v7. —- - —— . ... . ' 'I‘tl vuvvv- vvv-w—v--- 'I“'v' - V... v--- -- -" '."~O'..D... ' " ~ .90. q .. . .. ' -o'.. .._ 7 . , . ..¢ ". O. . g 0 O ... i ‘0'. . y o 0 Q r. ’ .ocl- 00.0 o :0- -o.4 '0 ‘- ”qu - .xtuu‘ C 0 'cv’l v HQ «31.; . o «a a .94 .g. " .rwa . u". '\ ‘.§I-O I‘.‘ ‘ but-.1 u" 1.0 ‘ u»....—.. I ‘ I . 'uoc'l” . . ‘ ' ‘ . ‘ c. I191‘0.~0 ‘ . .. fll‘. ':O .- ~ .- -. ; . .-;JCO4 0’- t‘-. .Hlt; 0-90-)0~ 5-030 .- I ‘-o-a l' '0’ 0 . — -.. - mm. 5 E H T- ABSTRACT AEROBIC BACTERIAL GINGIVAL FLORA OF THE DOG BY Dennis Armin Saphir Gingival scrapings from dogs were collected in order to isolate and identify species of the aerobic gingival flora. Scrapings were inoculated into selective and general purpose media and placed in a C02 incubator for a minimum of 48 hours. Various tests were then used to identify the organisms isolated. Members of the following genera were found: Streptococcus, Staphylococcus, Actinomyces, Escherichia, Corynebacterium, Pasteurella, Caryophanon, Mycoplasma, Acinetobacter, Moraxella, Neisseria, Enterobacter, and Bacillus. Of particular interest was the frequent recovery of three unclassified groups of aerobic gram-negative bacteria, IIj, EF-4, and M-5, pre- viously associated with human infections resulting from dog bites. Although no set pattern seemed to exist between the variability and consistency of gingival microbiota as related to age, sex and breed of dog, a certain characteristic flora may be predicted in the gingiva of the healthy dog. AEROBIC BACTERIAL GINGIVAL FLORA OF THE DOG BY Dennis Armin Saphir 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 1975 Dedicated to my wife Karen, my mother and father, and Barbara and David ii ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. Gordon R. Carter for his guidance, patience, and critical suggestions during the preparation of this thesis. Grateful acknowledgement is also made to Mr. Harold A. McAllister and Mrs. Mary Grace Shue for sharing their experiences in the clinical microbiology laboratory and providing me with an invaluable insight into the field of veterinary diagnostic microbiology. I would also like to thank Mrs. Dorothy Boettger for preparing the necessary supplies used in this research project. I am indebted to Dr. R. E. Weaver of the Center for Disease Control, Atlanta, Georgia, for providing me with cultures of Group IIj and Group EF-4. I would also like to thank Dr. R. J. Moon and Dr. R. R. Brubaker for their critical comments and suggestions. I also acknowledge my fellow graduate students working under the direction of Dr. G. R. Carter, for their assistance and suggestions in the prepa- ration of this thesis. A special acknowledgement goes to my wife Karen, whose patience, support, and understanding were instrumental factors in the completion of this degree. iii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF THE LITERATURE. . . . . . . . . . . . . . . . . . . . 3 PART I: THE ORAL MICROBIAL FLORA AND THEIR HOSTS. . . . . 3 The Development of the Indigenous Oral Microbiota of Man . . . . . . . . . . . . . . . . 3 Variability . . . . . . . . . . . . . . . . . . . . 5 Mechanisms of Retention . . . . . . . . . . . . . . 11 l. Adhesion . . . . . . . . . . . . . . . . ll 2. Mechanical Entrapment. . . . . . . . . . 13 Physiology. . . . . . . . . . . . . . . . . . . . . 14 Microbial Relationships . . . . . . . . . . . . . . 16 PART II: ORGANISMS IN THE ORAL CAVITY WHICH MAY BE ASSOCIATED WITH DISEASE. . . . . . . . . . . . . . . . . 20 Pasteurella . . . . . . . . . . . . . . . . . . . . 20 Miscellaneous Gram-Negative Bacteria. . . . . . . . 27 1. Group EF-4 . . . . . . . . . . . . . . . 27 2. Group IIj. . . . . . . . . . . . . . . . 27 3. Group M—S. . . . . . . . . . . . . . . . 3O Actinomycetes . . . . . . . . . . . . . . . . . . . 30 Staphylococci . . . . . . . . . . . . . . . . . . . 32 Streptococci. . . . . . . . . . . . . . . . . . . . 33 Neisseria . . . . . . . . . . . . . . . . . . . . . 34 LITEMTURE CITED 0 O O O O O O O O O O O O C O O O O O O O O O O 3 5 ARTICLE: AEROBIC BACTERIAL GINGIVAL FLORA OF THE DOG . . . . . 43 SUMMARY 0 O O O O O I O O O O O O O C O O O . O O O 4 3 INTRODUCTION 0 O O O C O O I O O O O O O O O C I O O 4 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . 44 RESULTS 0 O O O O O O O O O O O O O O O O O O O O O 4 5 DI SCUS S ION O O O 0 O O O O C O O O O I O O O O O O O 5 3 LITERATURE CITED. . . . . . . . . . . . . . . . . . 59 APPEme. O O O O O O O O O C O O O O O O I O O O O O C O O O I 61 iv Table LIST OF TABLES Page REVIEW OF THE LITERATURE Composition and distribution of oral flora in the adu 1 t O O I O O O O O O O O O O O O O O O O O O O O O 6 Organisms of the human gingival crevice region . . . . . 8 Microorganisms and products which may be associated in gingival diseases . . . . . . . . . . . . . . . . . . 21 Differential characteristics of Pasteurella multocida and aerogenic Pasteurella multocida- like organisms . . . . . . . . . . . . . . . . . . . . . 26 Characteristics of unclassified gram-negative aerobic oral flora as reported in the literature . . . . 28 ARTICLE Summary of identification reactions of some unusual aerobic microorganisms found in gingival scrapings from dogs. . . . . . . . . . . . . . . . . . . . . . . . 47 Frequency of isolation of aerobic bacteria recovered in gingival scrapings from 50 dogs . . . . . . 49 Frequency of isolation of aerobic bacterial species recovered in gingival scrapings from 50 dogs . . . . . . 50 Results of antibiotic susceptibility tests on Group IIj. . . . . . . . . . . . . . . . . . . . . . . . 54 Results of antibiotic susceptibility tests on Group EF-4 O 0 0 O O O I I O O O O O O O O O O O I O O O 55 APPENDIX A summary of the identification data used in studying the gingival flora of 50 dogs . . . . . . . . . 62 Identifiable aerobic microorganisms isolated from gingival scrapings of 50 dogs. . . . . . . . . . . . . . 64 V Table A-lO A-ll A-12 Page Frequency of isolation of aerobic gram—negative bacteria recovered in gingival scrapings with respect to gender. . . . . . . . . . . . . . . . . . . . 69 Frequency of isolation of aerobic gram-positive bacteria recovered in gingival scrapings with respect to gender. . . . . . . . . . . . . . . . . . . . 71 The occurrence of aerobic microorganisms isolated from 6 sporting dogs . . . . . . . . . . . . . . . . . . 73 The occurrence of aerobic microorganisms isolated from 4 hounds I O O O O O O O O O O O O O O O O O O O O O 74 The occurrence of aerobic microorganisms isolated from 15 working dogs . . . . . . . . . . . . . . . . . . 75 The occurrence of aerobic microorganisms isolated from terriers (1), toys (2), and non-sporting dogs (2) . 77 The occurrence of aerobic microorganisms isolated from 20 mongrels . . . . . . . . . . . . . . . . . . . . 79 Isolation of aerobic gingival bacteria as related to the ages of male and female dogs. . . . . . . . . . . 81 Isolation of aerobic gingival bacteria as related to the ages of male dogs . . . . . . . . . . . . . . . . 84 Isolation of aerobic gingival bacteria as related to the ages of female dogs . . . . . . . . . . . . . . . 87 vi LIST OF FIGURES Figure Page APPENDIX 1 Gram stain of Caryophanon. . . . . . . . . . . . . . . . 61 vii I NTRODUCTI ON The study of bacterial flora isolated from the oral cavity of domestic animals is virtually an unexplored field compared with the data available on human oral flora. Researchers investigating the gingival microbiota in humans faced two problems, one of sampling, and the second involving the "technical task of enumerating a diverse, poorly described, difficult to cultivate, complex group of micro- organisms" (84). Those organisms which were successfully isolated from the gingival crevice region could not always be categorized into acceptable classification tables. Studies of gingival flora in the dog are of particular value since this animal has a gingival crevicular epithelium similar to that found in humans (31). Few investigators, however, have concen- trated their efforts on describing gingival flora in dogs. Most certainly the problems of sample collection are enhanced with these animals, and many of the organisms which can be isolated are not recognizable using present classification schemes (personal observation). The organisms found within the oral cavity of domestic animals consist of many potential pathogens and commensals (77,84,88). Disease may occur as a result of local infection by the microorganisms which usually inhabit the mouth in large numbers. However, for an organism to produce lesions, dynamic alterations involving the host and/or the microorganisms must occur. These may include a local or general reduction of vitality of the host, a lessened resistance of the host's tissue, physiological changes in the host, an increase in the number of organisms, or an increase in the virulence of the microorganisms. Irritation, trauma, penetration of foreign bodies such as bones or sticks, carious teeth, and neoplasms may all con- tribute to bacterial invasion (30). It has also been shown that certain organisms of the oral cavity have been recovered in wounds resulting from animal bites. For example, Pasteurella multocida infections in humans have for some time been associated with animal bites (2,19,20,28,43,44,47,49,60,70,89,92). The microorganisms within the oral cavity consist of the resident and transient flora. Resident flora can be continuously demonstrated by culture, staining, and immunologic techniques. The transient flora represent those organisms which are occasionally isolated from the oral cavity. Knowledge of the resident and transient oral flora is essential in determining which organisms cause or are associated with different clinically recognized oral diseases of man and animals (69). The purpose of this investigation was to isolate and identify to species, where possible, the aerobic bacterial flora found in gingival scrapings of dogs. Particular attention was given to those organisms previously reported to be associated with human infections resulting from dog bites. REVIEW OF THE LITERATURE PART I: THE ORAL MICROBIAL FLORA AND THEIR HOSTS The Development of the Indigenous Oral Microbiota of Man Research workers investigating the oral flora in man are in disagreement as to when bacteria first appear in the mouth. Some believe the oral cavity is basically sterile at birth (46,66) with no evidence of microbial growth seen prior to the initiation of the respiratory process (1). Other reports indicate the newborn first comes in contact with the microbial flora of the mother's vagina, and then with the organisms of the external environment (11,69), where the bacterial count increases rapidly (10.59.61). During the first few days of the child‘s life, his mouth is highly selective towards the type of bacteria that can establish themselves. Subsequently, bacterial composition is quite diversified within this time period (11,88). Aerobic and facultative aerobic bacteria dominate the oral flora prior to the appearance of teeth (1,66,69,75). Some of the representative flora include: streptococci, pneumococci, micrococci, staphylococci, coliforms, Bacillus subtilis, Neisseria, Actinomyces, and Corynebacterium (10,11,66,69,75). Although the anaerobe Veillonella alcalescens has been frequently recovered from infants over one week old (66,69), other anaerobes are seen in low numbers in the child's initial year (5,10,51,6l,66,7l). 4 There are both qualitative and quantitative changes in the oral cavity with the appearance of teeth. Such anaerobes as Leptotrichia, spirochetes, fusiform bacilli, spiral forms, and Vibrio increase in number significantly (69). Dental carious lesions, which frequently appear in adolescents, create an entirely new environment in the oral cavity for new microbial growth. These lesions provide new attach— ment sites for organisms which previously had difficulty in adhering to surfaces in the mouth (88). The bacterial flora in man is subjected to changes in diet and habitats through tooth extractions and use of dentures. Subsequently, bacterial flora remains diversified, and competition among organisms with respect to nutrients and attachment sites is high. For example, Streptococcus salivarius is provided with an adequate nutritional environment in the mouth of an infant. This organism primarily resides on the tongue rather than teeth (10,15,66,69). In contrast, spirochetes are not found in the edentulous infant. Spirochetes have strict nutritional demands and require the gingival crevice area as their habitat (88). However, Carlsson et a1. (15) have found that S. salivarius cannot compete successfully with other organisms in the adult mouth unless dietary sucrose is provided. The percentage of S. salivarius in the oral cavity, therefore, decreases with a decrease in dietary sucrose. Due to individual variation, it is difficult to describe a basal flora for the oral cavity of all healthy dentulous humans. However, a broad pattern of the oral bacterial flora may still be predicted. A review of the literature indicates at least 29 different species 5 of microbes found within the oral cavity (11). Table 1 depicts the composition and distribution of oral flora in the adult. The proportion of diphtheroids and gram-negative anaerobic rods increases in plaque and gingival sulcus. Neisseria, Bacteroides melaninogenicus, and spirochetes are frequently seen. Lactobacilli, staphylococci, and filamentous forms comprise approximately 1% of the population. The incidence of Candida, coliforms, and Mycoplasma