products produced (large amounts of lactic acid with trace amounts of acetate and succinate), and negative results for esculin hydrolysis, mannose and sucrose. Strain #32 was tentatively identified as E, ggggg because it produced major amounts of propionate and minor amounts of acetate and failed to utilize most substrates tested. Anaerobic Cocci Seven clinical cases yielded five strains of Peptostrepto- coccus intermedius, and two strains of E, anaerobius. E, intermedius was isolated from a fistulous tract infection in a dog (as noted earlier), along with a number of other anaerobic and facultative organisms. It was isolated, in association with strains of E, perfringens, from cases of canine sinusitis and equine dermatitis. E, intermedius was isolated in pure culture from cases of equine sterility and chronic cystitis in a cat. E, anaerobius was isolated 104 from an aborted bovine fetus and a swine abscess. In both cases, it was found in association with several other anaerobic organisms (see Table 17). The two strains of E, anaerobius were similar in morphology (relatively large Gram-positive cocci in chains) while the E, intermedius isolates showed considerable variation in cell size. One E, intermedius strain (#40) occurred as clusters of cells rather than in chains. All five E, intermedius strains were obligate anaerobes on primary isola- tion although they became aerotolerant on subculture (see Table 18). The biochemical and chromatographic test results are shown in Table 21 and Figures 25 and 26. Although strain #40 varied from the other E, intermedius isolates in weakly fermenting esculin and melezitose, this was still within the range of results previously reported for this species (63). Strain #43a of E, anaerobius fermented maltose, which is atypical, but has been observed previously (63). 105 TABLE 21. Biochemical Characteristics and Acid Metabolic Products of Gram-Positive, Anaerobic Cocc1.a m 3 U U 0 U m U) U) on O 3 m 3 3 U" 3 4..) ’I"' 3 0r- 'l- .2 '1'- as a '5 a a a a Lw- E O E E O E “'0 S- 8. S— S- $- 030 CD 0) d) m (D O) DEA 44 on .9 4; 2A 4; 3383-0 SE 50 EB ---P~ can: --r'~ “W" .° .3 .52 .° .2 .2; gi-Eb (3.1:; (LP mp AIS-I mp a. Canine Bovine Equine Swine Feline PY Growth - - - - - - - Cellobiose A A - A H - A Esculin pH - - - - w - _ hydrolysis + + - + + - + Fructose A A H A H H A Glucose A A A A H A A Lactose A A - A A - A Maltose A A - A H A A Melezitose - — - - w - - Sucrose A A - A H - A Gelatin - - - - - - - Indol - - - - - - - Nitrate - - + + - - - Catalase - - - - - - - PYG-Tween - S S S S - _ Threonine - - p - - p _ Acid End Products aL aL Apibbiv . aL aL Apibbiv aL icls icls aFor explanation of symbols refer to p. ix. 106 —i fiat—A Fig. 25. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E, intermedius (#23d). For explanation of symbols, refer to p. x. 107 4 Fig. 26. A gas chromatogram showing the volatile (left) and non- volatile (right) acid end products of E. anaerobius (#30b). For explanation of symbols, refer to p. x. 4 V. DISCUSSION Recently there have been several studies pertaining to the isolation and identification of anaerobes in diseases of animals (7, 9, 38, 39). Berg and Fales (7) investigated the anaerobic flora of selected canine lesions. They reported that anaerobic bacteria of the following genera were commonly isolated: Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, and Propionibacterium. Hhile they did not report precise numbers, they did report a relatively high incidence of Clostridium species. Berkhoff and Redenbarger (9) in a similar study, investigated the incidence of anaerobes in a variety of veterinary clinical specimens obtained primarily from dogs, cattle, horses, and swine. Anaerobes were isolated in the following propor- tions: Clostridium sp., 50%; Gram-negative NSF rods, 19%; Gram-positive NSF rods, 19%; Actinomyces sp., 10%; and anaerobic cocci, 1%. However, in this report no correlations were drawn between the animal source, the type of specimen, the kinds of anaerobes isolated, and whether they were present in pure or mixed culture. In agreement with the above studies, the results of the present investigation revealed a higher incidence of Clostridium sp. in infections of animals than has been found in man (35, 49, lll). Further, the incidence of anaerobic Gram-negative rods (particularly 108 109 .E. fragilis) was found to be much lower in animal infections than in humans. Many of the anaerobic species isolated during the course of this study have previously been reported to be a part of the normal gastrointestinal flora of these animals (18, 63) and have also been shown to be associated with human infections (35, 43, 44, 45, 111). Clostridial infections generally occur more frequenly in animals than in man because clostridia are ubiquitous in soil and animal quarters, and many animals harbor clostridial spores on their hair or skin (115). The clostridia may invade through breaks in the skin or open wounds and cause various superficial infections. E, perfringens was frequently isolated from a number of different clinical disorders in animals during the course of this study. This organism has also been the most common clostridial isolate from a variety of infections in humans (35, 111). E, er- fringens has been associated with a number of relatively harmless and self-limiting infections and has often been isolated in the absence of disease entirely (49). However, this organism has also produced serious and often fatal illnesses in both humans and animals (35, 111, l15). Dogs appear particularly susceptible to E, perfringens der— matitis, conjunctivitis, and otitis. There were no previous reports of isolation of E, perfringens from these sites in dogs or in other