THE BACTERIAL FLORA OF CANINE ANAL SACS Thesfs fox. Hm Degree of M. S. MICHIGAN STATE UNIVERSITY David '3‘homas Duncan 1958 LIBRARY Michigan State University THE BACTERIAL FLORA OF CANINE ANAL SACS by DAVID THOMAS DUNCAN A THESIS Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Division of Biological Science 1958 to ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. E. V. Morse for his guidance and aid. Sincere appreciation is also extended to Dr. R. G. Schirmer for his donation of the use of the animals in this study and for his many helpful suggestions. Many thanks, also, to Mr. John Drives for aid in obtaining the samples and the preparation of media. TABLE OF CONTENTS PAGE INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . 14 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 21 DISCUSSION OF RESULTS . . . . . . . . . . . . . . . . . . . . 24 SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . . . . 3O FIGURES . . . . . . . . . . . . . . . . . . . . . . . . 32 TABLES . . . . . . . . . . . . . . . . . . . . . . . . 40 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . 97 FIC D1 C.” -_l LIST OF FIGURES FIGURE PAGE 1. The percentage incidence of the respective micro- organisms isolated from 125 canine anal sacs . . . . 32 2. A comparison of the percent incidence of the respective microorganisms isolated from 124 canine anal sacs with the sex of the animals . . . . . . . . . . . . 33 3. A comparison of the percent incidence of the respective microorganisms isolated from 125 canine anal sacs with’the colors of the secretion . . . . . . . . . . 34 4. A comparison of the percent occurrence of the different A.K.C. groupings of dogs examined . . . . . . . . . 35 5. A comparison of the percent incidence of the respective microorganisms isolated from the canine anal sacs with the A.K.C. groupings . . . . . . . . . . . . 36 6. A comparison of the percent occurrence of the ages of the dogs examined . . . . . . . . . . . . . . . . . 37 7. Inhibition of total anal sac bacterial flora by various antibiotics . . . . . . . . . . . . . . . . 38 8. A comparison of the incidence of Proteus morganii found in the anal sacs with the dogs' ages . . . . . . . 39 TABLE II. III. IV. VI. VII. VIII. IX. XI. XII. XIII. XIV. XVI. LIST OF TABLES The microorganisms isolated from 125 canine anal sacs . . A summary of the classification data on the 125 dogs whose anal sacs were examined for their bacterial flora . . . . . . . . . . . . . . . . . . . . . . Summary of identification reactions for streptococci isolated from 110 canine anal sac samples . . . . . Summary of the identification reactions used for E. coli isolated from 101 canine anal sacs . . . . . . . . . Summary of identification reactions used for Aerobacter isolated from 71 canine anal sacs . . . . . . . . . Summary of Proteus identification reactions isolated from 41 canine anal sacs . . . . . . . . . . . . . Summary of the identification reactions used for Pseudomonas isolated from 9 canine anal sacs . . . . Summary of identification reactions for staphylococci isolated from 15 canine anal sacs . . . . . . . . . The occurrence of microorganisms isolated from 44 female dogs . . . . . . . . . . . . . . . . . . . . The occurrence of microorganisms isolated from 80 male dogs 0 O Q I C O I O O I O O I O O O O I The occurrence of microorganisms isolated from 72 white to light gray samples of anal sac fluid . . . . . . The occurrence of microorganisms isolated from 16 brown samples of anal sac fluid . . . . . . . . . . . . . The occurrence of microorganisms isolated from 10 green samples of anal sac fluid . . . . . . . . . . . . . The occurrence of microorganisms isolated from 27 gray samples of anal sac fluid . . . . . . . . . . . . . The occurrence of microorganisms isolated from 21 dogs or the hound Class C O O O O 0 O I O O O O O O O O O The occurrence of microorganisms isolated from 46 dogs of the sporting class . . . . . . . . . . . . . . . . PAGE 40 44 50 56 62 66 69 7O 71 72 73 74 75 76 77 78 TABLE XVII. XVIII. XIX. XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. XXIX. XXX. XXXI. The The The The The The The The The The The The The The occurrence LIST OF TABLES —— Continued occurrence of microorganisms isolated of the working class . . . . . . . occurrence of microorganisms isolated of the non-sporting class . . . . . . occurrence of microorganisms isolated of the terrier class . . . . . . . occurrence of microorganisms isolated mongrel dogs . . . . . . . . . . . . occurrence of microorganisms isolated 01d dogs 0 I O O I I O I O O I C occurrence of microorganisms isolated two "year 01d dogs 0 o o o o o o o o occurrence of microorganisms isolated three—year old dogs . . . . . . . . occurrence of microorganisms isolated four-year old dogs . . . . . . . . occurrence of microorganisms isolated five-year 01d dogs 0 o o o o o o o occurrence of microorganisms isolated six-year old dogs . . . . . . . . occurrence seven—year of microorganisms isolated old dogs . . . . . . . . occurrence eight-year of microorganisms isolated old dogs . . . . . . . . of microorganisms isolated nine-year 01d dogs 0 o o o o o o o occurrence of microorganisms isolated ten-year 01d dogs 9 o D I I o o o from 34 dogs from 8 dogs from 5 dogs from 10 from 21 one-year from 18 from 14 from 12 from 13 from 11 from 12 from 8 from 5 from 5 A summary of antibiotic sensitivity studies on the bacterial flora of 21 canine anal sacs PAGE 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 INTRODUCTION Two well developed sac-like pouches, termed anal sacs are located on the lateral margin of the anal orifice of the dog. These structures were described anatomically as early as 1805 by Cuvier, and subsequently by many others. Their Secretions have been analyzed as to chemical constituents by Hebrant (1899), Bruggeman and Rathsfeld (1937), and Montagna and Parks (1948). The pathological alterations associated with them have been cited and discussed by many authors (Saunders, 1910; McKenney, 1931; Coquot Bressow and Monet, 1933; Feldman, 1942; Brumley, 1943; and McCunn, 1953). A questionnaire sent to veterinarians in five different sec- tions of the country including the Lansing, Michigan, area, has shown that 60 to 100 percent of the dogs examined experienced some problem involving the anal sacs (Belding, 1957; Benson, 1957; Green, 1957; Grounds, 1957; Kirk, 1957; Peigh, 1957; McBride, 1957; Schirmer, 1957; Zeeb, 1957; and others). Techniques for rapid and safe removal of these structures have been developed by Theobald (1941), Wheat and Rhode (1951), and Blake (1954). The physiological importance of these sacs has been postulated by Hebrant (1899), and others. To date, little information regarding the bacterial flora of these structures is available. For this reason, research was undertaken to determine the bacterial flora of the canine anal sacs. An attempt has been made to analyze the data in such a manner that it may indicate a change in the microbial population as influenced by various factors. The effect of 12 antibiotics upon the sac flora was determined as part of the investigation. LITERATURE REVIEW There is a great deal of variation in the nomenclature of the many glandular elements in the canine anal region. The anal sacs have been termed by different authors, the anal pouches, the anal glands, the anal sacs, and the para-anal glands. The term anal sac will be used for the remainder of this thesis in accordance with a system of terminology proposed by Mladenowitsch (1907) and adopted by Ellenberger (1911). The gross anatomy of the canine anal sacs has been described in detail by Hebrant (1899). Coquot, Bressow and Monet (1933), Montagna and Parks (1948), and Neilsen (1953). These sacs are bilateral organs