varies. .Mycoplasma is not seen in the edentulous mouth which would indicate that these organisms inhabit the gingival sulcus (11). In addition, the edentulous mouth shows a considerable reduction in the number of spirochetes, lactobacilli, yeasts, Streptococcus mutans and Streptococcus sanguis (9,16,62,81). Although B. melaninogenicus and spirochetes are regular inhabitants of the adult gingival crevice region, neither organism is seen with any degree of regularity in children (25). A list of cultivable organisms in the gingival crevice area and the genera commonly found in this site is shown in Table 2. Variability Socransky and Manganiello (88) have suggested that oral bacterial flora differ between host species, within the same species, between sites in the same oral cavity, and within the same site in the same individual. Gordon and Jong (41), reporting on bacterial flora from human saliva, indicated that gram-positive facultative cocci represented the largest single group (46.2%), with streptococci comprising 41% of the salivary isolates. Gram-negative anaerobic cocci made up 15.9% of the total isolates, with most strains belonging to the genus Table 1. Composition and distribution of oral flora in the adult Gingival Dental crevice plaque Tongue Saliva (is) (’3) (is) (is) Gramrpositive facultative cocci 29 28 45 46 Streptococcus spp. 27 28 38 41 S. mutans L** L—H L L S. sanguis M H M M S. salivarius L L H H Enterococci M L L L Staphylococcus salivarius*** L L H H Related nonpathogenic staphylococci L L H H Gramrpositive anaerobic cocci Peptostreptococcus, Peptococcus 7 13 4 l3 Gramrnegative facultative cocci Neisseria spp. <1 0 3 l Gram-negative anaerobic cocci veillonella spp. ll 6 16 16 Gram-positive facultative rods and filaments . Nocardia, Rothia, Corynebacterium, 15 24 13 12 Bacterionema****, Lactobaccillus Gram-positive anaerobic rods and filaments Actinomyces, Propionibacterium, 20 18 8 5 Leptotrichia Gram-negative facultative rods 1 <1 3 2 Table 1 (continued) Gingival Dental crevice plaque Tongue Saliva (%) (%) (%) (%) Gram-negative anaerobic rods 16 10 8 5 Fusobacterium 2 4 <1 <1 Bacteroides oralis 6 5 5 2 Bacteroides melaninogenicus 5 <1 <1 <1 Vibrio sputorum 4 l 2 2 Spirochetes Treponema microdentium, T. 1 <1 <1 <1 denticola, T. oralia Modified from Microbiology. Davis et al. (eds.), Harper and Row, Maryland. 1973. (24) * Approximate percentage of total cultivable flora present in each area. ** Approximate proportions found: L=1ow, M=moderate, H=high. *** This organism is not a member of the Staphylococcus genus. It grows poorly and ferments glucose weakly (6). Bergan et al. (4) have suggested it is a micrococcus. **** Belonging to the family Actinomycetaceae. 8 Table 2. Organisms of the human gingival crevice region Group Approximate percentage of Genera and/or species cultivable microbiota commonly found in this site Gram-positive Gram-positive Gram-positive Gram-positive Gram-negative Gram—negative Gram-negative Gramrnegative facultative cocci anerobic cocci facultative rods anaerobic rods facultative cocci anaerobic cocci facultative rods anaerobic rods Spiral organisms 28.8 7.4 15.3 20.2 16.1 1 to 3 Staphylococci, enterococci, S. mutans, S. sanguis, S. mitis Peptostreptococcus Corynebacterium, Lactobacillus, Nocardia, A. vis- cosus, B. matruchotii A. bifidus, A. israelii, A. naes- lundii, A. odonto- lyticus, P. acnes, L. buccalis, Corynebacterium Neisseria V. alcalescens V. parvula B. melaninogenicus, B. oralis, V. spu- torum, F. nucleatum, S. sputigenum T. denticola, T. oralis, T. macro- damnm,B. vincentii Modified from S. S. Socransky (84). 9 Veillonella. Gram-positive facultative rods comprised 11.8% of the total number of organisms. Bowen (7) showed that the salivary flora of monkeys is very similar to humans although quantitative differences do exist. Strep- tococci comprised 72% of the total aerobic population. Veillonella represented only 3% of the total anaerobic organisms or approximately one-seventh of that found in human saliva. The appearance of coliform bacteria was greater in monkeys than humans, and the lactobacilli count was ten times greater than that reported from human saliva. Actinomyces was not cultured from the saliva of any of the monkeys (7), in contrast to its occasional recovery from human saliva (41). MacDonald et al. (64) observed that the flora in the rice rat periodontium is composed primarily of aerobic, gram-positive bacteria comprising mostly enterococci and diphtheroids. A limited number of actinomycetes were found while no spirochetes were reported. Of note was the infrequent recovery of Bacteroides or Fusobacterium, organisms indigenous to man. Courant et al. (21) indicated similarities in the relative abundance of bacteria from gingival debris recovered from beagle dogs and humans. However, significant differences were apparent following limited bacterial identifications. Coliform organisms were found to be minor components of dog crevicular flora. However, a comparison between Gibbons'et al. (38) report on gingival crevice flora in man with Courant's et al. (21) survey indicates that B. melaninogenicus is twice as prevalent in the dog's gingival flora. 10 Table 1 supports Socransky's and Manganiello's report (88) on the variability of oral flora among different sites within the oral cavity. There is considerable evidence which supports their findings on variability of microbial flora within the same site in the oral cavity at different periods of time. The flushing action of saliva results in the swallowing of l to 2.5 grams of bacterial cells each day (33). Actions performed by the tongue, lips, mucous membranes of the cheeks, and the mechanism of chewing can remove bacteria from dental surfaces. Organisms from the gingival crevice areas are removed by fluids which originate from the submucosal capillaries. Epithelial cells are constantly being shed, resulting in the removal and subsequent swallowing of microorganisms in the saliva (69). Carlsson (12), Ritz (75), and Socransky and Manganiello (88) examined various sites in the oral cavity at different periods of time. Ritz reported that as plaque development occurs, an anaerobic environment emerges which establishes favorable conditions for the significant increase of such organisms as Actinomyces, Corynebacterium, and Fuso- bacterium. Subsequently, the proportion of streptococci and Neisseria decrease with increasing plaque formation (75). The difficulty in establishing a new species of the oral flora in the mouths of older animals may be due to a failure of the organism to break into an established ecosystem (88). During the first few years of life, the oral cavity is exposed to a myriad of microorganisms. As physiologic changes occur in the host, certain organisms become established in their own niches. As more organisms become established, a certain degree of stability may be seen in the adult and there are 11 less openings for the establishment of other species. The later appearing organisms must be able to compete with the already estab- lished flora, or a major alteration as may occur through antibiotic treatment, must take place. This explains why, although the oral cavity is continuously exposed to such a variety of microbes, many are not able to find permanent residency there. Those organisms for which there is no permanent niche make up the transient oral flora. Generally, the organisms best adapted to a given site and circumstance will survive (88). Mechanisms of Retention If an organism is to survive in the oral cavity, a mechanism of retention as well as certain types of nutritional and physicochemical environments are required. Retention in the mouth usually occurs through adhesion or mechanical entrapment (88). l. Adhesion Microorganisms have the capacity to adhere either to dental surfaces and/or to each other (37,88). Gibbons and Nygaard (37) tested 23 strains of representative plaque bacteria for their ability to agglutinate. Noting that 18 of the 23 strains agglutinated, they concluded that there are plaque bacteria which can adhere to the surface coatings of specific strains of other plaque species. They reasoned that this may explain how some species are able to adhere together in dental plaque. Dental plaque has been defined as a product of microbial growth, tenaciously attached to the surfaces of teeth and exhibiting a definite histological architecture (31). 12 A second mechanism for interbacterial adhesion has been demon- strated using strains of Streptococcus mutans, an organism which produces an extracellular polymer in the presence of sucrose. This extracellular polysaccharide, similar in structure to dextran (24), is involved in the attachment of the organisms to the teeth, and has been shown to hold the organisms together both in vivo (58) and in vitn9(35,57). Fitzgerald et al. (29) demonstrated that dextranase, the enzyme responsible for dextran degradation, can prevent plaque formation in a hamster model system. It was observed that this enzyme did not inhibit the growth or survival of the streptococci. Therefore, they concluded that the anti-caries effect was brought about by the use of dextranase, which prevented the streptococci present in the oral cavity from colonizing on the tooth surfaces. Kelstrup and Gibbons (58) reported that a strain of Streptococcus salivarius produced large quantities of levan and smaller amounts of a glucose-containing polysaccharide when grown in the presence of sucrose. Since plaque formation was not observed from those cells grown in sucrose broth supplemented with dextranase, they concluded that the dextran-like polysaccharide, rather than levan, was essen- tial for the development of plaque formation in vivo and in vitro. In addition, it has been shown that levan is rapidly hydrolyzed by levanase, an enzyme produced by many human plaque streptococci (22,93). Gibbons and Fitzgerald (36) noted that although S. mutans was capable of agglutinating with dextran, other bacterial species, including other dextran-forming organisms, could not. They reasoned that S. mutans agglutinated in the presence of dextran because of 13 specific receptor sites on the surface of the organism which are capable of binding dextran molecules. Agglutination would be observed if the molecule was large enough to allow several cells of S. mutans to bind to the same polymer. A third method of interbacterial adhesion can take place through the production of polymers by the host. Although the exact mechanism of this process is still under investigation, such organisms as Streptococcus sanguis, Actinomyces naeslundii, and Actinomyces viscosus agglutinate when mixed with salivary polymers (39,88). In addition to interbacterial adhesion, oral microorganisms can be retained by attaching to the epithelium or dental surfaces (88). However, the attachment of a microbe to a particular site in the oral cavity is highly specific. Note from Table l, the preference of certain species for specific sites in the human oral cavity. Observe, for example, how S. mutans and S. sanguis prefer hard surfaces, while S. salivarius has an affinity for epithelial cells (88). 2. Mechanical Entrapment Another mechanism of retention in the oral cavity is through mechanical entrapment. Possible sites of nonadhesive retention of bacteria include carious lesions, gingival crevices, and periodontal pockets (88). Spirochetes such as Treponema denticola, and various gramrnegative anaerobic rods such as Bacteroides melaninogenicus, have been recovered primarily from periodontal pockets or gingival crevices (24). A greater number of lactobacilli have been recovered from denture-wearers than from edentulous adults. This suggests that dentures mechanically retain organisms. l4 Spirochetes are reduced in numbers in the edentulous adult with or without dentures (59,78), and in infants without teeth (10, 59,61). Spirochetes do not appear in plaques, on teeth, or on tissue surfaces. Loesche (63) showed that spirochetes' unique nutritional requirements for growth could only be met by retention in the gingival crevice region. This shows how nutrition plays a dominant role in combination with mechanical entrapment (88). Physiology Nutritional sources for organisms residing in the oral cavity include the host's diet, the host's tissues or secretions, and/or secretions by other microorganisms (88). Both the quantity of flora and kinds of microbial populations are influenced by the diet of the host (8,12,14,26). De Stoppelaar et al. (26) reported that human subjects put on a carbohydrate-free diet for 17 days showed signifi- cant decreases in the percentage of Streptococcus mutans in dental plaque. Simultaneously, the percentage of Streptococcus sanguis increased. Reinstitution of the normal diet resulted in the return of these organisms to their original proportions. The presence and increase of S. sanguis during the carbohydrate-free period indicates that this organism does not require the synthesis of extracellular polysaccharides and the presence of sucrose for its establishment on tooth surfaces. The importance of sucrose in the establishment of S. mutans in dental plaques of humans and animals has already been discussed (24,35,57,58). The amount of plaque in man and animals increases with the sucrose rather than glucose content in the diets (12,14,38). Carlsson and Sundstrom (17) observed alterations in 15 population densities and carbohydrate to nitrogen ratios due to the production of extracellular polysaccharides by certain plaque organisms. Socransky et