animals. Interestingly, veterinarians specified anaerobic culture in these cases, with screening for E, perfringens often requested. In a preliminary sampling, neither E, perfringens nor any other anaerobe could be detected in fresh swabs from the conjunctivae of three healthy dogs. E, perfringens was previously isolated from human cases of 110 pyodermatitis and conjunctivitis and was shown to be frequently associated with otitis media and mastoiditis (35). The results suggest that E, perfringens may be very significant in infections of the eyes, ears and skin of dogs. E, perfringgns was isolated in pure culture, with no other anaerobic or facultative organisms present, in cases of pododermatitis and thoracic effusion in dogs. The isolation of E, perfringens in canine pododermatitis may have been significant as the organism could well have gained access by minor trauma, into the animal's foot. In the case of thoracic effusion, the isolation of E, perfringens may be of more interest. E, perfringens has been isolated from a number of thoracic infections of man, particblarly those infections following surgical or accidental wounds (35, 111). There was nothing in the clinical history of this animal to indicate whether trauma or some other pre-disposing factor was involved. However, considering the pathogenic potential of this organism in humans and animals, it s isolation in this case appears significant. As no other organisms were cultured in either of these cases, it is unlikely that the E, er- fringens isolates were picked up in sampling as contaminants. Of the other clostridial isolates in dogs, E, ggEgj§.and E, sordellii have previously been reported in animal infections (18, 115). E, sordellii is more commonly found in liver infections of cattle and sheep, but has been found in contaminated wounds of these species as well (115). E, sphenoides has not been reported in animals, but was isolated from infected wounds in man (18). The significance of its presence in extracted toOth material of a dog cannot be determined at the present time, although it is generally believed that clostridia are 111 not present as a significant portion of the normal flora in the mammalian oropharynx (35). E, E35331, also known as E, paraperfringens, has never been reported in clinical conditions in man or animals. Isolation of this organism in pure culture from an ear infection in a dog suggests that this organism may occur as a natural pathogen in canines. Consistent with previous reports (18, 115), E, perfringens was found in several cases of suspected bovine enterotoxemia and in one case of malignant edema. From one case of suspected entero- toxemia (#44), E, botulinum (non-prot. BEF), E. tertium, and E, bifermentans were isolated, in addition to E, perfringens, of which no one organism predominated in the specimen. The isolation of E, perfringens is of dubious significance in this case, as an apparently normal spectrum of clostridia and facultative anaerobes was found in the intestinal contents of this animal. E, perfriggens and E, botulinum (non-prot. BEF) are both found in the normal intestinal flora of a wide variety of birds and mammals (18, 63). E, tertium has been found in human feces (18) and may well be present in the normal flora of other animal species. However, this is the first reported isolation of this organism from the GI tract of an animal. E, bifermentans is known to occur in the normal flora of ruminants, and has been isolated from several human and animal infections (63). . E, perfringens was also isolated from several cases of bovine mastitis. Bovine mastitis due to E, perfringens has not previously been reported. This organism appears particularly significant in case #17. In this case, anaerobic culture alone was requested and clostridial infection was strongly suspected by the clinician. 112 Although E, perfringens predominated in the specimen, E, glycolicum was also isolated. E, glycolicum has never been reported from in- fections in animals although it has been isolated from soil and certain human clinical infections (18). In this case, it is not clear whether E, glycolicum was acting as a pathogen, opportunist, or a contaminant picked up in sampling. The significance of the isolation of E, perfringens from an aborted bovine fetus (#14); is difficult to assess because it was present in association with E, butyricum, E, anaerobius, and large numbers of Pasteurella sp. E, perfringens has been found in the normal flora of the human female genitourinary tract, as well as in numerous genital infections (46, 49, 71). E, butyricum has been found both in human wounds of traumatic origin and in animal feces (18). E, anaerobius is commonly found in the normal human vaginal flora and (less often) in infections of the female genital tract (26, 35, 36). The pathogenicity of this organism for animals has not been documented. The isolation of several clostridial species (E, perfringens, E, beijerinckii, and E, botulinum non-prot. BEF) from cases of bovine and equine infertility may or may not be significant depending upon the care with which specimens were taken. Several clostridial species have been found in the normal human vaginal and cervical flora, as well as in uterine infections (26, 35, 49). Normally, the human uterus is devoid of bacterial contamination and the same should be true of animals. Although E, perfringens has not previously been isola- ted from cases of human infertility, a low-grade infection with this organism might preclude conception in animals. E, beijerinckii, often found in soil, has also been reported as a rare clinical isolate in 113 humans (18). The significance of E, botulinum (non-prot. BEF), isolated from a case