from a hazel nut to a walnut in size situated lateroventrally to the anus. They lie between the external sphincter muscle of the anus and the longitudinal muscle of the rectum (Bradley, 1943) and between the white internal and red external sphincters of the anus (Montagna and Parks, 1948; Neilsen, 1953). They are pouch-like and form a passive reservoir irto which apocrine and sebaceous glands epen. Each sac opens ventrally on the lateral margin of the anus by a single duct which is approximately 3/16 inches from the anal orifice (Kirk, 1953). They are considered by Neilsen (1953) as nothing more than sacculations of the skin forming a passive reservoir and excre- tory duct for the complex of glandular tissue comprising the true parenchyma of the organ. Cornified stratified squamous epithelium lines the sac and its duct. Subadjacent to this lies a thick mantle of glandular tissue embedded in a connective tissue stroma. This stroma is rich in diffuse lymphatic tissue and, some lymphatic nodules may be present. Coiled apocrine sudorifercus tubules are found surrounding the fundus of the sac and communicating with its lumen. In addition to the apocrine glands, large sebaceous acini are also present. In general the sebaceous glands are found close to the neck of the anal sac and the apocrine glands are concentrated in the fundus. Morphologically, it is difficult to distinguish the sudorifer- ous and the sebaceous glands of the sac from the corresponding cutaneous glands. The former are present in larger numbers, and the individual glands seem larger than the glands of the skin (Montagna and Parks, 1948). Simple columnar epithelium lines the highly convoluted sudori— ferous tubules. Spirally arranged elongated myoid cells form a wall between the columnar cells and the prominent basement membrane of a tubule. The myoid cells seem to fit together like barrel staves when observed in a longitudinal section. They are very similar to smooth muscle fibers. Some of the tubules are lined by tall columnar cells whose apices are often frayed and protrude into the lumen, giving the appearance of a smaller lumen. The epithelium may also be of the low columnar or cuboidal type. These types give the tubules the appearance of being greatly dilated. In the apices of the tall columnar cells, argyrophilic granules are revealed in abundant quantities when tissues are pre- pared by the DeFano method (Montagna and Parks, 1948). In the low columnar cells, there is a sparse distribution of granules. The entire cytoplasm of some tall columnar cells is highly argyrophilic while morphologically similar adjacent cells show only discrete blackened granules. In the low columnar cells, the Golgi apparatus is a flattened filamentous mass. Generally it encircles the distal half or third of the nucleus but may occasionally encircle it completely or lie at one side. It is as large or larger than the nucleus of the tall columnar cells, especially those with frayed outer surfaces situated in the apical cytoplasm. In rare cases, filamentous strands may descend along the sides of the nucleus toward the proximal pole of the cell (Montagna and Parks, 1948). The mitochondria of the columnar cells are situated in the supranuclear cytoplasm but occasionally, they may encircle the nucleus completely. They are usually in the form of rounded bodies arranged to reveal a definite axial polarity of the cells. The mitochondria are found to be abundant throughout the apical cytoplasm in the tall columnar cells, but are restricted to a narrow supra-nuclear band in the low columnar cells. The apically frayed cells are heavily laden with mitochondria in their distal cytoplasm. This would suggest that these are the most active cells metabolically (Montagna and Parks, 1948). The lumen of the apocrine tubules appears to contain a slightly acidophilic, colloidal material. Numerous vacuoles appear at the periphery where the tall columnar cells are in contact with the colloid. These vacuoles seem similar to the chromophobic secre- tion droplets of the type found in the thyroid gland follicle (Montagna and Parks, 1948). According to Montagna and Parks (1948), the anal sacs are filled with a viscid, malodorous, acidic secretion. Coquot, Bressow and Monet (1933) describe the normal secretion as a cloudy liquid, grayish white, "gooey," or viscuous, to a pasty, gray-brown material. Hoare (1915) described the normal secretions as brown, butter-like in consistency and acid in reaction, composed chiefly of fatty material with an offensive odor, Hebrant (1899) stated that the secretion contains cholesterol and leucine and ascribes the character- istic odor and acidity of the sacs to "fermentation" which results in the production of butyric acid, skatoles and indoles. He found the ash residue of the secretion to contain abundant amounts of calcium. sodium and potassium. Bruggemann and Rathsfeld (1937) indicated that the secretion of the anal sacs was made up of approximately 87.8 percent water and 12.2 percent drystuff. The drystuff was in turn made up of 96 percent organic matter and 4 percent inorganic matter. The lipid fraction com— prised 13.2 percent of the organic drystuff and was found to contain 2.21 percent cholesterol and variable amounts of phospholipids. The inorganic ash contained 11 percent total phosphorous. Montagna and Parks (1948) stated that there exists within the glandular complex of the anal sacs some neutral fats, Cholesterol, lipine and plasmal as well as Fischler-positive substances, which may be fatty acids. These workers found that two types of glands are responsible for the secre- tion of these substances. They are the sebaceous glands located in the dermis of the excretory ducts of the sacs and the apocrine tubules located within the sacs. The apocrine tubules are very numOus' Of tr.»lpor1‘-’nI Wh apocrine C61 fireplets “(hi "zh 5ULian I {rut Men .11 The Bake“ ac Mind dflpl There 15 no mough gran“ granules ma) hese 'Orker sible for th anal Sac sec SinCe columnar C91 lined that montagna 8" The 69 renaming 11 the bulk of inch are at substances. the sebum 0f With” canine tut hands and s numerous, of a serous nature, and probably secrete the water-like component which makes up the greatest part of the secretion. The apocrine cells of the glands of the anal sac also contain lipid droplets which Montagna and Parks (1948) indicate are not stained with sudan III and sudan IV. but do stain with sudan black. This is true even after the secretions have been immersed in lipid solvents. The Baker acid-hematein and Smith-Dietrich tests indicate that these liquid drcplets may be lipines (Baker. 