al. (85) reported on the differences in microbial flora in rats fed high protein diets or Purina lab chow diets. The rats on high protein diets showed twice as many gram-positive facul- tative pleomorphic rods. It was believed that these rods were responsible for the increased incidence of calculus formation in these rats. Studies using dogs given a soft diet showed a higher incidence of gingivitis than those on a hard diet (27). Subsequent studies by Carlsson and Egelberg (13) revealed no differences in the fairly rapid accumulation of plaque in dogs whether a soft diet composed of protein or fat was used with or without added sucrose. Polysaccharide- producing streptococci, as found in human mouths, are not found in the oral cavities of dogs. This may account for the differences in plaque formation in response to sucrose in the two hosts. Many organisms residing in the oral cavity are not influenced by the host's diet. Such organisms as Bacteroides molaninogenicus and Treponema denticola obtain their nourishment directly from the saliva, gingival crevice fluid, or mammalian tissue cells (88). Treponema denticola requires alpha 2 globulin compounds for survival. This compound is found only within the oral cavity in mammalian tissues or secretions (86). Loesche (63) has postulated that other sources of nutrition for bacterial growth may come from local tissue cell destruction. l6 Theilaide et al. (91) described variations in microbial flora which occurred as a result of alterations in the normal flow of gingival crevice fluid. They described a close correlation between the amount of plaque development and gingivitis, and concluded that plaque formation influences gingivitis. Socransky (88) has postulated that the reverse may be true; i.e., inflammation of the gingiva may encourage plaque formation. Some bacteria can survive in the oral cavity only if certain other microbes which would supply them with their essential nutrients are present. For example, Treponema microdentium is capable of sur- viving in the oral cavity only if organisms secreting isobutyrate and polyamines are present (87). Bacteroides melaninogenicus requires a vitamin K—like substance for growth which can be provided by Staphylococcus aureus (69). Different organisms have varying oxygen-tension requirements. The oral cavity can house aerobic, microaerophilic, and anaerobic organisms. As the amount of oxygen present within a particular site in the oral cavity changes, so do the microorganisms which inhabit that location. Plaque formation simulates an anaerobic environment and, subsequently, the number of aerobes such as Neisseria and streptococci decrease and anaerobes such as Actinomyces and Fusobac- terium increase (75). Microbial Relationships Microbial populations in the oral cavity are directly influenced by the interrelationships among its members and the oral environment. The associations between different microorganisms and their hosts are 17 described as symbiotic, commensal, opportunistic, synergistic, or pathogenic. Microorganisms can establish similar types of relation- ships among themselves (69). Many organisms found within the oral cavity resemble potential pathogens. Alpha-hemolytic streptococci, staphylococci, and spiro- chetes of the canine mouth resemble well known potential pathogens. Evolutionists have postulated that the human normal flora was initially pathogenic but in time established a passive relationship with man (24). Loesche (63) and Socransky et al. (87) reported that the presence of certain organisms which secrete isobutyrate and polyamines, as well as the type of environment provided by the gingival sulcus region, are required for the growth of Treponema microdentium. Certain aerobic organisms utilize atmospheric oxygen and subsequently reduce the oxidation-reduction potential, thus favoring the growth of anaerobes (69). Ritz (75) reported that the growth of anerobic organisms such as Neisseria and Nocardia, while high initially, declined with plaque development. Anaerobes such as Fusobacterium and Veillonella increased in proportions as plaque grew. Bacteroides melaninogenicus was dependent upon organisms in the oral cavity which secrete a vitamin K—like substance. This commensal-type relationship can be seen on blood agar with B. melaninogenicus growing as a satellite colony within a Staphylococcus aureus colony (69). The oral flora may benefit the host in several ways. Components may compete with certain pathogens in such a way as to restrict the number of the latter in the mouth. This has not been the case in 18 those individuals whose normal flora has been altered through the use of various antibiotics. The normal microbial populations may also act as an antigenic stimulus as these organisms commonly enter the blood stream in small numbers. This results in the host having low levels of circulating antibodies which may cross-react with various pathogens (24). The antibodies in saliva are almost all of the secretory IgA class of immunoglobulins, which function especially well on mucosal surfaces (31). As a result of antibody interaction with plaque microbial antigens, certain processes such as enhanced phagocytosis, bacterial lysis, neutralization of enzymes or toxins of the bacteria, or interference with bacterial metabolism or growth may result (31). The oral flora may also play a substantial role in initiating many serious diseases. These microbes can gain access to tissues in man and cause abscesses in the lungs, brain, and extremities. Actinomycosis, candidiasis and subacute bacterial endocarditis are examples of infections initiated by oral flora (24,31,69). The accumulation of certain types of oral bacteria in the mouth can have serious consequences such as carious lesions or periodontal diseases, e.g., gingivitis and periodontitis. Dental plaque can induce pathological changes in periodontal tissues as a result of microbial products and components such as enzymes and endotoxins, immunopathologic processes, and the release of endogenous histolytic enzymes from the host tissues as a result of microbial action (31). Endotoxin has been associated with pathologic changes in oral tissues, but may also induce nonspecific resistance 19 to local and systemic infections involving human oral bacteria. Endotoxin-stimulated polymorphonuclear cells have increased capacities for intracellular killing of oral bacteria in vitro (54). Therefore, it is thought that endotoxin-stimulated polymorphonuclear leukocytes could be involved in phagocytosis of the gingival sulcus (42). Jenson et al. (55) have reported on the capacity of the Veillonella endo- toxin to induce inflammation as well as enhance phagocytosis. Gustafson et a1. (42) have found that the endotoxin produced by Leptotrichia buccalis has a greater potency than the endotoxin of Escherichia coli. This organism has potent lethal, pyrogenic, and leukopenic activity in rabbits. Endotoxins from gram-negative plaque bacteria may cause the rupture of polymorphonuclear leukocytes. This could result in the release of lysozomes from leukocytes which release free acid phosphatases, esterases, and other enzymes that affect host gingival tissue and may cause periodontal disease (69). Baboolal et al. (3) pointed out that the quantity of bacterial endotoxin and the clinical degree of inflammation are directly correlated. Past studies have indicated that spontaneous periodontitis and gingivitis are not widespread in animals. Studies of periodontal disease in monkeys and dogs are of particular value since these species have a gingival crevicular epithelium similar to that found in humans (31). Saxe et al. (79) have reported that gingivitis and destruction of the deeper periodontal tissues in the beagle dog were correlated with the accumulation of dental plaque. However, plaque formation increased in response to sucrose in the human diet, whereas in the dog there was no difference in plaque formation in response to diets with or without sucrose (13). 20 Periodontal disease is the main cause of tooth loss in the adult and has been attributed to certain organisms residing in the gingival crevicular area (31). Actinomycetes, Rothia, Nocardia, Corynebacterium, and certain streptococci all have the ability to form large quantities of subgingival plaque. The exact mechanism for this disease is unknown, but it has been shown that removal of microbial plaque may help prevent periodontal disease (11). No specific organism has been shown responsible for gingivitis. The types of organisms isolated from healthy gingiva have already been listed (Table 2). In gingivitis, there appears to be a marked increase in the number of organisms present, as well as a shift in the prevalent types. Gram-negative cocci and bacilli, fusiform bacilli, spirochetes, and vibrios all increase in number, but invasion by new organisms does not occur (80). A number of products produced by various microorganisms are responsible for the breakdown of normal tissues. Table 3 lists some of the microbial products that may contribute to the invasiveness and virulence of the organisms, and those organisms which may be associated with pathogenicity in gingival disease. PART II. ORGANISMS IN THE ORAL CAVITY WHICH MAY BE ASSOCIATED WITH DISEASE Pasteurella Pasteurella multocida has been known as an important animal pathogen for many years and its occurrence as a human pathogen has been increasingly noticed (l9). Pasteurella multocida is now con- sidered a commensal usually found in the upper respiratory and 21 Table 3. Microorganisms and products which may be associated in gingival diseases Microorganisms pro— Products Action ducing these products ENDOTOXIN A) interferes with essential Gram-negative bacilli metabolic functions neces- sary to maintain cellular integrity B) may cause extensive tissue destruction C) causes generalized physio- logic changes in the host D) appears to have no tissue selectivity HEMOLYSINS A) breakdown of red blood Streptococci and cells and periodontal staphylococci, et tissue ultimately al. resulting in the spread of infection STREPTODORNASE A) liquifies purulent Gram—positive cocci (deoxyribonuclease) exudates HYALURONIDASE A) breaks down the intra- Staphylococci, strep— cellular substance tococci, pneumococci, hyaluronic acid diphtheroid bacilli, B) facilitates the spread at al. of infection through tissues PROTEASES A) aid in invasion of normal Anaerobic strepto- tissues cocci Table 3 (continued) 22 Products Action Microorganisms pro- ducing these products COLLAGENASES A) B) hydrolyzes collagen destroys collagen fibers Clostridium sp. Bacteroides sp. EDEMA-PRODUCING A) causes edema Pneumococci FACTOR LEUKOCIDIN A) destroys polymorpho— Staphylococci, nuclear leukocytes streptococci Modified from Oral Microbiology. C. V. Mosby Co., St. Louis, Mo. A Clinical Approach With Basic Science Correlation. St. Louis, Mo. 1973. William A. Nolte (ed.). 1973: and Scopp, I. W. Oral Medicine. C. V. Mosby Co., 23 digestive tracts of domestic animals. It can be readily transmitted by animal bites (83). Smith (82) studied the bacterial flora in the nose and tonsils of 111 healthy dogs, and found that 54% of them harbored P. multocida in their tonsils and 10% in noses. He also reported that P. multocida was frequently the dominant bacterium ' recovered from the tonsils of dogs. Hawkins (43) reported on a survey regarding the incidence of P. multocida in tonsils and gums of healthy dogs and cats. Fifty-two percent of these cats and 14% of the dogs harbored these organisms. Lee and Buhr (60) noted that P. multocida was the most common infecting organism in their 1960 report of 69 patients who had been bitten by dogs. Hubbert and Rosen (49) reported that the infection rate resulting from cat bites was approximately ten times greater than that for dog bites. Cats may initiate this infection by biting and/or scratching. The sharp- ness of the cat's claws, coupled with its habit of grooming by licking its paws, appears to be of significance in infections (92). Variations in populations of P. multocida, like the pneumococci, tend to increase during the cold and damp seasons. In some dogs, P. multocida may function as a secondary invader, resulting in pene- tration of the mucous membrane, with secondary pneumonia following recovery from such viral diseases as distemper (82). Lee and Buhr (60) and Smith (82) reported an increase in the incidence of P. multocida infections from dog bites during the winter months. This was attributed to the greater incidence of respiratory diseases such as canine distemper in the winter. Smith (82) reported a high inci- dence of P. multocida in the nose and tonsils of dogs between 1 and 3 24 years of age, with a tendency towards nonfatal cases of viral infec- tions associated with the respiratory tract. Smith did not recover P. multocida in dogs oNer' 8 years of age and he thought this reflected a general simplification of the nasal and tonsillar flora with increas- ing age in dogs. Put forward as possible explanations were changes in the mucous membranes due to senility and the development of full immunity against certain organisms (82). Pasteurella multocida seems to have developed a high degree of adaptation for a commensal existence as evidenced by its spread and abundant occurrence in dogs without usually causing disease. The dog accommodates a large number of these organisms in its throat and yet is usually able to withstand invasion of tissues (82). Three clinical patterns of P. multocida infection in man are usually seen. These include: a) local infection following animal bites or scratches; b) chronic pulmonary infection with P. multocida, either as the primary pathogen or in association with other organisms; and c) systemic infections with meningitis or bacteremias (28,43,89, 92). Tindall et al. (92) reported that the most common type of human infection stemmed from injuries caused by animals, especially cat and dog bites as well as cat scratches. Within 18-24 hours, the resultant infection may be quite severe and painful to the touch, swollen, and red, with a gray-colored serous or purulent discharge emanating from the puncture wound. Considerable cellulitis is usually found in the surrounding area (28,44). If the wound infection is deep, osteomyelitis, an inflammation of the underlying bone caused by pus-forming bacteria, may result (44,67,89,92). Tissue damage may 25 be extensive and human infections are characteristically slow in healing as granulation is delayed. Frequently, infections are aggra- vated by the use of sutures in the wounds, thus requiring prolonged treatment, and resulting in unsightly scars (60). These organisms may also invade the blood stream of debilitated individuals and persons with a general lowered resistance, producing a septicemia followed by chills, high fever, severe prostration, and occasionally death (44,70,92). Pasteurella multocida is a small, gram-negative rod or cocco- bacillus, nonmotile, nonsporeforming, and fermentative (20). However, some differences in cellular morphology, although not diagnostic, have been noted in strains from different animals. Pasteurella multocida recovered from dogs varied from small coccobacilli to long filaments. Bovine strains tended to be pleomorphic as compared to the nonpleo- morphic, porcine strains (83). Weaver (94) described characteristics of P. multocida which differentiate it from various aerogenic P. multocida-like organisms (Table 4). Forty-four strains of Pasteurella- like organisms were described as producing some gas from glucose (strains designated P. "gas"). Of these, 17 strains had been recovered from humans bitten by dogs and 2 from humans bitten by cats (94). Talbot and Sneath (89) reported isolating a strain of P. multocida recovered from a human wound resulting from a dog bite which produced small amounts of gas from sucrose, maltose, mannitol, and trehalose. Rogers and Eldes (76) isolated an aerogenic P. multocida strain asso- ciated with purulent leptomeningitis in a dog. Winton and Mair (95) isolated what was reported to be the first strain of Pasteurella pneumotropica recovered from a dog bite wound. 26 Table 4. Differential characteristics of Pasteurella multocida and aerogenic Pasteurella multocida-like organisms Pasteurella multocida Pasteurella "gas" Gas from glucose - - or + Beta hemolysis? Carbohydrate base used >11 Glucose Xylose Mannitol Lactose Sucrose Maltose Levulose Catalase ++>'<3’<<<>'11 l Oxidase MacConkey - - Gelatinase - ' TSI Sl/butt A/A A/A gas/H25 —/- V/- Motility - - Indole + + Nitrate - - Urea - + NO3 reduction + + Modified from R. E. Weaver (94). + = positive - = negative A = acid V = variable F = fermentative 27 Miscellaneous Gram-Negative Bacteria The late Elizabeth 0. King and her colleagues at the Center for Disease Control (CDC) in Atlanta, Georgia, have made considerable progress in establishing the identity of over 35,000 cultures from human clinical specimens between 1949 and 1973. Of these, over 4,000 organisms have been classified into what is today known as the "miscellaneous gram-negative bacteria" (90). Particular attention is directed in this literature review to those organisms frequently associated with human infections resulting from dog bites. 1. Group EF-4 This group of bacteria consists of small gram-negative, short, rod- to coccoid-shaped organisms. Their action on carbohydrates is fermentative. Colonies average l-2 mm in diameter and appear circular, entire, opaque, convex, and mucoid. They are not hemolytic although an occasional greening on blood agar has been observed after 24 hours. Some of the typical biochemical reactions are shown in Table 5. CDC has reported isolating 85 strains of this type; 66 of these were recovered from humans and, of these 66, 32 were from humans who had been bitten by dogs or cats. EF-4 has also been isolated from extra- intestinal sources in humans: 11 isolates were reported from gums, mandibles, and lungs of dogs (90). 2. Group IIj Following an incubation period of 24 hours, colonies of this type are 0.5 mm in diameter, circular, entire, translucent, smooth, glossy, and butyrous. No hemolysis is seen in blood agar plates, TafleS. 28 flora as reported in the literature (90) Characteristics of unclassified gram-negative aerobic oral Test or substrate EF-4 IIj M-S * Catalase + (100%) + (94%) + (100%) Oxidase + (100%) + (100%) + (100%) Growth on MacConkey (+w) or - 0(65%) — (0%) or - (85%) Urease - (0%) + (100%) I (100%) Indole - (0%) + (100%) - (0%) Simmon's citrate - (0%) - (0%) — (0%) Motility - (0%) — (0%) - (0%) Gelatin - or (+) 0(25%) + (94%) - (0%) Oxidation-fermentation F (100%) or NG (100%) I (100%) Glucose + or (+) 82(18%) - (0%) - (0%) Maltose - (0%) - (0%) - (0%) Lactose - (0%) - (0%) - (0%) Mannitol - (0%) - (0%) — (0%) Sucrose - (0%) - (0%) — (0%) N03 reduction: NO3 + NO2 + or - (26%) - (0%) - (0%) N03 + amine - or + (15%) - (0%) - (0%) Unreduced nitrate - (4%) + (100%) + (100%) 29 Table 5 (continued) Test or substrate EF-4 IIj M—S Pigment - (0%)a + (94%) + or - (44%) TSI sl/butt K or A'w/A or N' K7/N or N/N K/K or K/N H28 on TSI agar - (0%) - (0%) - (0%) * (%) = percent positive based on 85 cultures of EF—4, 35 cultures of IIj, and 41 cultures of M-S. w = weak positive reaction or light growth or + = most strains negative, some are positive or - = most strains positive, some are negative inactive NG = no growth + = positive - = negative (+) = most strains positive K = alkaline A = acid H + II a . . . . Some discrepanCies in the literature. 30 although occasional greening may be observed. These bacteria are most often medium length rods, although some filamentous forms have been reported. Of the 36 cultures isolated at CDC, 17 had been recovered from infected human lesions resulting from bites or scratches of dogs or cats. Eleven cultures, obtained from human spinal fluid, blood, and sputa, were of unknown host origin. Seven isolates were recovered from lower animals, including dogs and cats (90). Two strains were isolated in the Clinical Microbiology Labora- tory at Michigan State University, one from the conjunctiva of a dog, and the second from the periodontium of a cat. The biochemical characteristics of IIj are listed in Table 5. 3. Group M—S Members of this group resemble bacteria of the genus Meraxella. They produce either a yellow or tan pigment and are rod-shaped or coccoid, and usually occur in pairs or chains. Filamentous forms have also been observed. Table 5 lists some of the biochemical reactions of this group. Of 41 cultures studied at CDC, 25 had been isolated from infected wounds caused by dog bites. Four strains were recovered from the tongue, gums, and trachea of dogs. The other 12 isolates recovered from wounds in humans were of unknown origin (90). Actinomycetes Members of this genus are gram-positive, irregularly staining bacteria which are nonacid-fast, nonsporeforming, nonmotile, and anaerobic or microaerophilic. Many species exhibit filaments with true branching (6). A catalase positive, aerobic, nonacid-fast 31 actinomycete isolated from hamster dental plaque has been extensively studied. Howell et al. (48) officially named this organism Odontomyces viscosus; it was later renamed Actinomyces viscosus (6,32). This "hamster organism" was shown to induce periodontal disease (48). Actinomyces viscosus has been isolated from the oral cavity of hamsters, rats, and man, but its pathogenicity for man has not been established (6). Actinomycosis is a chronic, suppurative infection, most often found involving the oral cavity and cervicofacial region of domestic animals as well as man (69). The principal causative agent of human actinomycosis is A. israelii (72,80), although A. naeslundii and A. eriksonii have occasionally been implicated (69). Actinomyces vis- cosus was the infective agent in 6 cases of actinomycosis diagnosed in dogs (23). Actinomyces israelii is a gram-positive anaerobic to microaerophilic organism, normally found in the human mouth (6,80). It is unable to penetrate intact mucosa and requires a break in the tissues to establish itself (69). Actinomycosis may result from tooth extractions or injury to the mouth or throat (65), surgical or accidental trauma, e.g., a compound jaw fracture (52), periodontal disease (18), or an infected root canal of a carious tooth (40). Actinomycetes may also be aspirated into the lungs causing pulmonary actinomycosis, or be swallowed and invade the intestinal mucosa resulting in abdominal actinomycosis (69,94). Transmission by human bites, although rare, has been reported (69). Actinomyces israelii gains entrance to the soft tissues and, as the lesion grows, multiple abscesses develop. These may break through the skin and cause a 32 characteristic pattern of multiple sinuses. Within these abscesses are found sulfur granules consisting of microcolonies of filamentous and branching actinomycetes (72). Currently attempts are being made to answer the question, why do some actinomycetes, which are normal inhabitants of the oral cavity, become pathogenic? In cases involving cervicofacial actino- mycosis in particular, actinomycetes are found in association with organisms such as streptococci, fusobacteria, Bacteroides corrodens, or Actinobacillus actinomycetemcomitans. Some researchers think it is the association of aerobes and anaerobes acting synergistically which contributes to the pathogenicity of the actinomycetes (45). Others hypothesize that an allergic reaction may have a role in the development of lesions (69). Staphylococci Micrococci occur universally in the human oral cavity, but are not usually dominant numerically (73). Coagulase positive Staphylococcus aureus may be found regularly in the oral cavity of man (56). Staphylococcus epidermidis is found in greater frequency than S. aureus in the healthy mouth. However, S. aureus predominates over S. epidermidis in cases involving open suppurative lesions such as periodontitis (53). The pathogenicity of staphylococci may relate to their ability to produce such extracellular factors as hemolysins, leukocidin, enterotoxins, coagulase, and hyaluronidase (69). Staphylococci have been implicated in a number of oral and dental lesions such as osteomyelitis of the jaw, parotitis (an infection characterized by a 33 discharge of purulent exudate), and facial cellulitis (69). Tooth extractions may result in bacterial endocarditis under certain circumstances (74). Streptococci Streptococcus species comprise approximately 30 to 60% of the bacteria residing on the surfaces of teeth, cheek, tongue, and saliva. The majority of these organisms belong to the viridans or alpha-hemolytic group (34). Streptococcus mutans appears to be the most virulent of the alpha-hemolytic streptococci and has been shown to have the capacity to initiate dental decay affecting smooth enamel surfaces. The cariogenic potential of this organism may be associated with its ability to adhere on tooth surfaces forming large microbial dental plaque deposits as previously described (24,31,34, 35,57,58). The two species found most frequently in the oral cavity are S. mitis and S. salivarius. These have been found in abscesses, ulcerative stomatitis, root canals, periodontal pockets, calculus, and carious lesions (69). The appearance of beta-hemolytic streptococci in the oral cavity is of considerable interest because of their possible role in the spread of infection. Group A beta-hemolytic streptococci have been implicated in causing septic sore throat, scarlet fever, and rheumatic fever in human beings. Certain extracellular products such as hyaluronidase, streptokinase, deoxyribonuclease, and hemolysins (69) contribute to the pathogenesis of streptococcal diseases. 34 Neisseria Neisseria species have been found in the oral cavity and upper respiratory tract of healthy humans (6): however, some of these organisms have been identified as the only microorganisms present in some serious infections. Hudson (50) isolated N. pharyngis from a patient who had developed subacute bacterial endocarditis. Neisseria catarrhalis (Branhamella catarrhalis) has been implicated in an infec- tion of the parotid gland following a blow from a fist (69). Addi- tional evidence suggests that pigmented dental plaque may be produced by certain oral chromogenic Neisseria organisms (69). Carious mouths have a characteristic population of nonpigmented Neisseria organisms which contain catalase and do not produce a copious amount of poly- saccharide on 5% sucrose agar. Noncarious mouths, in contrast, have been shown to contain Neisseria organisms which do not produce catalase but do produce a copious amount of polysaccharide on 5% sucrose agar (68). LITERATURE CITED 10. 11. LITERATURE CITED Allen, Paul W., D. F. Holtman, and L. A. McBee. 1941. Microbes Which Help or Destroy Us. C. V. Mosby Co., St. Louis, Mo. Allott, E. N., and R. Cruickshank. 1944. Infections of cat- bite and dog—bite wounds with Pasteurella septica. J. Path. Bact. 56:711. Babooloal, R., R. N. Powell, and A. S. Prophet. 1970. Hydro- lytic enzymes in developing gingival plaque. J. Perio- dontics 41:87. Bergan, T., K. Bovre, and B. Hovig. 