of equine infertility, is not clear as this has been the only reported isolation from an animal so far. The organism is not known to be associated with any human infections. E, perfringens has been reported to be a cause of enterotoxemia in foals (115) and in this study was isolated from a young horse suffering from diarrhea. E, perfringens was also found in an eye disorder and dermatitis in older horses. This probably reflects a susceptibility of equines to skin and eye infections by E, perfringens, similar to that noted earlier in dogs; however, it is conceivable » that the isolates were soil contaminants acquired during specimen collection. E, perfringens was isolated twice from the hock joints of two necropsied swine. In the first case, E, ramosum was also isolated but no aerobic growth was obtained in either case. E, perfringens is known to occur in a number of soft tissue, muscle, and bone infections in man and animals, particularly following trauma (35, 104, 108). E, ramosum is second only to E, perfringens in the frequency with which it is isolated from clinical cases in humans (35). Reportedly, this organism also occurs in animal infections (18) and in the normal flora of swine (63). Therefore, the isolation of E, perfringens in pure culture in one case and in mixed culture with E, ramosum in a second case may be of considerable pathogenic significance. It is also possible, however, that both of these organisms were post-mortem invaders. The isolation of E, perfringens from a case of feline conjunc- tivitis may reflect the predisposition of cats to eye infections by this organism, similar to that observed in dogs and horses. 114 In the rabbit and chicken necropsy cases, the significance of the isolation of E, perfringens cannot be determined. The isolates may represent post-mortem invaders which were originally a part of the normal flora or may have caused a fatal septicemia or toxemia in these species. Throughout the course of this study, typing of E, perfringens was not done due to a lack of facilities here. Toxin typing would have been of considerable help in determining the pathogenicity of the E, perfringens isolates. The anaerobic NSF bacteria isolated in this study included organisms previously isolated in conjunction with animal disease (63, 115) as well as several Species which have never before been reported to occur in animals. E, fragilis is reported to be the most frequently isolated anaerobe from soft tissue infections of humans (35). A similar Bacteroides sp. was isolated from a case of canine conjunctivitis. Berkhoff and Redenbarger (9) reported isolations of several Bacteroides sp. in their study, but did not indicate the source animals, the clinical conditions or the biochemical characteristics for any of their isolates. As the recognition of anaerobic infections in animals increases and stricter anaerobic techniques are utilized, more isola- tions of this organism may be reported from animals. Regrettably, however, veterinary diagnostic laboratories still identify anaerobic rods found in clinical specimens primarily on the basis of morphology. Gram-negative cells with pointed ends are usually reported as Fusobacterium sp., while those with rounded ends are reported as Bacteroides. 115 Several anaerobic, Gram-negative NSF rods (E, clostridiiformis ss. clostridiiformis, E, varium, and E, necrogenes) were isolated from a fistulous tract in a dog. The infection apparently stemmed from a blow to the side, which resulted in a swelling that later opened and began draining. Hhen surgically debrided, the fistulous tract was traced back to the abdominal cavity. E, necrogenes was isolated again from a post-operative specimen collected from this animal. E, clostridiiformis ss. clostridiiformis (now thought to belong in the genus Clostridium [18]) has been found in abscesses and the lower intestinal tract of various animal species as well as man (18, 63). E: varium has been reported from a number of purulent infections in man (notably necrotizing fascitis) but this is the first time that it has been reported from an animal infection. E, necrogenes was first isolated from a necrotic abscess in a chicken but has not been reported in any animal infection. Both of the above fusobacteria have reportedly been found in the intestinal flora of man and several animal species (18, 63). The cultivation of E, necrophorum from a bovine stifle joint and a swine abscess (#43), is consistent with the kinds of infection reportedly caused by this organism in man and lower animals. E, necrophorum is found in many necrotic lesions in a variety of warm- blooded animals, with occasional joint involvements (16). It is also a part of the normal GI flora in ruminants and swine (63). Character- istic infections include bovine liver abscesses and foot-rot in cattle and sheep (8, 99, 106, 107); although this organism causes a number of different animal infections collectively termed "necrobacilloses." In man, E, necrophorum is the most common anaerobic isolate in joint 116 infections, although it is also associated with a number of other purulent, necrotizing and/or metastatic diseases of man (35). E, anaerobius and two strains of Actinomyces were also isolated from the above mentioned swine abscess (#43). The location of the abscess was not stated in the clinical history; however, a large volume of pus was submitted for examination, indicating an abscess of substantial size. The known pathogenic role of E, anaerobius in purulent infections of both man and animals (35, 63), as noted above, makes the isolation of this organism significant. Actinomyces species are known for the production of chronic low-grade infections. These are characterized by