1946; Montagna and Parks, 1948). There is no cholesterol or neutral fat in these columnar cells even though granules stainable by the Fischler technique are found. These granules may be fatty acids according to Montagna and Parks (1948). These workers also believed that the tall apocrine cells are respon— sible for the mineral salts that appear in the ash residue of the anal sac secretions. These salts include potassium. calcium amdsodium. Since ribonuclease abolishes the cytoplasmic basophilia of the columnar cells of the apocrine tubules of the anal sac. it was be- lieved that this cytoplasmic basophilia represents ribonucleic acid (Montagna anvaarks, 1948). The sebaceous glands were believed to be responsible for the remaining lipids found in the anal sac secretions, and contribute the bulk of these substances. This would include cholesterol esters which are abundant, as are unsaturated glycerides. Fischlermpositive substances, plasmal and lipine. All of these substances appear in the sebum of the sebaceous glands. Within the myoid cells and the basement membrane of the apocrine tubules alkaline phosphatase is abundant. The sebaceous glands and sebum of the excretory ducts contain moderate amounts of alkaline phosphatase while the epithelial cells contain only small amounts of this enzyme. Acid phosphatase was localized only in the apical cytoplasm of the active apocrine cells in large quantities according to Montagna and Parks (1948). The biological significance of the combined secretion of the tubules of the anal sac and the sebaceous glands associated with the anal sac's excretory duct are unknown. Hebrant (1899) and Smith (1940), along with many others, believed that the anal sac secretions may serve to aid in the passage of feces and to protect the anal area. Bradley (1943) stated that the anal sacs are located adjacent to the sphincter ani internis whose action is to asist the an; externis. These two muscles are responsible for the lifting of the dog's tail, the closing of the anus, the constriction of the anal sacs, the retraction of the penis, and the compressing of the vagina. As defecation occurs, firm feces distend the rectum and anal orifice. At the same time, the muscles mentioned above are in a state of semi- contraction. This results in the discharge of the contents of the sacs through their ducts onto the anal orifice. It was postulated that the anal sac secretions coated the feces thereby making defecations easier (Hebrant, 1899; Smith, 1940). Another function of these secretions may be to form a protective layer between the tissues of the area, and the irritating materials present in the feces (Hebrant, 1899). These workers correlated the ideas mentioned above with the fact that there are similar glands to those of the anal sacs elsewhere in the body and that in these cases the function of the glands is local. Montagna and Parks (1948) did not hold these views because the openings of the ducts of the erratic 1933; Kn nrkers ire to t The secr during 1 5f the c‘ 9C8 con iffretic This ler bEen deE tides 10H 0’ anal sacs are at the very margin of the anus. This they felt would result in very little actual lubrication or coating. Another function proposed was that the anal sacs have an errotic role (Hetrant, 1899; Hoare, 1915; Coquot, Bressow and Monet, 1933; Knappenberger, 1940; McCunn, 1953). It was felt by these workers that the secretions of the sacs have an odor which is attract- ive to the opposite sex and is individually specific for each animal. The secretions may serve as a means of identification and stimulation during the time of heat. This has been referred to as the "spoor" of the dog by McCunn (1953). Comparative anatomists favor a third theory. In the skunk, sacs comparable with those of the dog, contain a pungent odorous secretion which this animal uses for protection (TheobaJd, 1941). This lends to the possibility that the anal sacs of the dog may have been designed for this same purpose but through lack of use, the muscles that enable voluntary discharge of the sac contents have been lost or are vestigial (McBride, 1957). It may be proven in the future that any one or all of the theories that have been proposed to explain the biologic significance and physiological role of the anal sacs of the dog are correct. A new theory for anal sac existence in the dog may evolve and hence none of the present theories may be valid. Veterinarians are well aware of the various types of involve- ments and pathological disturbances that occur in and around the anal sacs of dogs. Hebrant (1899) describes inflamed anal sacs as warm, painful, fluctuating swellings, which sometimes give rise to frequent 10 and violent efforts at defecation. The ability to express pus from these structures and the fact that they sometimes become ulcerated has been well established (Hebrant, 1899, 1910; Saunders, 1915; Hoare, 1915; and others). The inflammation or irritation that develops is believed to cause the animal to chase its tail, lick its anus, or drag its rear quarters on the ground or floor. Many workers believe that these actions are either an attempt to evacuate the sacs. an attempt to bring relief from the irritation, or both (Saunders, 1915; Hoare,1915; Coquot, Bressow and Monet. 1933; Knappenberger, 1939; McClelland, 1942; Brumley. 1943; Lacroix, 1947; McCunn, 1953). Disease of the anal sacs is reported to be especially common in dogs confined to the house as pets as Opposed to those living in the open, i.e.. on a farm, etc., according to Hoare (1915), Saunders (1915). and others. ThiS‘iS believed to be due chiefly to the diet and to a lack of exercise. A diet which lacks the necessary material to make feces firm or hard enough to bring about the discharge of the anal sacs of their contents serves as an example (Hoare,l915; McCunn, 1953; McBride, 1957). This is also true of dogs denied sexual inter— course and those suffering from senile changes (Hoare,1915; Saunders, 1915). Other causes of anal sac difficulties are helminthiasis, proctitis, and retention of the anal sac secretion which "ferments" in the sac (Coquot, Bressow and Monet, 1933). The secreting membrane often becomes inflamed and irritated from constipation, foreign bodies, and infections, all of which may change the character of the secretion to a thicker mass which partially or completely occludes the anal sac ducts according to Brumley (1943). This results in the retention of ll 0 the secretions of the glands of the sac and their subsequent swelling and redness. In some cases, an increase in the secretion may result in an accumulation of the discharge on the hairs and margin of the anus thus setting up an irritation of the entire anal area. The Merck Veterinary Manual (1955) states that the retention of the material in the sacs sets up an inflammation and irritation, and this the dog attempts to relieve. A retention cyst is sometimes formed when soft feces fail to stimulate discharge of the gland com- pletely. The soft feces may then occlude the outside of the duct while the glandular secretions produce internal occlusion (McCunn, 1953). Since the openings of the ducts of the sacs are directed upward, it is possible for fecal material to be forced in and act as a foreign body. In this case, the glands act as "natural incubators" (Smith. 1940). McBride (1953) stated that in puppies, one or both of the anal sacs frequently become occluded, however infection is rare. It is possible that a thickening of the secretion and/or granules formed in the secreticn occurs causing the occlusion of the ducts by making it difficult for the secretion to be forced out. In older dogs, occlusion cr impaction may result from obesity, lack of exer- cise, and/or lack of muscle tone (Hoare, 1915; Saunders, 1915; McCunn, 1953). In such cases the secretion remains within the sac and becomes hard. This hard material acts as a foreign body. Infection may then become established and complications result (Runnells, 1954). Acute infection of the anal sacs is common. This may in turn extend to the surrounding tissue and result in abscess formation and chronic infection (McBride, 1953). These factors predispose the 12 wall of the sac to infection, and with infection, the wall of the sac may rupture (Theobald, 1941). Lacroix (1941) warned that unless the owner of the animal is observant and concerned with the dog's welfare, the abscesses may rupture spontaneously, heal, and again rupture with the ultimate formation of fistulus tracts. The reaction on the part of the dog to relieve the discomfort probably aids in this rupture. With the development of an opening to the outside, organisms that were originally limited to the outer surface of the skin can now infect the underlying tissue, thereby bringing about a deeper and more acute irritation. Once the abscess is broken the organisms present may