1970. Present status of the species Micrococcus freundenreichii Guillebeau 1891. Int. J. Syst. Bacteriol. 29:249-254. Berger, U., M. Kapovits, and G. Pfeifer. 1959. Zur beseidlung der kindlichen mundhohle mit anaeroben mikroorganismen. Z. Hyg. 145:564. Bergey's Manual of Determinative Bacteriology. 8th Edition. 1974. Edited by Buchanan, R. E., and N. E. Gibbons. Williams and Wilkins Co., Baltimore, Md. Bowen, W. H. 1965. A bacteriological study of experimental dental caries in monkeys. Int. Dent. J. 15512. Bowen, W. H., and D. E. Cornick. 1967. Effects of carbohydrate restriction in monkeys (M. irus) with active caries. Helv. Odont. Acta 11:27. Bradel, S. F., and J. R. Blayney. 1940. Clinical and bacterio- logic studies on dental caries. J. Am. Med. Assoc. 31: 1601. Brailovsky-Lounkevitch, Z. A. 1915. Contribution a l'etude de la flore microbienne habituelle de la bouche normale (nouveau-nes, enfants, adultes). Ann. Inst. Pasteur 29:379. Burnett, George W., and H. W. Scherp. 1968. Oral Microbiology and Infectious Disease. Williams and Wilkins Co., Baltimore, Md. 3S 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 36 Carlsson, J. 1967. Presence of various types of nonhaemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odont. Revy. 16:55. Carlsson, J., and J. Egelberg. 1965a. Local effect of diet on plaque formation and development of gingivitis in dogs. 11. Effect of high carbohydrate versus high protein fat diets. Odont. Revy. 16:42. Carlsson, J., and J. Egelberg. 1965b. Effect of diet on early plaque formation in man. Odont. Revy. 16:122. Carlsson, J., H. Grahnen, G. Johnsson, and S. Wikner. 1970. Early establishment of Streptococcus salivarius in the mouths of infants. J. Dent. Res. 22:415. Carlsson, J., G. Soderholm, and I. Almsfeldt. 1969. Prevalence of Streptococcus sanguis and Streptococcus mutans in the mouth of persons wearing full dentures. Arch. Oral Biol. 14:243. Carlsson, J., and B. Sundstrom. 1968. Variation in composi- tion of early dental plaque following ingestion of sucrose and glucose. Odont. Revy. 12:161. Caron, G. A., and I. Sarkany. 1964. Cervicofacial actinomycosis. Brit. J. Derm. 26:421. Carter, G. R. 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Advances in Veterinary Science 11:321—379. Carter, G. R. 1973. Diagnostic Procedures in veterinary Microbiology. 2nd Edition. Charles C. Thomas, Springfield, Ill. Courant, P. R., S. R. Saxe, L. Nach, and S. Roddy. 1968. Sulcular bacteria in the beagle dog. Periodontics 6:250. daCosta, T., and R. J. Gibbons. 1968. Hydrolysis of levan by human plaque streptococci. Arch. Oral Biol. 16:609. Davenport, A. A., G. R. Carter, and R. G. Schirmer. 1974. Canine actinomycosis due to Actinomyces viscosus: Report of six cases. Veterinary Medicine/Small Animal Clinician Nov. 1974:1442. Davis, B. D., R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and W. B. Wood. 1973. Microbiology. 2nd Edition. Harper and Row, New York, N.Y. deAraujo, W. C., and J. B. MacDonald. 1964. Gingival crevice microbiota of preschool children. Arch. Oral Biol. 2:227. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37 deStoppelaar, J. P., J. vanHoute, and O. Backer-Dirks. 1970. The effect of carbohydrate restriction on the presence of Streptococcus mutans, Streptococcus sanguis, and iodophilic polysaccharide-producing bacteria in human dental plaque. Caries Res. 3:114. Egelberg, J. 1965. Local effect of diet on plaque formation and development of gingivitis in dogs. I. Effect of hard and soft diets. Odont. Revy. 16:31. Eisenberg, Jr., H. G. George, and D. C. Cavanough. 1974. Pasteurella. Page 246 in Manual of Clinical Microbiology. Edited by Lennette, et al. American Society for Micro- biology, Washington, D.C. Fitzgerald, R. J., P. H. Keyes, T. H. Stoudt, and D. Spinell. 1968. The effects of a dextranase preparation on plaque and caries in hamsters, a preliminary report. J. Am. Dent. Assoc. 26:301. French, Cecil. 1906. Surgical Diseases and Surgery of the Dog. Washington, D.C. Genco, Robert J., Richard T. Evans, and Solon A. Ellison. 1969. Dental research in microbiology. J. Am. Dent. Assoc. 16: 1017. Georg, L. K., L. Pine, and E. M. A. Gerencser. 1969. Actinomyces viscosus comb. nov., a catalase-positive, facultative member of the genus Actinomyces. Int. J. Syst. Bacteriol. 12:291-293. Gibbons, R. J. 1969. Significance of the bacterial flora indigenous to man. Page 27 in American Institute of Oral Biology, Twenty—Sixth Meeting. Gibbons, R. J. 1972. Streptococci and Streptococcal Diseases. Recognition, Understanding, and Management. L. W. Wannamaker and J. M. Matson, eds. New York Academic Press, N.Y. Gibbons, R. J., K. S. Berman, P. Knoettner, and B. Kapsimalis. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch. Oral Biol. ll:549. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran-induced agglutination of Streptococcus mutans, and its potential role in the formation of microbial dental plaques. J. Bacteriol. 26:341. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. SO. 38 Gibbons, R. J., and M. Nygaard. 1970. Interbacterial aggrega- tion of plaque bacteria. Arch. Oral Biol. 16:1397. Gibbons, R. J., S. S. Socransky, S. Sawyer, B. Kapsimalis, and J. B. MacDonald. 1963. The microbiota of the gingival crevice area of man. II. The predominant cultivable organisms. Arch. Oral Biol. 6:281. Gibbons, R. J., and D. M. Spinell. 1970. Salivary induced aggregation of plaque bacteria. Page 207 in Dental Plaque. W. D. McHuah, ed. E. & S. Livingstone, Ltd., Wynnwood, Pa. Gold, L., and E. E. Doyne. 1952. Actinomycosis with osteo- myelitis of the alveolar process. Oral Surg. 6:1056. Gordon, D. F., and B. B. Jong. 1968. Indigenous flora from human saliva. J. Appl. Microbiol. 16:428. Gustafson, R. L., et al. 1966. The biological activity of Leptotrichia buccalis endotoxin. Arch. Oral Biol. 11: 1149. Hawkins, L. A. 1969. Local Pasteurella multocida infections. J. Bone Joint Surg. 51A:363-366. Herrell, Wallace E., ed. 1969. Pasteurella multocida infection. Clinical Medicine, 79(9):11-15. Hertz, J. 1960. Actinomycosis. Borderline cases. J. Int. Coll. Surg. 62:148. Hoffman, H. 1966. Oral microbiology. Advance in Applied Microbiology 6:195. Holmes, M. A., and G. Brandon. 1965. Pasteurella multocida infections in 16 persons in Oregon. Public Health Records 66:12. Howell, Jr., A., H. V. Jordan, L. K. Georg, and L. Pine. 1965. Odontomyces viscosus, gen. nov., spec. nov., a filamentous microorganism isolated from periodontal plaque in hamsters. Sabouraudia 2:65-68. Hubbert, William T., and M. N. Rosen. 1970. I. Pasteurella multocida infection due to animal bite. Am. J. Pub. Health 66:6. Hudson, R. 1957. Neisseria pharyngis bacteriaemia in a patient with subacute bacterial endocarditis. J. Clin. Path. 10:195. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 39 Hurst, V. 1957. Fusiforms in the infant mouth. J. Dent. Res. 36:513. Hylton, R. P., H. S. Samuels, and G. W. Oatis, Jr. 1970. Actinomycosis: Is it really rare? Oral Surg. 66:138. Ikeda, T., A. Isoda, and T. Iidako. 1964. A study on staphylo- cocci isolated from the acute suppurative diseases in the oral area with reference to their comparison in pathogenicity. J. Nipon Univ. Sch. Dent. 6:88. Jensen, 8. B., F. V. Jackson, and S. E. Mergenhagen. 1964. Alterations in type and bactericidal activity of mouse peritoneal phagocytes after intraperitoneal administration of endotoxin. Acta Odont. Scand. 1:71-93. Jensen, S. B., et al. 1966. Influence of oral bacterial endo- toxin on cell migration and phagocytic activity. J. Periodont. Res. 1:129 (no. 2). Jordan, H. V., R. J. Fitzgerald, and J. E. Faber, Jr. 1956. Studies on the aciduric oral micrococci. J. Dent. Res. 35:404. Jordan, H. V., and P. H. Keyes. 1966. In vitro methods for the study of plaque formation and carious lesions. Arch. Oral Biol. 11:793. Kelstrup, J., and R. J. Gibbons. 1970. Induction of dental caries and alveolar bone loss by a human isolate resembling Streptococcus salivarius. Caries Res. 3:360. Kostecka, F. 1924. Relation of the teeth to the normal develop- ment of microbial flora in the oral cavity. Dental Cosmos. 66:927. Lee, M. L. H., and A. J. Buhr. 1960. Dog bites and local infec- tion with Pasteurella septica. Brit. J. Med. 6:169—171. Lewkowicz, X. 1901. Recherches sur la flore microbienne de la bouche de nourrisons. Arch. Med. Exp. et Anat. Pathol. 13:633. Lilienthal, B. 1950. Studies on the flora of the mouth. III. Yeast-like organisms: Some observations on their inci- dence in the mouth. Australian J. Exp. Biol. Med. Sci. 28:279. Loesche, W. L. 1968. Importance of nutrition in gingival crevice microbial etiology. Periodontics 6:245. MacDonald, J. B., S. S. Socransky, and S. Sawyer. 1959. A survey of the bacterial flora of the periodontium in the rice rat. Arch. Oral Biol. 1:1. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 40 Martin, W. J., and D. R. Nichols. 1961. The mycoses as they affect man. Vet. Excerpts 21(2):33. McCarthy, C., M. Snyder, and R. B. Parker. 1965. The indigenous oral flora of man. I. The newborn to the one-year-old infant. Arch. Oral Biol. 16:61. Meyers, B. R., B. L. Berson, M. Gilbert, and S. Z. Hirschman. 1973. Clinical patterns of osteomyelitis due to gram— negative bacteria. Arch. Int. Med. 131:228-233. Morris, E. O. 1954. The bacteriology of the oral cavity. British Dent. J. 66:259. Nolte, William A., ed. 1973. Oral Microbiology. 2nd Edition. C. V. Mosby Co., St. Louis, Mo. Normann, B., B. Nilehn, J. Rajs, and B. Karlberg. 1971. A fatal case of Pasteurella multocida septicemia after cat bite. Scandinavian J. Infect. Dis. 6:251—254. Onisi, M., K. Kolke, and Y. Tachibara. 1960. Modes of estab- lishing fusobacteria, lactobacilli, and streptococci in the human mouth. D. Abs. 6:470. Pindborg, J. J. 1973. Atlas of Disease of the Oral Mucosa. W. B. Saunders Co., Philadelphia, Pa. Pike, E. E., J. H. Freer, G. H. G. Davis, and K. A. Bisset. 1962. The taxonomy of micrococci and Neisseriae of oral origin. Arch. Oral Biol. 1:715. Quinn, E. L. Personal communication. Ritz, H. L. 1967. Microbial population shifts in developing dental plaque. Arch. Oral Biol. 11:1561. Rogers, R. J., and J. K. Eldes. 1967. Purulent leptomeningitis in a dog associated with an aerogenic Pasteurella multocida. Australian Vet. J. 66:81-82. Rosebury, T. 1972. Distribution and development of the micro- biota of man, in Microorganisms Indigenous to Man. McGraw—Hill, New York. Rosenthal, S. L., and E. H. Gootzeit. 1942. The incidence of Bacteroides fusiformis and spirochetes in the edentulous mouth. J. Dent. Res. 61:373. Saxe, S. R., et al. 1967. Oral debris, calculus, and perio- dontal diseases in the beagle dog. Periodontics 6:217. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 41 Scopp, Irwin Walter. Oral Medicine. A Clinical Approach with Basic Science Correlation. C. V. Mosby Co., St. Louis, Mo. Shklair, I. L., and M. A. Mazzarella. 1961. Effects of full- mouth extraction on oral microbiota. D. Progress 1:275. Smith, J. E. 1955. Studies on Pasteurella septica. I. The occurrence in the nose and tonsils of dogs. J. Comp. Path. 66:3. Smith, J. E. 1959. Studies on Pasteurella septica. II. Some cultural and biochemical properties of strains of dif- ferent host species. J. Comp. Path. 66:315-328. Socransky, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res., Supplement to No. 2,66:203-222. Socransky, S. S., P. N. Baer, and P. H. Keyes. 1969. Relations of diet, oral microbiota, and rate of calculus formation in conventional rats. IADR, 47th general meeting #282 (Abstr.). Socransky, S. S., and C. Hubersak. 1967. Replacement of ascitic fluid or rabbit serum requirement of Treponema dentium by aglobulin. J. Bacteriol. 66:1795. Socransky, S. S., W. J. Loesche, C. Hubersak, and J. B. MacDonald. 1964. Dependency of Treponema microdentium on other oral organisms for isobutyrate, polyamines, and a con- trolled oxidation reduction. J. Bacteriol. 66:200. Socransky, S. S., and S. D. Manganiello. 1971. The oral micro- biota of man from birth to senility. J. Periodont. 61: 485-494. Talbot, J. M., and P. H. A. Sneath. 1960. A taxonomic study of Pasteurella septica, especially of strains isolated from human sources. J. Gen. Microbiol. 61:303-311. Tatum, Harvey W., W. H. Ewing, and R. E. Weaver. 1970. Miscel- laneous gram-negative bacteria. Pages 191-198 in Manual of Clinical Microbiology. J. E. Blair, E. H. Lennette, and J. P. Truant, eds. American Society for Microbiology, Washington, D.C. Theilade, E., W. H. Wright, S. J. Borglum, and H. Loe. 1966. Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J. Periodont. Res. 1:1. 92. 93. 94. 95. 42 Tindall, John P., C. M. Harrison, and M. S. Durham. 1972. Pasteurella multocida infections following animal injuries, especially cat bites. Arch. Derm. 105:412-416. Van Houte, J., and H. B. Jansen. 1968. Levan degradation by streptococci isolated from human dental plaque. Arch. Oral Biol. 16:827. Weaver, R. E. 1970. "Unclassified" groups of aerobic gram- negative bacteria isolated from clinical specimens. Seminar on Current Topics in Clinical Microbiology, 70th Meeting, Am. Soc. Microbiol., Boston, Mass. Winton, F. W., and N. 8. Hair. 1969. Pasteurella pneumotropica isolated from a dog bite wound. Microbios. 