granulomatous lesions that break down to form draining abscesses (16). Actinomyces have been isolated from suppurative processes in humans in association with a number of microaerophilic and/or anaerobic organisms; one of the more common being Fusobacterium sp. (98). In swine, Actinomyces commonly localize in the mammary gland, although pulmonary actinomycosis has also been reported (16). A, Eggjé, isolated as the predominant organism from a case of subepithelial keratopathy in a dog, may have been significant in the pathogenesis of this condition. This animal was reported to be immunologically deficient and subsequently responded to a combined steroid—antibiotic therapy. Actinomyces have not previously been reported from eye infections in animals; however, this organism has been reported in lacrimal canal infections in man (35). A, bovis was also isolated from a case of lymphadenitis in sheep in this study. Actinomyces are known to cause chronic nodular bronchopneumonias in sheep, which may have contributed to the lymphadenitis observed (16). The significance of the isolation of an Actinomyces sp. from 117 the intestinal contents of a necropsied sheep could not be determined as no clinical history was available. The eubacteria isolated in this study were all found in association with E, perfringens. E, cylindroides, isolated from a case of canine pyoderma, has occasionally been reported in human clinical material (35, 63). This organism has been reported in the canine GI tract, as a part of the normal flora (63). Berkhoff and Redenbarger (9) reported that 2.6% of their anaerobic isolates from animal infections were eubacteria but did not mention the type or source of specimen or the characteristics of the organisms isolated. An organism similar to E, lgAEEm_was isolated from a case of canine conjunctivitis, in conjunction with E, fermentum. The animal suffered from chronic facial dermatitis and a strain of E, perfringens was isolated from the animal's ear. Neither species, E, lentum or E, fermentum, has been reported from any animal infections before. E. lentum commonly occurs in a number of purulent, inflammatory pro- cesses of man, as well as in the normal human flora (35), but has not been reported in any animal species. E, fermentum is a rare isolate in human disease and has been found in the normal flora of man and several animal species (35, 63). Considering the pathogenicity of these organisms in man, their isolation in this case is significant, despite the fact they have not previously been reported in conjuncti- vitis. E, limosum, isolated from a case of bovine mastitis, normally occurs in the rumen. This is the first reported occurrence of this organism in animal infections, although it has been reported from a number of cases of human peritonitis and other abdominal disorders 118 (35). In this particular case of mastitis, E, perfringens and large numbers of E, coli (non-hemolytic) were also isolated. The significance of the isolation of E, limosum is uncertain at this time since E, coli by itself has previously been shown to be a causative agent of bovine mastitis (16). A case of fistulous withers in a horse yielded an organism similar to E, Egggg as the sole anaerobic isolate. Small numbers of non-hemolytic E, Egli_and alpha-hemolytic streptococci were also isolated from this specimen. Later, the animal was found to have brucellosis and was euthanized. These results are in agreement with those of Smith (111) who reported that E, gggg§_is almost always found in association with a Brucella sp. from fistulous withers in horses. All five strains of E, intermedius isolated during the course of this study were strictly anaerobic on primary isolation, although they rapidly became aerotolerant on subculture. Aerobic streptococci were not detected in any of these cases. In only one case (#15), a beta-hemolytic streptococcus was isolated by routine methods while a non—hemolytic E, intermedius strain was isolated anaerobically. The taxonomic validity of E, intermedius has been questioned (18), as the development of aerotolerance and variation in strains would seem to indicate a diverse group of anaerobic to microaerophilic streptococci. However, the designation may have clinical value considering the inadequacy of aerobic techniques for primary isolation. Although E, intermedius is often detected in human clinical material, its pathogenicity to humans has not been satisfactorily demonstrated. The organism is ubiquitous in the normal human flora and has been reported in animals as well (35, 63). As E, intermedius was 119 found in mixed culture with other potential pathogens in three cases (canine fistulous tract infection and sinusitis, and equine dermatitis) the significance of the organism in these cases is not clear. The organism may or may not have contributed to the pathologic conditions cited and could have been present as a contaminant from the normal flora only. In two cases, equine sterility and feline chronic cystitis, E, intermedius was isolated in pure culture and thus may be of greater significance. In the case of feline chronic cystitis, several aerobic cultures were negative while the anaerobic culture promptly yielded a pure culture of E, intermedius. The high incidence of Clostridium species, specifically of E, perfringens, noted in this study may be more a reflection on the types of specimens received for study than on the anaerobic techniques used. Specimens that would ordinarily be excluded from anaerobic culture, such as gastrointestinal contents, were examined at the request of clini- cians. The anaerobes found in the specimens processed are certainly valid isolations. 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