be capable of establishing new foci of infection elsewhere in the body (Hirshman, 1931; Smith, 1940; Zepp, 1945; McBride, 1953). Proctitis, and inflammation of the rectum, may in many cases have an origin in pathologic conditions of the anal sacs (Theobald, 1941). Infected anal sacs have also been linked to pruritis ani and various adenomas (Hoare, 1915; Feldman, 1932; Knappenberger, 1939; Theobald, 1941, 1942; McClelland, 1942). If infection involves the crypts of Morganii, the ensuing conditions of cryptitis, ulcers, fissures, abscesses or fistulas may occur (McKenney, 1931). To further increase the scope of difficulties of anal sac origin, the severe constipation that often occurs may cause an absorption of toxins from the digestive tract as well as from the foci of infection. Convulsions, lameness, paraplegia, neuritis, auto— intoxication, and muscular pain may develop (Smith, 1940). Zepp (1945), Biejers (1954), Visintine (1954), and others indicated that 13 the anal sacs, due to tfiue bacterial flora, may serve as a source of origin for certain types of dermatitis, i.e., acne, furunculosis, inter-digital infectious eczema, eczema of the anus and surrounding parts, acanthosis nigricans, and similar conditions. Because of these facts, the cause of these ailments must be determined and cor- rected at the origin and preventive measures taken if a permanent cure is to be effected (Zepp, 1945). 14 MATERIALS AND METHODS The dogs for this study were obtained through the courtesy of Dr. R. G. Schirmer of the College of Veterinary Medicine, Michigan State University. The equipment used for the collection of each of the samples consisted of a sterile 5 cc syringe and a blunt, lfi-inch, 18-gauge needle. Sterile 0.85 percent sodium chloride solution (saline) was also available. Sterilization of these materials was accomplished by autoclaving at a temperature of 121' C.at 15 lbs. pressure for 45 minutes. The following information pertaining to each dog was obtained: owner's name, breed, identification number, age, sex, and the preliminary diagnosis or reason for hospitalization. After the sample was obtained, the color of the anal sac secretion was noted. A general anaesthetic, Surital (Parke, Davis and Company), was administered to relax the muscles of the anal area. One attendant held the tail in an elevated position, and the anal area was swabbed with 1:1000 Nolvasan (Parke, Davis and Company), an anti- septic solution. Using sterile technique, the blunt needle was in— serted into the anal sac. Extreme care was employed in this operation to be sure that the needle did not tauch any of the adjoining tissue and thereby produce a contaminated sample. Once the needle was well within the sac, aspiration of the sac contents was effected. By this technique it was possible to obtain material for bacterio- 10gical examination, even when secretions were hard or of a waxy 15 consistency. Approximately 1 cc of the secretion was mixed with 1 ml of the sterile saline. The samples were then taken to the laboratory for culture using the various selective and differential media. The interval from the time of collection to the time of plating on media varied from 30 to 60 minutes. All culture media used in this study were obtained from the Difco Laboratories, Detroit,Michigan. Sterile defibrinated bovine blood, to give a final concentration of 5 percent, was used in all the blood agar media. One 5 mm loop-full of the saline-secretion suspension was streaked over 2 eosin methylene blue agar plates (EMB). 2 aside blood agar plates, and 2 blood agar plates. Three 5 mm 100ps of the saline suspension were inoculated into 1 tube of ethyl violet azide broth (EVA). The remaining amount of the sample was poured into 10 cc of selenite broth. One of each set of plates was incubated aerobically, while the other plate was incubated anaerobically at 37° C. In this manner isolations of both aerobic and anaerobic organisms was possible. Eosin methylene blue agar was chosen because it is recommended as a differential plating medium for the detection and isolation of gram negative intestinal bacteria and at the same time gives a sensi— tive accurate and stable differentiation between the fecal and non-fecal types of the colon-aerogenes group (Holt—Harris and Teague, 1916; Levine, 1918). Blood agar is recommended as being distinctly advantageous for culturing pneumococci, streptococci, and staphylococci. At pH 6.8, very clear zones of ehmolysis are evident and the hemolytic characteristics of colonies are readily discernible. Azide blood agar is recommended as the medium of choice for the isolation of l6 streptococci from stools, sewage. and other materials (Snyder and Lichstein, 1940; Mallmann, Boatwright and Churchill, 1941; Lichstein and Snyder, 1941). Sodium azide was first used by Hartman (1937). to suppress the growth of gram negative bacteria while allowing the growth of streptococci. Azide blood agar is also recommended for the isolation of staphylococci. A selective medium containing ethyl violet and sodium azide is recommended as specific for the growth of enterococci (Litsky, Mallmann and Fifield, 1953). The ethyl violet inhibits the growth of gram positive bacteria with the exception of the enterococci at a concentration of 0.00083 grams per liter. while the sodium azide atra concentration of 0.4 grams per liter inhibits the growth of gram negative bacteria. Ethyl violet azide broth was used as an isolation and confirmatory medium to demonstrate the presence of enterococci. Selenite broth is recommended as an enrichment medium for the isolaticn of various intestinal pathogens (Leifson, 1936). Sodium selenite possesses properties which have a differential in— hibiting effect on the growth of various microorganisms. The isola- tion of intestinal pathcgens of the Salmonella group from feces. urine, and infected tissues, is facilitated by the use of media containing this chemical (Leifson, 1936).! Following incubation for 24 and 48 hours, plates of the.media were examined. Gram stains were made from isolated colonies. The colonies obtained from the eosin methylene blue agar plates were of four types. The typical Escherichia coli type and the typical Aerobacter aerogenes type (Levine, 1918), the atypical coli—aerOgenes 17 type, and a fourth type which had the characteristics of the genus Proteus. If the colonial morphology was similar to that of E. coli, Aerobacter, or not typical of either and yet was composed of gram negative rods, but was not of the Proteus type, the "I. M. Vi. C." tests were conducted (Dubos, 1952). If the results of the "1.1LHVi.CL" test indicated that the colony isolated was E. 3211, this evidence was considered sufficient. If the organism isolated gave a positive reaction for the Aerobacter group, it was further determined whether the species was A. aerogenes or A. cloaceae.1 If the colonial morphology was similar to that of Proteus, a subculture was made on a urea agar slant. A red butt and slant were indicative of active hydrolysis and probably indicated a member of the Proteus group (Stuart, Van Stratum and Rustigian, 1945; Christensen, 1946). All cultures believed to be Proteus were subcultured on tryptose agar slants until further identification was possible. If the colonies were gram negative rods and did not prove to be Escherichia, Aerobacter, or Proteus, they were likewise cultured on tryptose agar. If the culture was mixed, it was suspended in a drop of sterile saline and restreaked over an E.M.B. plate. The selenite broth tube was incubated for 6 hours and three loopfulls were then streaked on a Salmonella-Shigglla agar plate. Salmonella-Shigella agar is recommended as a selective medium for the isolation of Salmonella and Shigella from feces and other materials. (Hardy, 1942; Rose, 1942) Three types of colonies are 1The ability to liquefy gelatin is sometimes very slow (Breed, Murray and Hitchens, 1948), and sometimes lost by Aerobacter cloaceae (Kligler, 1914). For this reason, the genus Aerobacter is used in this thesis instead of the genus species. 