1:155-162. ARTICLE AEROBIC BACTERIAL GINGIVAL FLORA OF THE DOG BY D. A. Saphir and G. R. Carter SUMMARY Gingival scrapings from dogs were collected to isolate and identify species of the aerobic, bacterial gingival flora. Of par- ticular interest was the frequent recovery of three unclassified groups of aerobic gram-negative bacteria, IIj, EF-4, and M-5, pre- viously associated with human infections resulting from dog bites. Although no set pattern existed between the variability and consis- tency of gingival microbiota as related to age, sex, and breed of dog, certain characteristic flora can be predicted in the healthy canine gingiva. Members'of the following genera were found: Streptococcus, Staphylococcus, Actinomyces, Escherichia, Corynebacterium, Pasteurella, Caryophanon, Mycoplasma, Acinetobacter, Moraxella, Neisseria, Entero- bacter, and Bacillus. INTRODUCTION Studies of gingival flora in the dog are of particular value as this animal has a gingival crevicular epithelium similar to that found in humans (7). Few investigators, however, have concentrated their efforts on describing gingival flora in dogs. The organisms found within the oral cavity of domestic animals consist of many potential pathogens and commensals (14,17,18). Disease may occur as a result of local infection by microorganisms which usually inhabit the mouth in large numbers. However, for an organism 43 44 to produce lesions, dynamic alterations involving the host and/or the microorganisms must occur. Irritation, trauma, penetration of foreign bodies such as bones or sticks, carious teeth, and neoplasms contribute to bacterial invasion (6). It has been shown that certain organisms of the oral cavity were recovered in wounds resulting from animal bites. For example, Pasteurella multocida infections in humans have for some time been associated with animal bites (2.5.10). The purpose of this investigation was to isolate and identify to species, where possible, the aerobic bacterial flora found in gingival scrapings of dogs. Particular attention was given to those organisms previously reported to be associated with human infections resulting from dog bites. MATERIALS AND METHODS Collection of material. Gingival scrapings were taken, using sterilized gauze pads, from 50 dogs in the East Lansing, Michigan, area. Care was taken to select only healthy dogs. Cultural and identification procedures. Immediately following collection, gauze pads were immersed in sterile flasks containing 10 ml of nutrient broth (Difco). All samples were thoroughly agitated, then streaked 15 minutes after collection for isolation on blood agar plates, 4% blood agar, MacConkey plates, and PPLO agar (Difco). Plates were incubated in a 4% CO2 incubator for 24 h, examined, and reincubated for an additional 24 to 48 h. Each different colonial type was subcultured. Colonial and cellular morphologies of all strains were recorded and biochemical characteristics of the organisms 45 were determined. Each organism was identified, when possible, using present classification tables (1,19). Antibiotic susceptibility tests. Antibiotic susceptibility tests were done on several isolates of Group IIj and Group EF-4 using the standard Kirby-Bauer procedure (19). Group EF-4 was placed on Mueller- Hinton agar media (Difco), and antibiotic susceptibility patterns for Group IIj were done using a tryptose agar (Difco) plus yeast extract (Difco) base. RESULTS Fifty dogs were used in this survey. The organisms isolated from gingival scrapings included Group IIj, Group EF-4, Moraxella (including M. phenylpyruvica, M. nonliquefaciens, M—4, and M—S), Enterobacter aerogenes, Escherichia coli, Acinetobacter calcoaceticus (var lwoffi and anitratus), Pasteurella (including P. multocida and Pasteurella-like organisms), Neisseria, Staphylococcus aureus, Staphylococcus epidermidis, and other Micrococcaceae, a-, B-, and y-hemolytic streptococci, Corynebacterium, Actinomyces (including A. viscosus), Bacillus, Caryophanon, and Mycoplasma. Only those organisms capable of growing aerobically on blood agar and/or MacConkey agar plates, and PPLO medium (Difco) within 72 h at 37 C in a 4% CO2 incubator were reported. Group EF-4 Bacteria of this type consisted of small gram-negative, short rod- macoccoid-shaped organisms. Colonies averaged 1-2 mm in diameter and appeared circular, entire, opaque, convex, and mucoid, sometimes 46 producing a yellow pigment. They were nonhemolytic, although an occasional greening of blood agar was observed. Typical biochemical reactions are shown in Table 1. Thirty percent of the dogs examined harbored these organisms (Table 2). Group IIj Colonies of this group were circular, entire, translucent, smooth, glossy, and butyrous. and very sticky, making them difficult to remove from solid media. Growth in broths was poor. No hemolysis was seen on blood agar plates, although greening was occasionally observed around colonies. Organisms were gram-negative, medium length rods. Much of the viability of this organism was lost within one week. Table 1 lists the differential reactions of Group IIj. A relatively large inoculum on Christensen's urea agar showed rapid hydrolysis. Group IIj was seen in 38% of the dogs examined. Mbraxella Members of this genus were rod-shaped or coccoid, usually occurring in pairs or chains. 0f the 40% of the canine samples har- boring bacteria of this type (Table 2), M-5 was the most frequent isolate (Table 3). M-5 resembled other bacteria of this genus. They produced a yellow or tan pigment, were nonhemolytic, and were identi- fied by the biochemical reactions shown in Table 1. Pasteurella and Pasteurella-like organisms These gram-negative coccoid to small rod-shaped organisms were found in 22% of the dogs (Table 2). Half of these isolates identified from the reactions shown in Table 1 consisted of organisms typical of 47 msmoomwS + I I I I d d « Adv m + mmumsonwuum zmmwommmz I I I I m >¢ > < m + Eswumuomnmcmhou I I | U ' H I | ' ' | ' l ' 3+ \ Smxu H + + «m z >+ > 3+ \ >«\M m m + + «e mm o I + I + I I + I\I Z\>Z I I l I l 02 + + a flHH mfiouw u. u T. m .b S n I I m m s T. .b o o o e T. u o a W T. 1 S S e zmum w 3 o. 1 T. o a I I w m. m w m. n H. W mzmH o .L 1 o T. e D e 1 1 1 o m. o. e s 1 T. T. m. u ,b s 3 s s s s u. s e T. e 3 u I e T. o a a a a .A a s s A a s s w" T. m. a 1 .A // a o H n e P T. 7. 3 3 m n S 3 a 1. v. a. T: 1. D. o a s u a n S a o. Hm>wocwo ca ochM mEmHommuoouows ownoumm accuses mEOm mo macauummn oowumuwmausoofl mo mumesbm moon sown mmcwmmuom .H wanna 48 .ommmHmo n o xo>wummwc mcwmuum umoE u AIS xw>fluflmom mcfimuum umos u A+V «manmwum> > xw>fiufimom gums u 3 “wave u a «moaamxam u M onAMHAM> haamcoflmmooo n > xo>aumoaxo u o am>aumucwsumm m «Humane: n z «cuaoum on n 02 «Hma ow mom on .mem& mumumom owed co cowuommu +v I mum «noomoum um mammflufiucoow you use om>nwwno aaucmsooum Emwcmmuo ad “Away .Hm um .Esuma .3 .m Bonn ommmwooz « a II t I I I e+ I I I «I. z\z 3 3 3 3 3 mo + + «Insocxco mxaa + I I I I I I I\I «\m >m m I m m + + Inmamnsmummm unaccuwss + + I I I I I I\I «\m > >AIV m >AIV m m + + mmmmuumummm > I I I > + >+ > I\I Z\M I >I A+V + o I + usumuuwnm Hm> > I I I >I + > > I\I z\x I I I I H I + weenie um> meowumomouflmo Hmuomnoumswow . m s T. .o o o o u. w. m w “w m" mm m m m m e n e T. e x e mamuzaomo m i p .. I o m a I I w n n m. m .. m a T. W W. U m... m 0 P I. O O O O W. p. T. K 1. T. T. U u 5 S 1. S S S S U. S a. S a 1. U N. I E T. O 8 a 3 8 .A a S T. rA a S S 1W T. p a S 1 .A / I a O H n p. D. I. Z 1. 3. n n. S 3. a D e a. n“ u a m a a n S 8 D. Aomsaapcooo H magma 49 Table 2. Frequency of isolation of aerobic bacteria recovered in gingival scrapings from 50 dogs GRAM-NEGATIVE No. of + Percent GRAM—POSITIVE ‘ No. of + Percent canine of inci- canine of inci- samples dence samples dence 1) Moraxella 20 40.0% 1) Strepto- 41 82.0% cocci 2) Group IIj 19 38.0% 2) Micrococca- 30 60.0% 3) Group EF-4 15 30.0% ceae 4) Escherichia 11 22.0% 3) Corynebac— 13 26.0% coli terium 5) Pasteurellaa 11 22.0% 4) Actinomycetes 7 14.0% 6) Caryophanon 10 20.0% 5) Bacillus 6 12.0% 7) Neisseriab 10 20.0% 8) Acinetobacter 5 10.0% calcoaceticus 9) Enterobacter 1 2.0% OTHER 1) Mycoplasma in 35 out of 41 dogs 85.4% aIncludes Pasteurella-like organisms. bIncludes Branhamella catarrhalis. 50 Table 3. Frequency of isolation of aerobic bacterial species recovered in gingival scrapings from 50 dogs GRAM-NEGATIVE No. of + Percent GRAM-POSITIVE No. of + Percent canine of inci- canine of inci- samples dence samples dence l) MOraxella - l) Strepto- 21 isolates cocci - M. phenylpyru- 3 14.3% 45 isolates vica a-hemolytic 36 80.0% M. nonlique— 2 9.5% streptococci faciens B-hemolytic 5 11.1% M-5 9 42.8% streptococci M-4 1 4.8% y—hemolytic 4 8.9% M-"species" 6 28.6% streptococci 2) Pasteurella 2) Micrococca- - 12 isolates ceae - 35 P. multocida 6 50.0% isolates P-like 6 50.0% Staph. aureus 9 25.7% Staph. epider- 16 45.7% 3) Neisseria midis - 10 isolates Other species 10 28.6% B. catarrhalis* 5 50.0% Neisseria 5 50.0% 3) Actinomycetes "species" - 8 isolates A. viscosus 5 62.5% 4) Acineto- A. "species" 3 37.5% bacter calcoaceticus - 5 isolates var lwoffi 4 80.0% var anitratus 1 20.0% * Present classification - Branhamella catarrhalis formerly - Neisseria catarrhalis 51 Pasteurella multocida. However, organisms similar in colonial and cellular morphology, but differing in key biochemical reactions from P. multocida, were also found (Table 1). These organisms, which appeared to belong to the Pasteurella genus, did not conform to any recognized species by available classification tables. Caryophanon This motile, filamentous, multicellular eubacterium was found in 10 out of 50 dog samples (Table 2). Some discrepancies exist in the literature relating to the gram-reaction of Caryophanon, but the organisms recovered in this survey were distinctly gram-negative. Neisseria Isolates of Neisseria catarrhalis, more recently reclassified as Branhamella catarrhalis (12), were found in 5 dogs (Table 3). The other 5 isolates identified as Neisseria were gram-negative, oxidase positive cocci that produced a yellow pigment. They could not be identified with known species. Actinomyces Five isolates of Actinomyces viscosus were found in gingival scrapings (Table 3). The organism was identified on the basis of delayed growth pattern (48 to 72 h), a crumbly, off-white, and dry colonial morphology, gram-positiveness, nonacid-fastness, filamentous to "Y—shaped" cellular configuration, and the biochemical reactions shown in Table 1. Three isolates with similar colonial and cellular morphology but different biochemical characteristics were also recovered. 52 Corynebacterium Twenty—six percent of the canine samples contained diphtheroids. They were all nonhemolytic, 0.5 to 1.0 mm in diameter, and white, but varied in biochemical reactions. Glucose and maltose were fer— mented in all cases, but lactose and sucrose fermentation varied (Table l). Mycoplasma These organisms, isolated on PPLO medium (Difco), had a charac- teristic "fried egg" appearance. Colonies were pleomorphic, extremely small on the agar medium, Opaque, with a yellowish central area. These organisms were recovered in 35 out of 41 dogs sampled. Miscellaneous unidentified bacteria 1. A number of organisms isolated from gingival scrapings in dogs produced a yellow pigment, were nonmotile, weakly or nonfermenta- tive, and catalase positive (Table 3). These bacteria were gram- negative, small, thin, rod- to coccoid-shaped cells and seemed to resemble species of Flavobacterium. 2. Granular, dry, nonhemolytic, rhizoid colonies were frequently recovered from dogs. Colonies were pinpoint in 24 h, but increased in size following further incubation. These filamentous organisms stained gram—variably, and were inactive biochemically. Streptococci and staphylococci Alpha-hemolytic streptococci were isolated more frequently than other organisms, with the exception of Mycoplasma, and made up a large part of the flora of gingival scrapings from these dogs (Table 2). 53 Isolates of beta- and gamma-hemolytic streptococci were much less frequent in comparison (Table 3). Of the 35 isolates of Micro- coccaceae, over 45% consisted of Staphylococcus epidermidis and 25% were yellow or white pigmented, beta-hemolytic Staphylococcus aureus (Table 3) . Twenty-three female and 27 male dogs were examined. No dif- ferences were found in gingival scrapings between males and females. Also, no differences in aerobic flora were observed when dogs were grouped according to the American Kennel Club classification of breeds. The results of the antibiotic susceptibility tests on Groups IIj and EF-4 are presented in Tables 4 and 5, respectively. DISCUSSION The gingival crevicular epithelium in man and dogs is quite similar (7). Perhaps the establishment of the oral flora in dogs and man is also similar. If an organism is to survive in the oral cavity of an animal, some mechanism of retention, as well as a suitable nutritional or physicochemical environment are usually required (18). The development of teeth provides a new attachment site for bacteria in carious lesions and gingival crevice areas. The oral cavity can sustain aerobic, microaerophilic, and anaerobic organisms. As the amount of oxygen present within a particular site in the oral cavity changes, so do the microorganisms which inhabit that location. Plaque formation simulates an anaerobic environment and, subsequently, the number of aerobes such as Neisseria and streptococci decrease and anaerobes such as Actinomyces and Fuso- bacterium increase (13). 54 Table 4. Results of antibiotic susceptibility tests on Group IIj Antimicrobial agents Ampicillin Chloramphenicol Gentamycin Lincomycin Neomycin Nitrofurantoin Penicillin Polymyxin B Sulfadimethoxine Tetracycline c d e f g S S S S S S S S S S I S S S S S t S S S S S E E R s s . S S S S S E E S S S R R S S R R R S S S S S S S S S = sensitive 50 II ['11 ll resistant equivocal 55 Table 5. Results of antibiotic susceptibility tests on Group EF-4 Antimicrobial agents a b c d Ampicillin S S S S Cephalothin S R S S Chloramphenicol S S S S Gentamycin S S S S Lincomycin R R S S Neomycin R R R R Nitrofurantoin S S S S Novobiocin S S S S Penicillin R R S R Polymyxin B S S S S Sulfadimethoxine S S S S Tetracycline S S S S S = sensitive R = resistant 56 The gingival flora seems to be as diversified or even more variable in the puppy than in the adult dog. Variability in the puppy, as in the human child, may be due to failure of a particular organism to find a suitable attachment site, the absence of an essential nutrient required for growth, or an unsatisfactory oxygen relationship to mention only several possibilities. Older animals develop an established ecosystem, making it more difficult to incor— porate a new bacterial species. During the first few years of life, the oral cavity is exposed to a myriad of microorganisms. As physio- logic changes occur in the host, certain organisms seem to establish themselves in particular locations. As organisms settle in these niches, a certain degree of stability ensues, especially in the adult, and there are fewer vacancies for new species. The latter must compete with the resident flora, and they frequently have dif- ficulty breaking into an established ecosystem (18). Based on the data of this survey, indications are that the young dog acquires gingival flora,similar to that found in the adult, at an early age. Most of the younger dogs examined had only limited contact with humans and were frequently housed among other members of their own species. It would therefore seem likely that the puppy acquires its adult-like gingival flora by association with other dogs, including its mother. The Center for Disease Control in Atlanta, Georgia (CDC), reported isolating 36 cultures of Group IIj, 17 from human lesions resulting from bites or scratches of dogs and cats (19). There is as yet no evidence that Group IIj causes disease in the dog. Its 57 role may be that of a secondary invader. Without guanine-cytosine percentages and base homologies one can only speculate on generic classification at this time. Considering the characteristics described in Table l and its non—fermentative nature, Group IIj appears to resemble the genera Moraxella (specifically M. phenyl- pyruvica) and Brucella (specifically B. canis) most closely. CDC reported isolating 85 strains of Group EF-4; 66 of these were recovered from humans and, of these 66, 32 were from humans who were bitten by dogs or cats (19). Group EF-4 strains have not been incriminated as a cause of disease in the dog. On the basis of the preceding characteristics described in Table 1, Group EF-4 appears to resemble most closely the genera Pasteurella and Actinobacillus. Group M—5 resembles bacteria of the genus Moraxella (19) on the basis of biochemical reactions (Table 1) and cellular and colonial characteristics. Of 41 cultures studied at CDC, 25 were recovered from infected wounds caused by dog bites. There is yet no evidence that suggests M-S is associated with disease in the dog. Caryophanon was described as a gram-positive, motile, large rod or filament, and was originally isolated from cow dung (8). Caryophanon was also described as a gram-negative organism (15) which concurs with the results of this study. Further studies described this organism in water, intestines of arthropods and vertebrates, and in decomposing organic material (11). Caryophanon has been seen not infrequently in Wright-stained smears from oral mucosa of dogs (3). An attempt was made to recover Caryophanon from several dogs that yielded it the 58 first time. The organism was not reisolated. It may be that Caryophanon is unable to establish permanent residence in the gingiva and is only a part of the transient flora. The organisms classified as Pasteurella-like differed from P. multocida in their negative indol and occasionally acid from maltose reaction. Smith (16) reported that dog strains of P. multocida frequently possessed such special characteristics as acid production from maltose but not xylose and mannitol, low pathogeni- city for mice, saline and acid sensitivity, and absence of capsules. Other Pasteurella-like organisms recovered from humans bitten by dogs or cats were described as producing some gas from glucose (20). Actinomyces viscosus was implicated as the causative agent of periodontal disease with subgingival plaque in hamsters (9). Experi- mentally induced infections were produced in mice from hamster strains (9). Although pathogenicity for man is yet undetermined, A. viscosus has been isolated from the human oral cavity (1). In this study, A. viscosus was found in the gingiva of 5 dogs with clean teeth and healthy gums. Actinomyces viscosus has been reported as the cause of 6 cases of actinomycosis in dogs (4). Although there was considerable variation in the oral flora among individuals, a number of organisms were recovered with fair consistency. These included streptococci, staphylococci, and Mycoplasma, 3 gram-negative bacteria associated with dog bites in humans (Groups IIj and EF-4, M—5), and occasionally such potential pathogens as Pasteurella and Actinomyces. Caryophanon, Neisseria, Acinetobacter, Corynebacterium, and Bacillus were sporadically iso- lated from the canine gingiva. 10. 11. 12. 13. 59 LITERATURE CITED Bergey’s Manual of Determinative Bacteriology. 8th Edition. 1974. Edited by Buchanan, R. E., and N. E. Gibbons. Williams and Wilkins Co., Baltimore, Md. Carter, G. R. 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Advances in Veterinary Science 11:321-379. Coak, R. Personal communication. Davenport, A. A., G. R. Carter, and R. G. Schirmer. 1974. Canine actinomycosis due to Actinomyces viscosus: Report of six cases. Veterinary Medicine/Small Animal Clinician Nov. 1974:1442. Eisenberg, Jr., H. G. George, and D. C. Cavanough. 1974. Pasteurella, p. 246. 12_J. E. Blair, et al. (ed.), Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C. French, Cecil. 1906. Surgical Diseases and Surgery of the Dog. Washington, D.C. Genco, Robert J., Richard T. Evans, and Solon A. Ellison. 1969. Dental research in microbiology. J. Am. Dent. Assoc. 16:1017. Gibson, T. 1974. Caryophanon, p. 598. 1p_R. E. Buchanan and N. E. Gibbons (ed.), Bergey's Manual of Determinative Bacteriology, 8th Edition. Williams and Wilkins Co., Baltimore, Md. Howell, Jr., A., H. V. Jordan, L. K. Georg, and L. Pine. 1965. Odontomyces viscosus, gen. nov., spec. nov., a filamentous microorganism isolated from periodontal plaque in hamsters. Sabouraudia 6:65—68. Hubbert, William T., and M. N. Rosen. 1970. I. Pasteurella multocida infection due to animal bite. Am. J. Pub. Health 66:6. Pelczar, Jr., Michael J., and R. D. Reid. 1972. Microbiology. McGraw-Hill, New York, N.Y. Reyne, Alice. 1974. Branhamella, p. 432. 12_R. E. Buchanan and N. E. Gibbons (ed.), Bergey's Manual of Determinative Bacteriology, 8th Edition. Williams and Wilkins Co., Baltimore, Md. Ritz, H. L. 1967. Microbial population shifts in developing dental plaque. Arch. Oral Biol. 16:1561. 14. 15. 16. 17. 18. 19. 20. 60 Rosebury, T. 1972. Distribution and development of the micro- biota of man. 16_Microorganisms Indigenous to Man, McGraw-Hill, New York, N.Y. Skerman, V. B. D. 1967. A Guide to the Identification of the Genera of Bacteria, 2nd Edition. Williams and Wilkins Co., Baltimore, Md. Smith, J. E. 1955. Studies on Pasteurella septica. I. The occurrence in the nose and tonsils of dogs. J. Comp. Path.‘66:3. Socransky, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res., Supplement to No. 2, 66:203-222. Socransky, S. S., and S. D. Manganiello. 1971. The oral micro- biota of man from birth to senility. J. Periodont. 61:485—494. Tatum, Harvey W., W. H. Ewing, and R. E. Weaver. 1970. p. 191-198. 16_J. E. Blair, et al. (ed.), Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C. Weaver, R. E. 1970. "Unclassified" groups of aerobic gram- negative bacteria isolated from clinical specimens. Seminar on Current Topics in Clinical Microbiology, 70th Meeting, Am. Soc. Microbiol., Boston, Mass. APPENDIX 61 Figure 1. Gram stain of Caryophanon. 62 wwaaoo m .05 m om 0mmuamz m .05 m ma News: cmflumnam m .03 m mm Hmumcoz : .mxz v 55 30£OI30£O m .05 m on Hmuosoz 2 .mx3 v ma ocdooxam cmammsuoz m .05 v mm kumooz z .muh m ma mammmm z .05 m Nm Hmumcoz h .muh e vH uwauuoa coumom z .05 N am mammmm m .mum NH ma vasomoomo m .05 N om Hmuocoz m .5» a NH madaoo m .05 m mN oncommnm cm5umo m .mx3 5 Ha umpumm nowaucm z .05 m mN 00mmxoou z .mum N 0H Hmumcoz z .mu» m hm nouumm amauH z .05 ma m mama ammuo 5 .mx3 55 mm “Shoo swam: 2 .mu» m m Hmumcoz m .05 o mN Hmumcoz m .05 m h Hmumaoz z .mu» NH em wauamam m .05 a o Hmumcoz m .mu> m MN HOUMHQMA Z .05 Ha m nonomcoom mudumacHz z .mxa m NN umxom m .m5> m v Hmumcoz 2 .mx3 0 HN oumcumm .um m .05 ON m Hmumsoz z .mx3 m 0N Hmumcoz z .mu> N N Hmuocoz 2 .mx3 m ma umucaom oouamouoom cm5umo m .muw v H ommum xmm mom mooo ommum xmw 0o< woou mmoo om mo monm Hm>amcflm on» mcfl>ocum ca com: mumo cowumoamaucmoa on» no >56555m 4 .HId manna 63 mauamnm m .05 o om Houmcoz m .05 o no muonmoom cm5500 z .mu» m we Houmcoz z .05 m Nv Hmuocoz z .05 h we Hoacmmm meoou z .05 m av opossum .um z .mnm e no kuumm nmflHH z .05 m cm Hmumcoz m .05 h we mwaaou z .05 m mm Hmumcoz z .mua v we cowcmum5om z .05 m mm oumcmocm omeuoo z .mu» N we Osaxmm smoH505< m .05 N mm ommum xom 0mm 0600 oooum xmm 0mm mooo lemonaucooc HIa manna Identifiable aerobic microorganisms isolated from gingival scrapings of 50 dogs Table A-2. 64 ewsefdoofiw uoueqd05193 snITToeg “saToedsu saofiwourqo snsoosTA saofiwouraov n°dsuwnrxanoeqeufiiog Tooooondaias oTQATomaq-L Toooooidaxqs OllKIOWBH-S Toooooqdeiqs quATomeq-o seas -eoDOQOJDIW “IBQQOu Srprwlaprda 'qdezs snaJne snoooootfiqdeas “seToedsu—PIJQSSIBN SITEQJJPJEO 'g "axTIH-erralnaised epTooqrnw 'd snoraaoeooreo Janoeqonaurov Ttoo equTJaqosg saueEOJae '3 “seToedsu-W V-W S-W suaToegaantuou 'u eoTAnJfidIfiuaqd °u v-as duels EII dnozs CODE 10 Table A-2 (continued) 65 ewserdoofiu uoueqd6fiieo snrrroeg "seToadsu saoflwourqov snsoosTA saofimournoy ,°ds“wnTJaqoeqaufizoo Toooooadaiqs quATomeq-A Toooooqdexns Dinfitomau-g Toooooqdaxas oTnxtomaq-n seas -eoooooxorw “JBQQOu srprwlaprda 'qdeas SHGJHP snooooorfiudezs "saToedsu—erxassrau srreqrzeqeo '3 “BXTIu-PIIGJHQQSPd epTooqrnw ‘d snoraaoeooreo Jaqoeqoaauroy Tron equTJaqosg sauefioxae '3 “SGIDGdSu-N V-W S-W suaroeganbrruou 'n eoTAnJfidrfiuaqd °w p—Jg dnoxs £11 dnozs CODE 11 12 13 14 15 16 17 18 19 20 21 Table A-2 (continued) 66 ewserdoofiw uoueqdofileo snrrroeg “saToedsu seofiwournoy snsoosTA saofimourqoy u'dsuwnrlaioeqaufilog Toooooqdezqs oTQAIomeq-k Toooooqdexqs Ornfitomeu-a Toooooqdeiis quKTomeq-n aeao -90000010IW "JBQJOu SIPIWIapIda 'qdeas SHSJHP snooooorfiqdeqs useToedsu-eTJassTaN spreqireqeo '3 II BXIT II 'PTTaInailSE’d epTooqrnm 'd snoriaoeooreo Jaqoeqonauroy Tron erqorraqosg sauaborae '3 II SGIDBdS II ’N V-W S-W suaToegaanruou 'W eoTAnzfidrfiuaqd 'w v-as duels EII dnozs CODE 22 23 24 25 26 27 28 29 30 31 32 Table A—2 (continued) 67 ewsefdoofiw NT NT uoueqdofiieg snttroeg “seToedsu saofiwournoy snsoosTA saofiwourqoy ,{dsumnrzanoeqaufixog Toooooqdexqs quATomeq-L Tooooondexqs Diufitomau-G Toooooqdexqs quATomeq—o seas -eoooooxo;n “Jaqnou srprwlaprda 'qdens snezne snooooorfiqdeas “seToedsu—ezrassran spreqrreaeo '3 II 83111: II-PITG-IUGQSPd sprooarnw 'd snorqeoeooreo Jaioeqoaeurov Troo equTJaqosg sauafiozae '3 “SGIOBCIS II 'W V-W S-W suaroeganbrruou °n eoTAnxfidtfiuaqd °n v-aa dhoxo III dnozo CODE 33 34 35 36 37 38 39 40 41 42 43 Table A-2 (continued) ewsetdoofin NT NT NT uoueqdofiieo snrfroeg “seToadsu saofiwournoy snsoosTA seofiwouyqoy u°dsuwnTJanoeqauflioj Toooooadezqs quKTomeq-L Toooooqdexqs atufitomaq-Q Tooooondexqs oTQAIomaq-n aeao -eooooororw “zeqnou sTpTwiapTda ‘qdeqs snaxne snooooorfiqdens “saToadsu-erxessrau sTrquJeneo °g "aXTIH-PITQJUGJSPJ sprooqrnw “a snorqaoeooreo Jaqoeqogaurov Troo equTJaqosg sauafiozae '3 “SGIOBdsu 'W V-W S-W suaToegaanTuou 'w eoTAnzfidffiuaqd °w 9-33 dnozs CII dnoxs CODE 44 45 46 47 48 49 50 Xa = Acinetobacter calcoaceticus var lwoffi. Xb = Acinetobacter calcoaceticus var anitratus. not tested. NT 69 Hwo.omv H Hwo.ov o mzumuuwcm Hm> Hwo.omv H “$0.00Hv m HMMOEH Hm> we.h N mo.MH m msowu00m00Hm0 H0u0mfl0umcwuw wN.NN o wh.HN m wHoo mwcowhmcomm w>.m H wo.o o mmcmmoamm hmuomnoumuzm lav.omc v lao.o~c m gmmHommmgIz ASH.mv H Awo.ov o vIz va.omv v Hmo.omv m mIz HwH.mv H Hwo.0HV H mcmwommmoowwcoc .2 HwH.mv H Awo.ONV N mewbskmmHmsocm .2 wo.hm «OH mm.mv 0H mwwmeHOE wo.mN m av.om h vam @9050 am.mm m mm.ma OH HHH maouw Hume magma Hmme mmuezmm my. .ww mm m...