18 to be found. One type is small, Opaque, and slightly raised. The second is similar to the first. but with a small black center, while the third type is large, raised and almost completely black. All three colonial types were subcultured on urea agar slants. Cultures which within 6 hours had developed red butts and slants were con- sidered to be of the genus Proteus (Stuart. Van Stratum and Rustigian, 1945; Christensen, 1944). Colonies which were urease negative were to have been cultured on Kligler's medium as well as the differential sugar broths. Cultures which resembled Salmonella or Shigella were to have been sent to the Michigan State Department of Health Laboratories. Lansing, Michigan. for serclogical confirmation. Gram negative organ- isms obtained from blood agar were suspended in sterile saline and sub- cultured on an E.M.B. plate. Confirmatory tests included the same procedures described above. All Proteus type cultures were sub- cultured on a urea agar slant and if shown to be urease positive were transferred to tryptose agar slants and held for further clas- sification. The classification of the Proteus group entailed the transfer of pure cultures to mannitol. sucrose, maltose. and indole broths (Breed. Murray and Hitchens, 1948). All gram positive cultures obtained from azide blood agar and ethyl violet azide broth (E.V.A.) were transferred to blood agar plates for further identification. Colonies of gram positive cocci which gave the colonial morphology and gram stain appearance of staphylococci were cultured on Staphylococcus Medium 110 (Chapman, 1946). This medium is selective for staphylococci due to its high sodium chloride concentration, and is well suited for pigment 19 production (Chapman, 1946). Colonial morphology, pigment production, ability to liquefy, gelatin, utilize NH4H2PO4, ferment mannitol, and coagulate hlood plasma were further used to classify the staphylococci (Breed, Murray and Hitchens, 1948). Those blood agar colonies believed to be streptococci on the basis of their colony morphology, gram stain, and growth in ethyl violet azide broth were further examined. The characteristics used to classify and differentiate members of the genus Streptococcus were colonial morphology, Gram stain, hemolytic activity, ability to grow in nutrient broth containing 6.5 percent sodium chloride, liquefication of gelatin, ability to grow in E.V.A. broth, and mannitol fermentation (Breed, Murray and‘ Hitchens, 1948; Dubos, 1952; Litsky, Mallmann and Fifield, 1953). An additional study dealt with the inhibiting effect of anti- biotics upon anal sac microorganisms 12.X3££9° Discs of the following antibiotics and their relative concentrations are listed in Table 31. The antibiotic sensitivity discs were obtained from Baltimore 0 Biological Laboratories, Baltimore, Maryland. Twenty-one of the 125 samples of the anal sac secretions were examined as to the complete aerobic flora sensitivity to antibiotics. Two blood agar plates were inoculated with 5 loopfulls of each sample. Antibiotics were arranged on a plate approximately equi- distant from each other, the center, and the edge of the plate. These plates were incubated at 37° C and were examined at 12, 24, 36, and 48-hour intervals. At each examination, the zone of inhibition was measured around each disc. The approximate inhibition ratings were as follows: + indicated that the zone of inhibition around the disc 20 had a radius of 3 to 6 mm; ++ referred to a zone of inhibition from the disc of from 6 to 9 mm; +++ indicated an almost complete lack of colonies in an area of from 9 to 12 mm; ++++ indicated a complete elimination of colcnies from 12 mm or more radius. 21 RESULTS There were 125 dogs used in this study. The organisms isolated from each of these dogs are listed in Table I. The identi— fication number, age, sex, color of the secretion, and reasons for clinic admittance of each of the dogs is listed in Table II. The cultures isolated included gamma, alpha, and beta hemolytic enteric streptococci, Staphylococcus albus, Staphylococcus epidermidis, Escherichia £213, Aerobacter, Proteus morganii, Pseudomonas aeruginosa, and some unidentified yeasts. The data obtained were analyzed for the incidence of each of the organisms isolated frcm the anal sacs. Enteric streptococci were isolated from 77.6 percent of the 125 dogs examined. Nonhemolytic enteric streptococci were isolated from 69.6 percent, alpha hemolytic enteric streptococci from 4.9 percent, and beta hemolytic enteric streptococci from 15.2 percent. The streptococci isolated were Streptococcus liquifacigns, and Streptococcus zymogenes. Table III gives the physiological reactions of the streptococci isolated and the sample numbers in which they were found. Coliform organisms were found in 93.6 percent of the dogs. Escherichia coli was found in 80.0 £ercent, while Aerobacter was isolated from 58.8 percent. The criteria for identification of E. coli are summarized in Table IV and for Aerobacter in Table V. Proteus was isolated from 72.0 percent of the dogs. The species' characteristics are summarized in Table VI. All of the Proteus 22 cultures were found to be Proteus morganii. Pseudomonas aeruginosa was obtained in 7.2 percent of the dogs. The identification of this species is summarized in Table VII. Staphylococci were found in 12.0 percent of the cases. Staphylococcus albus represented 8.8 percent and Staphylococcus epidermidis comprised 3.3 percent. The identification reactions for the staphylococci are summarized in Table VIII. Yeasts were obtained from 28.0 percent of the dogs. No attempts were made to identify the yeasts as to genus or species. Figure 1 compares the respective percentages of each of the organisms isolated. 0f the dogs examined, there were 44 females, 80 males, and one dog in which the sex was not recorded. The incidence of the organisms occurring in the females is found in Table IX, and for the males in Table X. Figure 2 compares the incidence of the respective organisms with the sex of the dogs. The secretions obtained from the anal sacs were of four color types: 1) white to light gray, 2) medium brown to dark brown, 3) medium green to dark green, and 4) medium gray to dark gray.' The white to light gray occurred in 57.6 percent of the dogs, the brown was found in 12.8 percent, the green occurred in 8.0 percent, and the darker gray in 21.6 percent. Figure 3 summarizes the distribution of color types and their respective occurrence, while Tables XI through XIV correlate the predominant microorganism with the color group. The breed of dog was grouped according to the American Kennel Club (A.K.C.) classification, and tabulations were made comparing 23 these groups with the organisms isolated. 