“ Hm... mm 31 a. u. 31 a. u. T o o T. T.o o T 0.3 T.o u o p.e T.o u o a u e I. a I. w W % I. e I. w 3 s.d s.d o .d s.d a o T.o m o a o T.o m o 1.. SS S 1.. SS S 0 T. .d I. o I. .d I. T.1 T.3 T.1 T.3 e T. a T. e I. a I. 3 A s A 3 A s A a e e a a a o. “v Hm>Hmch cH omum>oomu MHumuomo 0>HummmcI5mum 0Hnoumm mo soHumHomH m0 mucosuwhm Hoodoo ou uoommmu cqu mmsHmmuom .MI< 0Homa 7O mwNechNeueo ewummmHmz >HuoEHom «as .m 0» mo ooe 50: 0o mmueHOmH mmHoemm Heuou ecu .euom Immune .5mHsemHo 0xHHIeHHmusmumem e one eow00uH=5 eHthsmumem Anon oeo moo 0He5 0:0 *fi .oH on as ooe nos 0o meueH IOmH moHommm Heuou may .muOMoumca .vIz one e0w>shmmwmumcm .2 noon oen moo mHe5 moo .1 wN.NN o av.hH v socecmomheu Hwo.omv N Hwo.omv m :mmHommm: ewhmmmwmz Hwo.omv N Hwo.omv m *«ImHHeaaueueo eHHmfieaueHm wm.vH v wH.oN o ewhmmmwmz Hwo.0mv m Hwo.omv m 0xHHIeHHmhsmumem Hwo.omv m Hwo.omv m eoHUOuwzfi emehsmumem mm.mH «am aH.oN o eNthsmumem 2.3 mane: 3.8 $55.55 I d s N o N T d s N o N u a .d o e o u a .o o e o 3 I a O u 0 o J a O u 0 T o o T. t.o o I o.e T.o u o 0.3 T.o u o a u e I. a I. e u a I. a I. w 1. S S w .4. S d S .0 IA we .0 I. we 1.. S S S T: S S S O T... d 7.. O T.. 10 TI T.3. T.1 T.3 T.3 D. T? a T... D— T.. a T? 3 A s A 4 A s A a a a a a a P P Hooscwucoov MIm 0Hne9 71 Hwo.omv N Hwo.mNV H =mmHuomm: mmomEocwuow Hwo.omv N Hwo.mhv m nsmoomHS mmomsonwuow mm.eH v «o.mH m nm0u00>505Huo¢ mo.mN w wh.HN m EswumuoenmanOO Ham.mv N me.mv N Hoooooumouum 0Hu>H050£I> me.NHv m me.mv N HoooooumouUm owumHoeonIm HwH.mnv mH Awm.omv hH Hoooooummuum 0Hu>H050£Ia mm.Hm NN wo.Nm mH eHooououmouum Hmo.Hmv o Hwn.oNv e mmHommm 50:00 Hwo.HNv o Awh.oNv v msmhoe .2Qeum Hwo.hvv m Hwo.mvv h monEHmowmm .2Qeum wo.mm omH wN.mo mH 0e00e0000050H2 :8 meme: 3% $3255 T d s N o N T d s N o N u a .o o e o u a .d o e o o 1 e . u . o 1 a . u . T o o T. T o o _I 0.9 T.o u o 0.9 T.o u o e u a I. a I. e u a I. a J u 1 s u 3 s o .d 8.0 o .d s.w a o T.o m o a o T.o m T: S S S I... S S S o T. .d T. o T. .d T. T.1. T.1 T.1. T.1 e T. a T. P T. a T. 1 A s A 3 A s A a a a e e a 0. 0. Hoodoo 0» 0005mm“ SUH3 mmcheuom He>Hmon cH omuw>oomu eHHeuer 0>HuHmomI5eHm ownoume mo COHHeHomH m0 hocmswoum .vId mHneB 72 mmHoomm Heuou on» .muomouone .ouommuena moHommm Heuou on» .muowmuona .mH 0» m5 ooe no: 0o meueHOmH .emhu mHou mo 5mHsemuo 0:0 neon 0u05 oen mmoo Heum>mmo .m ou ms ooe 90: 0o moueHomH moHoomm Heuou ecu .EmHsemuo oxHHIueomsouwubw :e one msmoomwb umomaoswuum oen moo eHeaem 050 n .moHe5 mop 5H NN H0 mmHeawm may 5H mH on do ooe no: 0o meueHOmH .0500000um0Hum mo moHommm 050 neg» 0505 oes mmoo Heum>eme av.He HN\mH ao.ooH o~\o~ «amended»: «ease wm.mH m Hm.e H mSHHHuem RS mug RN» mange me am we an am we 01 a. u. 31 a. . 1.0 D T. 1.0 a T. P3 1.0 no 0.9 1.0 no an 81. 31.. an 31.. al. was. 3 S ml. 8 8 80 TM MM 80 1.1% WM 1. SS S 1: SS S o T. T. o T. .d T. T.3. .l 1. T.1. TIH+ e T. a T. e T. a T. 3 A s A 1 A s A 38 a 99 a 0. D. lemscHucooo vIe mHnea 73 .m 05 on ooe uon 0o m0ueH00H 00H00mm Heuou 0n» .0505050ne .0300000550550 50 00H00mm 0n0 neon 0505 oen HefiHne 0noe no . 8H oxe 52055855 mmmfio wo.om m nocecmom5eu Hwo.OOHV H =00H00mm= eflhmmmwmz 53w50uuenmnm500 mm.mm N mh.oH H ew50mmw0z .m05um .050nI> st.oHv H Hwo.00Hv H 00w335mmHmn0cm_.2 .3055... 65058 2558 m 3.3 H eHHexeuoz eHooooouo05um mm.mw m mh.oo v VImm @5050 mwowsfimowmo .m Hwo.00Hv N 0000e0000050H2_wm.mm N mm.mm N nHH @5050 NSHBHMOmI2 Ham.mmv H ao.mN H ew5ommw02 .50550 .050nIe wa.oov N Hoooooum05um wo.mh m “50.00HV H mIz mo.mN H eNN050502 mHeHsumeHne .0 130.0050 H 0000m0000050w2 Hmo.mN H wo.om N nHH @3050 MSHHHmomI2em0 m>aan052I2 Hao.omv H wkwoaw 50> mm.MH N mzuwumoeooqeo 5050en050nwon newcomwb .n Hwo.om. H Hao.o¢0 N 0xHHIeHH05smunem :m0Ho0mmsI.< Hao.omv H Hwo.oov m eoH005H35 eHH05n0umem mouoomaoaHuoe an.MH m 3m.mm m eHHmnumueem 53H5050e505m500 30.0N m «0.0 H 00:00050e 5050en050unm .mmuum .osenI> “55.00 H mm.mm m HHou eHnoH5enomm .mm5um .oemnIm Awn.mHv m .mouum .oseAIu Awo.mev NH Amo.ovo N =muHommm=Iz nHouououmeuum wm.mm 3H Amo.oev m mIz mm.mm m eHH0xe502 m0Ho0mm 50:50 Hwo.va m mHeHeumeHne .m Awe.Hvo m ao.o~ m «I55 5:050 030530 .0 Ham.mmv o ememoeouooouoH=.am.mk HH am.mm m H55 5:050 MthHmomI2¢MU MSHBNUNZI2QE0 u... i am 3 mm mm a: as u. a: o o T.o o T. T.o o T. 0.3 T.o u o 0.3 T.o u o 3 u 3 1. 3 I. 3 u 3 I. 3 I. w 1 s s w 3 s 3 3o Id md 30 1M mm .4. SS S 1. SS 8 o T. .d T. o T. .d T. T.3 T.3 T.1. T.1 e T. 3 T. e T. 3 T. 3 A s A 3 A s A 3 3 3 m.3 3 0. mmoo mon503 mH 505m o0ueHomH mBmHnem50050H5 0H5050e m0 00n0555000 0:9 .hIn 0Hnea 76 mwfiOmnmm H690“ USU. .OHOMOHOSB ooe uon 0o moueHomH 00Ho0mm Heuou 0n» .0505050n9 .03000005m05um mo 00H00mm 0:0 nenu 0505 oen mHeEHne He50>0m .uHNeHHHeueo ew5mmmw0z >H5055om l. .vH 05 on ooe uon 0o m0ueHomH a .HH on as .53050 mHnu nH m0H00mm 03» oen He5Hne 0noe ao.ooH HH\HH queeumoosz MM2HO an.MH N noneam0m500 Hao.omv N ImHHec55eueo eHH05enne5m Hwo.omv N :m0H00mmz ew50mmH02 no.0N v ew50mmw02 MSHEHWOmI2Humahom t .m0uum .oa0nuu Hao.oOHH H Hoooooummuum «o.ooH H 050330 .cmmum Hmo.ooHH H mmmuuoUOOOHowz 30.00H H «0.00H H wqoo mwaowuoaomm «H» mmmnmmms 30.00H N mxmdqmoumz aflfifio 30.00H N cocmcmOmth Hwo.00Hv H *mwwmnhumumo .m ao.om H mwnmmmwmz MbHHHmGNJZQMU MSHE :mmHommm=|.< Hwo.0Nv H mo.mH m usuwumomoowmo HmuomnOumcwuw mzmoomwb .< Hwo.omv v Umwumo>EOCHuom wo.0N w wo.mN m wNoo «wanHmnomm Ezwumuomnmmmhoo wo.mm N me.mmv v =m0Hommm=Iz Ham.mv H vs: .mmuum .osmsum Hmn.mHv m Aw>.va m ml: .mmuum .oEmnnv AmN.HmV NH me.mv H mcmwommmavano: .2 owoooooummuum wh.mm mH Ham.mv H MUH>ahsmwmzucm..z wo.om NH MNHmNMNoz 00Ho0mm 30330 HHN.H3V 3 033H3303330 .m Hwo.omv 0 Ho.mm 3 3-33 33030 030330 .33033 me.mv H n0m0umooououowz 30.00 NH Ho.om 0H HHH Adouu NbHRHMOmuzmmw MSHBQUNZszmw 3...... .HH mm. HH H3 H3 01 a. o a: a. u. To a T. T3 0 T. p.3 T.o u o 3.3 T.o u o 3 u 3 I. 3 I. 3 u 3 1. 3 I. u 3 s u 1 s 3 d 81% a d Sd 3 o T.o m 3 o T.o m o 1.. SS S 1: SS S 0T. dT. 0T. dT. T.1 T.1 T.3 T.1 ET. 9.... PT. 9.... 1 A s A 3 A s A 3 3 3 3 3 3 p. n. mHmumcoa oN Scum ovumHOmH mEmHGmmuoouoHE ownoumm mo muqmuudooo 0:8 .mld mHnma .mwwmahhmuMO mwummmwmz aHuuauom l .mH on m3 330 no: 03 0090HomH m0H00mm Hmuou 0:» .0Hom0uona .msooououQUMum 0Hu>H360nuwu0n 0 3:0 Imanm :0 can HMEHGM 0300 .NH on mi 330 no: 33 m0uwHomH m0Ho0mm H030» 0:» .0uomon0na .m0H00mm 0:0 30:» 0305 30: mHmaHQM H0u0>0mn .m on mi 330 no: 33 0030HOmH 00Ho0mm Hmuou 0gp .0uom0u0na .achmuuo 0xHH|0HHmnumummm 0 3:0 mfiwbouwna .m 0 can Huchm 0:00 80 wo.m H mifidflmdflhz $330 ao.m H cocmnmamNMU Hwh.mmv N «mwwmcuhmumo .m Ham.mmv H =m0Ho0mm: mwhmmmflmz ao.mH m mwhmmmwmz NSHSHmomlzdmu MSHH¢GN212¢NG HH HH mm «H HH HH 31 a. o 31 a. u. T 3 3 T. T.3 3 .t 3.3 T.o u o 3.3 T.o u o 3 u 3 I. 3 1. 3 u 3 I. 3 I. H. s s H. . s .0 :H HH .0 1H HH 1.. 88 S T... SS 8 o T. .3 T. o T. .d T. T.3 .l.+ T.4 T.3 nu HM an HM 3 3 3 m.3 3 3. H0033Hucoov 0-0 0H303 81 33.3 3 33.3 3 3H.HH H 33.0 3 303003030333 33.3 3 30.3 3 3N.NN N 33.0N 0 00303003300 33.33 H 33.3H H 33.33 m 30.3H 0 033000303 30.0 3 3H.Nm 0 33.33 m 33.3H m 00HH03033003 maowumomoono 33.3 3 33.0H H 30.33 0 30.3 3 3000000003300 33.3 3 33.0 3 33.33 m 33.0N 3 HH00 03303303003 H33.00 3 H33.330 3 H33.330 0 “33.030 0 .0030000=-.3 H33.33H0 N - H33.3N0 H .33.3mv 3 H33.330 N 3.2 33.33H N 33.HN m 33.33 N 33.3H 0 00HH030303 33.3 3 33.3N N 30.00 0 3H.0N 3 0-33 03033 30.30H N 33.N3 m 33.33 3 33.mN 3 HHH 03033 030320030 3333003212033 N N 0 N0 0003 30 033232 30303 330mm 3A 0300: mav 03003 MIN 3003 Nv haw 0H .HHHH. .HH .HHHH 0H,. HHmN 03 33mm 01 a. u. DJ 8. u. 01 a. . 01 8. . T.3 3 T. T.3 3 T. 1.3 3 T. T.3 3 T. Pa ....o no 0.8 To uo Pa T.o uo P8 1.0 00 mm... ““381... WWW W391. WW $1.31. WW $1.81. H. .3 m3 H. .3 .HH H. .3 m3 H. .H m3 1. SS S 1: SS 8 1: SS S 1.. SS 8 o T..d T. o T..d T. o T..d T. o I. .k 1.1 .L.+ 1.3. T.3 1.1. 1.2 1.1. 1.3 3 T. 3 T. 3 T. 3 T. 3 T. 3 T. 3 T. 3 T. 1A SA 3A SA 3A SA 4A SA 38 a 98 a 83 a 88 9 D. D. P D. moon 0H060u 3:0 0H0E Mo 0030 053 o» 3030H03 00 0H30uomn H0>Hmch ownou00 mo :OHUMHomH .0H14 0Hnms 82 33.33 H 33.3H H 3H.HH H 33.3 3 00333000 33.3 3 33.3H H 3N.NN N 33.NH 3 00033033303 33.3 3 3H.33 3 33.33 3 33.3 3 303303003033300 333.333 H 333.3H3 H .33.33 3 333.33 N .30330 .030313 333.333 H 333.3H3 H 333.33 3 H3H.HH. 3 .30330 .0303u3 A33.333 H .33.333 3 .33.33H3 3 .33.H33 NN .30330 .0303u3 33.33H N 33.33 3 33.33H 3 33.33 3N 0300000330330 .33.33 3 333.333 3 H33.333 3 H33.3H3 3 0030030 30330 333.33 3 333.333 N 333.3N3 H .33.N33 3H 03033300330 .0 333.33 3 H33.3H3 H 533.33 3 H33.N33 3 000300 .0 33.3 3 33.H3 3 33.33 3 33.33 HN 00000000000303: 030323030 33333003r3330 N 3 3 N3 0000 30 300302 33303 03003 3A 03003 313 03003 mus 3003 «V wow 33 Hmmm 3...... 33m... 33 33.3.3 33 H3m3 01 a. . 01 . . 01 a. . 01 a. . 1.0 o T. T.3 o T. T.o o T. 1.0 o T. 0.8 1.0 no P8 1.0 no Pa 1.0 no PW 1.0 no au 81.81. an 91.8.4. an 81.81: m.“ 81.31. ”1. S S m? S S w: S S 0.... S 8.0 .. ....H .HH .. .H 33 .. _..H H3 .. :H H. 1.. SS S 1. SS S 1.. SS S 1. SS S 0T. T. 01.03.. or.dT. onT. 1.3.1.3 1.11.3 1.11.1. 1.11.1 3.1.81. 9T.aT. 3.1.8? 3.1.8? 13A SA QASA SA SA QASA 83 a 88 a We a 89 a P P P .0003333003 3H-3 0H303 Table A-lO (continued) 83 dP aouapgoug 0 go gueozad 8 m H L. m m mt» pagetosg sagoads f SAIQISOd go 'ou N satdmes eutueo F? angggsod go °oN m aouapgoug 0 go guaozad a 8 m H m mt\ pagetos; sagoads o angggsod go °oN v sardines augueo : anxgtsod go °ou v 69 aouepgoug 0 <3 go guaoxad a o m H 0 sum pagetos; saroeds T angggsod go °oN ~* $81:de eugueo Q eAIggsod go “on m m eouapgoug m go guaozed g : mt» 31M pagetosg sagoads '3 angggsod go 'oN l‘ sardmes augueo Q eAIggsod go “on g I: ‘6 s: m § 2 E W q 0 § 0 23% a w H 2 Several animals had more than one species. a 84 00.0 o 00.0 0 00.0H H 00.0 0 nmuomnoumugm 00.0 o 00.0 0 00.00 N 00.00 0 nocmnmomumo 00.0 0 00.00 H 00.00 0 00.0 0 «whommwmz 00.0 0 00.00 0 00.0H H 00.0H m «Haousoummm msowumomoowmo 00.0 0 00.0 0 00.00 m 00.0 0 nouomnoumcwuw 00.0 o 00.0 00.0H H 0~.H0 0 0000 00000000000 A00.00 0 .A00.00H0 500.00. H0o.o00 H gmmHommm=u.z H00.00H0 H H00.00 H00.000 H00.o00 0n: 00.00H 00.00 00.00 0 00.0H 0 «Hmemuoz 00.0 00.0 0 00.00 00.00 0 010m ozone 00.00H 00.00 H 00.00 00.0H 00H myouo. mamaz0mcwm ownoumm mo cowumHOmH .HHlm «Home 85 00.0 0 00.00 H 00.0H H 00.0H 0 00000000 00.00H H 00.0 0 00.00 m 00.0 H mmomsocwuom 00.0 0 00.00 m 00.00 0 00.~H 0 auwumuumnmcmnoo “00.00 0 H00.0~0 H 100.00 0 H00.00 H .10000 .0500-» H00.00HV H A0o.0mv H “00.00 0 “00.00 H .mmuum .050010 100.00 0 H00.000 0 “00.00H0 0 H00.000 HH .mmnpm .oamnuu 00.00H H 00.00 0 00.00H 0 00.00 HH Hoooooummnum 100.00 0 100.000 H 100.00H0 0 H00.H~0 0 0000000 00000 H00.00 0 100.000 H .00.00 0 10H.000 0 mwowsqunwmu .0 “00.00 0 H00.000 H 100.00 0 H0¢.Hmv 0 msmuam .m 00.0 0 00.00 0 00.00 0 00.00 03 mmmomuoooouowz mamazwomo 00000001120mo 0 0 00 moon mo mwmzsz 00005 mummm m mummm mlv mummm MIN Hmmm H mom I.d SNON 1rd SNON Td SNDN 1rd SNDN ua mowo us @090 US .moeo ue .moeo 01 . . 01 . u. 31 . u. 01 . u. 110 d T. T.3 d T. 110 1.. 1.0 m... We T.o uo 0.8 To no P8 1.0 no P3 To 0 Um .HwJSI. WW $1.81: mm. «“381. MW 9.381. 3.... Km mm 00 mm mm 00 mm mm 0.... mm mm m I. I. o I..d I. o I..d I. o I. .E 1.1. 1.3 1.1. T.3 1.1. T.3 1.1. T.3 e I. a I. e I. a I. e I. a I. e I. a I. QASA QASA 1ASA QASA 99 a 88 9 98 a 88 a P P D. P HoonnHucooo HHu¢ oHnma Table A-ll (continued) 86 aouapIouI 3 go guaogoa 8 m .H H m 2... pa1etosI satdmes ‘0 aAI1Isod go 'ou A satdmes aqueo H \\ aAIgIsod go 'ou .4 an c: aouepIouI m 8 go guaozaa g '4 m ovv so po1etosI sotdmes 4 aAI1Isod go 'on H sotdmes aqueo :: eAIgIsod go °oN # c: aouapIouI m 8 go 1uaozaa g '4 0 one to pagetosI satdmes J‘ SAIggsod go 'ou In sotdmes aqueo ;? aAI1Isod go 'oN d? aouapIouI N r1 go guaozaa 3‘0 {3 Sufi "I pe1etosI sotdmes v aAI1Isod go °on ‘v satdmes aUIueo 04 ‘ In \ aAI1Isod go 'on § cn ts m m m S 1 § 2 s 3 h] 00 ‘1: O h § 0 ‘0’ : Several animals had more than one species. a 87 00.0 o 00.0 o 00.0 o 00.0 o amuomnonmugm 00.0 o 00.0 0 00.0 0 00.00 0 00000000000 00.00H H 00.0 o 00.0 0 0~.H0 0 mwnommHmz 00.0 0 00.00 H 00.00 m 00.0H 0 00000000000 maowumouoowuo 00.0 0 00.00 H 00.00 m 00.0 0 0000000000000 00.0 0 00.0 0 00.00 N 00.0H 0 0000 00000000000 100.0. 0 H00.000 0 100.00 0 H00.000 0 000000 H00.00H0 H H00.000 H H00.00H. m H00.0~0 H 0:: 00.00H H 00.00H 0 00.00 m 00.00 0 00000009: 00.0 0 00.00 0 00.00 m 00.0H 0 0-00 0:000 00.00H H 00.00 0 00.00 0 0~.H0 0 000 0:000 020020000 0000000212000 0 0 0 00 0000 mo 000202 00000 mummm ma 000mm mnv «noun mnH 0005 HV wow 00 00mm. 0...... $000 0.0. 00mm 00. mmmm 31 a. . DJ . u. 01 a. . 01 . . I.o o I. I.o o I. I.o o I. I.o o 1. Pa I.o uo Pa 1.0 no Pa I.o uo Pa I.o uo an 81:91. an 81.3.... an 31.8.... an 81.81.. 0.. .. .. 0.. .. .. 0.. .. .. 0.. .. .. ao 1.me 80 TM MM 80 TM mw 80 11% WM 1. SS S 1: SS S 1: SS S 1. SS S o I..d I. o I. I. o I..d I. o I..d I. T.3. T.1 T.3. T.3 T.1. T.1 1.1. T.3 e I. a I. a I. a I. a I. a I. e I. a I. 1A 8A JASA 3ASA QASA 98 a 83 a as 9 83 a P P D. P mmoo mesmu no mood on» on UwumHmH mm 0Humuomn Hm>Hmch UHQoumm mo GOHumHOmH .NHI< mHnma 88 00.00H H 00.0 00.0 0 00.0 0 00000000 no .0 o mm .mm wo .o o as. .3 0m mmomsocwuow 00.0 0 00.00 00.00 m 00.0 H 300000000000000 $0.005 H 30.8 30.8 22.2 H .1930 .9097» H00.00 0 100.00 H00.00 H00.0H0 0 .00000 .000010 H0o.00 o H00.00H0 A0o.0oH0 H00.000 HH .00000 .os0nnu 00.00H 00.00 00.00H 0~.H0 00H 0oooooum0uum H00.00 100.000 0 H00.000 HOH.00 H 0000000 u0nuo 100.00 0 “00.00. H 100.000 H00.00. 0 00005000000 .0 “00.00 .00.00 100.0. H00.000 0 000000 .0 00.0 00.00 00.00 00.00 HH 00000000000002 020020000 0500000010000 0 00 moon mo 000202 000o0 0.0005 0A 0.0063 wav 0.0065. n H. .0003 NV New Id SH on 11.4 S 0 d 8 ON 11:... SN ON 00 0.0... 0.0 0.00.0 M” 0.00... .00 03.0.. I.o o I. 1.0 o I. 1.3 o I. 1.3 o .} P9 1.0 no P8 1.0 no Po 1.0 no Po 1.0 no on 8.4.31. mu 9.4.91. on 61.31.. on 81.8.... 0.. .. .. .. .. . 0.. . .. 0... . . .2. 1.0 0.0 .2. 1.0 0.0 .2. 10 0.0 .2. 1.0 0.0 1... SS as 1. 88 OS 1. SS 68 1: SS SS 01. o.... OI. OI. OI. OI. or. or. 1.1. I.3 1.1. 1.3 1.1. 1.4 1.1 T.3 0000 00.00 0000. 0000 83 a We a so a 98 a D. D. P H00000ucooc NHn0 0Hn0e Table A-lZ (continued) 89 aouapIouI 3 go quaozaa 8' a H 3 pagetosI sanads 3"” aAIgIsod go °on ‘2 satdmes aqueo Q aAIquod go °on "" aouapIouI 3 go 11.130195 0 0° 0 0 g H mm pagetosI 3910363 .0 aAIgIsod go °ou 0', satdures aqueo Q aAIquod go °on m aouapIouI 8 go guaozad 0 c5 L. 0 8 H mm pagetosI sanads m “141806 J0 '05: 0'. sardines auItreo Q aAIgIsod go 'ou m aouapIouI 8 go guaozaa 8' 3' H 0 0 paqetosI sanads 3‘” aAInIsod go 'ou '5' m sardines aqueo Q aAInrsod J0 '0»: 3 AGE TOTAL NUMBER OF DOGS OTHER MYCOPLASMA Several animals had more than one species. a “0171471101:Minimum W