0f the dogs examined, 16.9 percent were of the hound class, 37.1 percent were of the sparting class, 27.4 percent were of the working class, 6.5 percent were cf the non-sporting class, and 4.0 percent were of the terrier. class. There were 8.1 percent mongrels. Figure 4 gives the inci- dence in which the dogs used in this study occurred in each of the different A.K.C. classification groups. Tables XV through XX summarize the correlation of the A.K.C. classification of breeds with the micro- organisms isolated. Figure 5 is a summary of Tables XV to XX. There were 120 of the 125 dogs for which the ages were available. The different age groups are indicated in Figure 6. Tables XXI through XXX summarize the relationship of age of dogs to microorganisms isolated. Table XXXI shows the data obtained from the antibiotic sensi- tivity studies. Sensitivity determinations for the microbial flora of the various samples are listed in Table XXXII. Figure 7 sum- marized the inhibitory effect of the 12 antibiotics used. Dihydro- streptomycin, chloromycetin, and neomycin appear to be most effective in inhibiting the organisms from the anal sacs. 24 DISCUSSION The bacterial flora of 125 canine anal sacs has been found to include Escherichia coli, Streptococcus liquefaciens, Streptococcus pymogenes, Proetus morganii, Aerobacter aerogenes, Staphylococcus albus, Staphylococggg epidermidis, Pseudomogas aeraginosa, and un- identified yeasts. These organisms have been divided into ten groups and compared with the following data: 1) the sex of the dog, 2) the age of the dog, 3) the color of the secretion obtained from the anal sac, and 4) the type classification of the dog as recommended by the American Kennel Club. The 10 groups of micro- organisms were: nonhemolytic enteric streptococci, alpha hemolytic enteric streptococci, beta hemolytic enteric streptococci, S. ELBEE’ S. epidermidis, E. coli, Aerobacter, E. morganii, P. aeruginosa, and yeasts. It was felt that subdividing the streptococci according to hemolytic activity would serve as a more practical means by which a clinician might estimate part of the flora of the sacs if any trends were observable. The comparison of the 10 organism groups with the sex of the dogs is listed in Tables IX and X, and summarized in Figure 2. There were 80 males and 24 females. In males the frequency of Proteus, was 18 percent greater than for females. Aerobacter was cflxmrved 10 percent more frequently in males than in the females. ‘E. coli showed a 15 percent greater incidence in the females than in the males. 25 The American Kennel Club (A.K.C.) recommendations for groupings of the breeds of the dogs was compared with the micro- organisms isolated. The data are listed in Tables XV through XX. The A.K.C. groupings included the hounds, sporting dogs, non-sporting dogs, the working type, and the terriers. A few mongrels were also present and these constituted an additional category. There were 124 animals whose breeds were known. The percent of each group for each organism isolated is summarized in Figure 5. The occurrence of the organisms isolated from each A.K.C. group is summarized in Figure 4. Although McBride (1957) suggests the possibility that screw tailed dogs may have a predisposition for inflammation and infection of the sac due to the unhygienic conditions around the tail, the evidence does not support this assumption. No specific trends are observable from the A.K.C. classification comparisons. The comparison of the color of the secretions obtained from the anal sacs with the microorganisms isolated is listed in Tables XI through XIV. Four colors were observed, i.e., a gray-white, a brown, a green, and a darker gray. Figure 3 compares and summarizes the oc— currence of the microorganisms isolated with the colors of the secre- tions. The gray-white colored secretions exhibited no specific trends. The green colored secretions indicated a higher incidence of alpha hemolytic enteric streptococci and Pseudomonas than did any of the other secretion types. In the brown colored secretions, Staphylococcus albus was predominant and Pseudomonas_sp. was not un- common. E. coli and Proteus occurred most frequently in the gray colored secretions. Although these trends are evident, many more 26 samples would be necessary before these facts could be considered to have statistical validity. It is still not plausible to predict unequivocally the anal sac flora from the color of the secretion. A comparison of the incidence of the microorganisms isolated and the ages of the dogs studied is listed in Tables XXI through XXX. Gamma hemolytic enteric streptococci were most often encountered in dogs one and ten years old. Alpha hemolytic enteric streptococci were most frequently encountered in seven and ten year old dogs. Beta hemolytic enteric streptococci occurred most often in dogs of five and ten years. Although the streptococci seemed to be present in high incidence in the tenth year, this may well be due to the low numbers of samples in this age group. For this reason there seem to be no demonstrable trends evident. This is also true of the staphylococci. Staphylococcus albus was encountered more often in the five and nine year old dogs, while Staphylococcus epidermidis demonstrated little variation when compared with the ages of the dogs. E. coli occurred most commonly in two, three, and nine year old dogs, while Aerobacter species demonstrated little variation when compared with the ages of the dogs. Pseudomonas was constant in occurrence when compared with age. Proteus demonstrated a slight increase in incidence with increasing age (Figure 8). The antibiotic sensitivity data are listed in Tables XXXI and XXXII, and summarized in Figure 7. Dihydrostreptomycin and chloromy- cetin appeared most effective in inhibiting the heterogeneous flora of the anal sacs and neomycin was almost equally effective. This 27 data compares favorably with that of Craige (1948, 1949) who reported that streptomycin when given orally was very effective against Proteus group organisms isolated from the intestinal tract of dogs. The results obtained from Schwenberg, Jacob and Rutenberg (1952) and Ferguson (1957) appear to agree with the results obtained with neomycin. The flora of the anal sacs seems very similar to the flora of the lower intestinal tract of the dog. Schwenburg, Jacob and Rutenberg (1952) reported isolating E. coli, Aerobacter, Clostridium welchii, enterococci, Proteus vulgaris, Staphylococcus aureus hemolyticus, beta hemolytic streptococci, Pseudomonas, and yeasts ‘from this area. It may well be true that in the uninfected anal sac, the flora is the same as that of the lower digestive tract. Whether this is true of the infected anal sac has yet to be determined. A comparison of the infected and non-infected anal sac might well dis- close a causative microorganism. 0n the other hand, the flora of the two may be the same. If this is the case, a comparison of the percent concentration of the organisms within the two may demonstrate an anal sac pathogen. Schirmer (1957) questions the ability of the clinician to ac- curately determine at all times whether the anal sac is definitely infected. He points out that the constant discharge from the sac makes this diagnosis extremely difficult. In general, a review of the literature indicates that the actual assurance that anal sac infection exists occurs only when certain conditions believed to be the result of the anal sac infection are improved by therapy. 28 Other possibilities to be included in considering that a microorganism pathogenic for the anal sacs is responsible for the disease would be the various serologic types of E. £211_and Proteusw There may well be existing within these groups a condition similar to that in infant diarrhea which in some cases is believed to be caused by certain serologic types of E. 5213 (Ferguson. 1957). Craige (1948), Cherry, Lentz, and Barnes (1946), Cooper, Davis and Wiseman (1941), and Gorham (1949) entertain the possibility that members of the Proteus group may be the causative agents in certain intestinal disturbances in dogs. Craige (1949) lists Proteus as one of the microorganisms believed to be responsible for dysentery in dogs. Cooper, Davis and Wiseman (1941) and Cherry, Lentz and Barnes (1946) have indicated that strains of Proteus mirabilis were responsible for an outbreak of gastroenteritis. Analysis of the data obtained from this study indicates that the incidence of Proteus is more frequent with increasing age, in the dark gray colored anal sac secretions, and in the males. This tends to indicate that Eggiegs may be responsible for the initial infection either before or after injury to the epithelium of the sac. The ability of members of the Eggtegg group to become enteric pathogens while residents of the intestinal tract of man or animals is open to question; such may occur under proper condi- tions, one must concede. g. 2211 occurred most frequently in females. in the gray colored secretions, and in two, three and nine year old dogs. These facts coupled with the generally high incidence of E. coli when compared with the other microorganisms may be interpreted 29 to indicate that an examination of the types of E. 2211 present in the secretion and the correlation of this data with infection of the sac may prove valuable. Other factors of importance in considering anal sac disease include a thorough understanding of the physiology and biochemistry of the sac and its glands. It may well be true that knowledge of these factors could uncover the entity initially responsible for anal sac disease. 30 SUMMARY AND CONCLUSION The bacterial flora of 125 canine anal sacs was determined accompanied with a procedure for the isolation of microorganisms from the sacs. The effect of 12 antibiotics on the heterogeneous flora of the sac secretions, and a review of anal sac anatomy, histology, pathology and physiology, was discussed. The organisms isolated included E. coli, Streptococcus lique- faciens, Streptococcus zymogenes, Proteus morganii, Aerobacter species ,, Staphylococcus albus , Staphylococcus epidermidis, Pseudomonas aeruginosa, and some unidentified yeasts. In an attempt to provide the clinician with an easier and more rapid means to analyze the data, the microorganisms isolated were grouped in the following manner: 1. Nonhemolytic enteric streptococci, 2. Alpha hemolytic enteric streptococci, 3. Beta hemolytic enteric streptococci, 4. Staphylococcus albus. 5. Staphylococcus epidermidis, 6. E. coli, 7. 7Aerobacter, 8. Proteus morganii, 9. Unidentified yeast, 10. Pseudomonas aeruginosa. Using this grouping of microorganisms, their occurrence within the anal sac was analyzed with relation to the sex of the dog, the age 31 of the dog, the color of the anal sac secretion, and the classifica- tion of breeds as recommended by the American Kennel Club. The anal sacs of the males showed a higher incidence of Proteus, Aerobacter, and yeasts while those of the female indicated a higher incidence of E. sell and Staphylococcus albus. The breed classification as recommended by the American Kennel Club demonstrated no correlation with the flora present. The color of the secretion indicated the possibility that certain microorganisms might be present. In the green colored secretions the incidence of alpha hemolytic enteric streptococci and Pseudomonas aeruginosa were highest. Stapgylgcoccgs 21222 was highest in incidence in the brown colored secretions. E. ggli and Proteus were highest in occurrence in the gray colored secretions. In general, these trends need corroboration with many more samples before they could be considered in any way definite. In comparing the age of the dogs with each of the organism groupings, only Proteus demonstrated any definite trend. The incidence of Proteus increased with age. The antibiotic inhibition of the anal sac flora cf 21 samples was determined. Dihydrostreptomycin and chloromycetin demonstrated the most marked inhibition while neomycin appeared to be the third most effective. nonhemolytic enteric streptococci alpha hemolytic enteric streptococci beta hemolytic enteric streptococci Staphylo- coccus albus Staphylo- coccus epidermidis Escherichia coli Aergbacter sp. Proteus morganii Unidentified Yeasts Pseudomonas aeruginosa Percent Incidence 5 0' K) o :3); Q 9 P j —' v1 1h db .1». .9393 [nun autueo 931 MOJJ paie[os; smstuafidooaotm 32 'I '313 aAtioadsaJ aqi JO eouaptout afleiuaoaad aqg nonhemolytic enteric streptococci 5 female Percent Incidence [0 —il—— a male alpha hemolytic enteric streptococci beta hemolytic enteric streptococci Staphylo- coccus albus Staphylo- coccus epidermidis Escherichia coli Aerobacter sp. ‘EL Proteus morganii l 1 Unidentified [f Yeasts ‘ m I . f Pseudomonas . aeruginosa «Dot 1. di- 4. ~stemyun aqi JO xas aqi qitm sons [eue autuao v31 moa; paietosr smstuaSJooaotm antioadsaa aqi JO aouaptout iuaoJad aqi J0 nostaadmoo v 33 '313 '8 Percent Incidence 1+001 0 9 O 4’9A nonhemolytic enteric rfil‘ay ‘1 b streptococci '1 O G d h—i alpha hemolytic enteric streptococci beta hemolytic enteric streptococci ‘b r ‘w ‘8 r w Staphylo- g coccus ' g albus b g_ P a e [Staphylo- g g coccus lb g epidermidis gr g . O Escherichia “I j : coli ”1* _J H. b J g i_. r 5* Aerobacter , sp. a T . 2 LP~ J g A r L *3 ‘f m LL L o Proteus S .i] H morganii b I g (D s: ] m I e Unidentified I #:j g Yeasts Hie o b l r: _ r l 8 w . Pseudomonas {3' l aeruginosa lgr 7 's: '3” 931 HOJJ paietost smstuaflaooaotm antioadsaa sq; JO aouaptou; iuaodud aqi JO nostaedmoo v 40-- 354 30- 20‘ 13‘ 10d Fig. 4. 35 A comparison of the percent occurrence of the different A.K.C. groupings of dogs examined. 1 Toy Terrier Non- sporting Mongrel Hound Working Sporting - I 4 u Percent Incidence N U O 01 ‘0 36 94 001 nonhemolytic enteric rking streptococci ounds on-s i errier P alpha hemolytic enteric streptococci non-s beta hemolytic enteric streptococci '313 0'9 Staphylo- COCCUS albus ‘-—" non-s t s Staphylo- w coccus h epidermidis Escherichia coli '3'X'V 3q1 QQIM 8398 [909 BUIUHO Aerobacter sp. aqi mad; paietost smstuafiaooaotm antioadsad aqi JO aauaptout iuaoJad aqi JO uostJedmoo v °sfiutdnoas Proteus norganii Unidentified Yeasts Pseudomonas aeruginosa 1;. 37 Percent Occurrence NG:. H9 H9 > coaumwwmon ow are tonnes» onncwwosom cm are mmmm on are mean exaswzoa. m a n m 0 Ho >mo w: flemfim P930 93F1°FQI10V Neomycin Penicillin Polymixin B Terramycin Tetra- cycline Triple sulfa Erythro— mycin Dihydro- strepto- mycin Chloromy-' cetin Carbomycin Bacitracin Aureomycin L-Qz Percent Effectiveness 0| 0 1 q 0' l -- OOI 'sotiotqtiun snotaen Xq eJoI; {atuaioaq oes {nun 19101 JO notitqtqul 38 '3Id 'L 14 39 Percent Incidence mam. m. > coaumdwmo: on are wnnwnmnnm o» vwoaesm Sonmmsw» mecca w: are mama nmom ewes 97o comm. swam. ~OOI. nut 00. NE a u a HG >mm M: manna 40 TABLE I THE MICROORGANISMS ISOLATED FROM 125 CANINE ANAL SACS unassu=hon mmcosousomm manner Umfimwacuvwza «assumes naoaoam .qw LocomnOAod mace nanowhosomm mwcfisaacwmo maouoooahnmmam manua ozoooooahnnoam “ooooOpaeuon ashoaco afiuhaoae: use: «cocoounohun afiaouco 03.323: «Ewan flooooouaoaon owaooce owahaoaonso: Number Sample 10 ll 12 14 . . . + . _ . . _ . + + . + + + _ .+ . + 4 + + . . + + + . . . + + + + i + _ + + . + + + + _ . + + . + + 15 16 17 18 19 20 21 22 23 24 25 26 27 29 41 Continued TABLE I mooanSLom mucoEovsmmm meadow Umwmfiacocfisa wasnuhofi eunuchm _.nm aoaomnoaod “Hoe. swnowhmnomm naeaaaeeamw nsoooooahnmuam nan”: msoooolonnaam «cocooaaoham essence owuhuoao; «yon wouoooaAoaum caboose owuhaoao: mamas “cocooanoham ofiaeucm owuhgoaogao: Sample Number 3O 31 32 33 34 35 36 37 38 39 40 4] 42 43 44 45 46 47 49 51 52 54 55 56 57' 59 60 61 42 Continued TABLE I snoswmshow mnsoaousmmm manna» wowmwucecfica «w:dflhos mamaoam .nm hoeumnonw< “Hoe nanofihononm mwpwaaoufifie insoooooahamaum mange msoooooawmmnum wuuooouaoaun enhance ow «macaw: as on «cocoouachan owhopao wafmaoao: mamas “cocooanonam ofiaoace t owuhmoaoncon Sample Number 62 63 64 65 66 67 68 69 .70 71 72 73 74 75 76 79 80 81 82 83 84 85 86 87 88 89 91 92 43 Continued TABLE I nmosfimzhom mmaoaovSomm mumaew flowhwaaovwcn «flammaos mseeoam .nn u¢aod£0ho< «Hoe nanoahunonm macsanmcfimo mzoooolonAMpm manan mzooooonsmnam woooooanmhum ofihouao owuhaoao: neon mwooooownonum ownmuco ow 9.32:2.— manna wooooounoaun oaaouco owahaosonsc: Sample Number 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 TABLE II 44 A SUMMARY OF THE CLASSIFICATION DATA ON THE 125 DOGS WHOSE ANAL SACS WERE EXAMINED FOR THEIR BACTERIAL FLORA Sample Color of Presumptive Number .Age Sex Breed Secretion Diagnosis 1 3 yrs Female Beagle cloudy normal white 2 4 yrs Female English cloudy pustular dermatitis , Setter white 3 2% yrs Male Boxer dark sprain, left foreleg green 4 8 yrs Male German cloudy dermatitis and possible short hair gray anal sac infection Pointer white 5 Male Beagle dark conjunctivitis brown 6 2% yrs Male Beagle cloudy parasitism gray white 7 8 yrs Male Irish gray dermatitis Terrier white 8 5 yrs Male Boxer gray nephritis - white 9 7 yrs Male Boxer gray arthritis white 10 9 yrs Male Pointer gray anal sac abscess white 11 l‘/2yrs Female Great Dane gray chronic enterocolitis white 12 2V2yrs Female Great Dane light enterocolitis brown 13 2 yrs Male Springer gray ulcerative otitis Spaniel white 14 '3 yrs Female Cocker dark gastroenteritis Spaniel brown 15 1 yr Male Cocker white, hemorrhagic colitis Spaniel cloudy packed 16 5 yrs Male Boxer light brown undiagnosed l7 8 yrs Male Dalmatian white, gout cloudy 18 Female Boston cream, distemper Terrier cloudy TABLE II -- Continued 45 Sample Age Sex Breed Color of Presumptive Number Secretion Diagnosis 19 3 yrs Female Mongrel white, cloudy infectious dermatitis 20 5 yrs Female German light brcwn urocystitis short hair Pointer 21 4 yrs Male Fox Terrier dark brown gastritis 22 7 yrs Male English gray white contusion Setter 23 3 yrs Male Collie gray white urocystitis 24 7 yrs Male Beagle cloudy white encephalitis 25 2 yrs Male Labrador cloudy white keratoconjunctivitis 26 1 yrs Female Labrador gray white undiagnosed 27 2 yrs Male Weimaraner cloudy white enterocolitis 28 English' cloudy white Setter 29 7 yrs Male English cloudy white prostatitis Setter 30 1 yr Male Hound dark green bladder neoplasm 31 6 yrs Male Beagle dark green arthritis 32 10 yrs Female Bulldog cloudy white abdominal neoplasm 33 9 yrs Female Springer cloudy white urocystitis, chronic Spaniel 34 8 yrs Female Cocker cloudy white infectious kerato- Spaniel conjunctivitis 35 5 yrs Female Cocker cloudy white dermatitis Spaniel 36 1 yr Male Cocker cloudy white gastroenteritis Spaniel 37 3 yrs Male Brittany brown tonsilitis Spaniel 38 4 yrs Female Boxer brown ancylostomiasis 39 1 yr Male Doberman cloudy white contusion 40 21/2yrs Male Beagle gray, cloudy enterocolitis 41 3% yrs Male German dark green Pannus Shepherd 42 1% yrs Female Beagle dark gray gastritis 43 ‘zfi yr Male Collie cloudy white dermatitis 44. 9 yrs Female Gordon dark grey arthritis Setter TABLE II -- Continued 46 Sample Age Sex Breed Color of Presumptive Number Secretion Diagnosis 45 1 yr Female Mongrel white contusion 46 lléyrs Male blue tick white neoplasm, skin hound 47 1 yr Female Mongrel gray dermatitis 48 8 yrs Male Beagle thick brown hypothyroidism 49 6 yrs Female Pointer cloudy white bacteremia 50 3 yrs Male English cloudy white dermatitis Pointer 51 21kyrs Male Boxer cloudy white abscess 52 5 yrs Male Hound brown ligament rupture 53 ‘%.yr Female Poodle cloudy white coccidiosis 54 ‘% yr Female Mongrel thick green mange, demodectic 55 4 yrs Male Boxer cloudy gray keratoconjunctivitis 56 3 yrs Male Coonhound cloudy gray sinusitis 57 9 yrs Male English cloudy white dermatitis Setter 58 2 yrs Male blue tick cloudy white neoplasms hound 59 Male Dalmation dark brown luxation, interverte- bral disc 60 Sléyrs Female Dachshund cloudy white urinary caliculi 61 'fléyrs Male Great Dane cloudy gray encephalitis 62 8 yrs Female Beagle green intestinal parasitism and fleas 63 Male Mongrel cloudy white osteomyelitis 64 2 yrs Male German cloudy white intestinal short hair parasitism Pointer 55 1 yr Male Labrador cloudy white mange, demodectic 66 8 yrs Male Cocker cloudy white dental tartar Spaniel 67 4 yrs Male Springer cloudy white dermatitis 'Spaniel 68 25§yrs Male Beagle cloudy white intestinal parasitism 69 5 yrs Female Boxer clear white tracheitis 70 4 yrs Male Poodle clear white dermatitis 71 5%yrs Female Terrier clear white luxation, interverte- bral disc 47 TABLE II —- Continued Sample Age Sex Breed Color of Presumptive Number Secretion Diagnosis 72 1 yr Female Brittany cloudy white wounds, lacerated Spaniel 73 6 yrs Male Poodle cloudy white lymphosarcoma 74 2%yrs Female Weimaraner cloudy white fracture ., left femur 75 9 yrs Male Cocker cloudy gray undiagnosed Spaniel 76 'fléyrs Male Ibrrier cloudy white conjunctivitis, chronic 77 2%yrs Female Labrador cloudy white dermatitis 78 1 yr Female Beagle cloudy white keratitis, ulcerative 79 10 yrs Male Labrador cloudy gray dermatitis, infectious 80 1 yr Male Boxer gray white neoplasm, skin 81 7 yr Male Boxer gray white neoplasm, skin 82 5 yrs Male Boxer gray white abscess 83 6 yrs Male English gray white muscle rupture Setter 84 6 yrs Male German gray white bacterial Shepherd pericarditis 85 4 yrs Female Cocker whitish gray conjunctivitis. acute Spaniel 86 1 yrs Female Mongrel gray white dermatitis, allergic 87 5 yrs Male Boxer gray white keratitis, ulcerative 88 Male Red Bone gray perianal fistula 89 3 yrs Male German grayish brown dermatitis Shepherd 90 7%yrs Male Boxer gray keratitis, ulcerative 91 2%yrs Male Weimaraner gray ancylostomiasis and coccidiosis 92 5 yrs Male English gray gastritis, chronic Pointer 93 3 yrs Female Beagle gray white contusion 94 3 yrs Male Irish gray yellow encephalitis Setter 95 4 yrs Male Spitz gray brown dermatitis, photo- sensitive 96 4 yrs Male Mongrel gray dermatitis 97 'flfiyrs Male blue tick gray abscess, postorbital hound TABLE II -- Continued 48 Sample Color of Presumptive Number Age Sex Breed Secretion Diagnosis 98 2%yrs Female Brittany gray brown undiagnosed Spaniel 99 6 yrs Female Doberman gray green chorea 100 6 yrs Female Irish gray otitis Setter 101 5 mo Male Dachshund clear white mange, demodectic 102 :fléyrs Male Beagle gray lugation, interverte- bral disc 103 10 yrs Male Boxer dark gray neoplasm, frontal sinus 104 7 yrs Male Boxer gray brown keratitis, ulcerative 105 1%yrs Female Boxer clear white dermatitis 106 2 yrs Female Cocker clear white urticaria Spaniel 107 6 yrs Male Mongrel clear white filariasis 108 7 yrs Male Boxer dark green keratitis, ulcerative 109 2 yrs Male English cloudy white intestinal parasitism Setter 110 4 yrs Male Poodle cloudy white stomatitis, ulcerative 111 2%yrs Male Springer clear white dermatitis Spaniel ‘ 112 10 yrs Male Boxer clear white neoplasm, frontal sinus 113 Myra Male Beagle cloudy white luxation, interverte- bral disc 114 7 yrs Female German dark green histoplasmosis Shepherd 115 7 yrs Male Collie clear white congenital dysplasia 116 6 yrs Male English gray keratitis, ulcerative Setter 117 10 yrs Male Boxer gray neoplasm, brain 118 5 yrs Female Cocker gray pseudopregnancy Spaniel ‘ 119 6%yrs Female English gray neoplasm, skin Setter 120 7%yrs Male Cocker gray castration Spaniel TABLE II -- Continued 49 5325:: Ase Sex Breed $2232.25. P32213532“ 121 4 yrs Male Collie gray undiagnosed 122. 3 yrs Female Collie gray fore leg paralysis 123 4 yrs Female Irish gray foreign body, nose Setter 124 5 yrs Female Collie cloudy white gastritis 125 £3 ;yrs Male French cloudy white otitis poodle 50 TABLE III SUMMARY OF IDENTIFICATION REACTIONS FOR STREPTOCOCCI ISOLATED FROM llO CANINE ANAL SAC SAMPLES cocci Sample Gram . Growth in Growth in Gelatin Mannitol Numb Stain Hemolysis Nutrient E V A Li ue- F t er' Broth With Broth. ragtion t:::en a- 6.5% NaCl 1 Positive gamma cocci 2 Positive gamma cocci 3 Positive gamma cocci 4 Positive gamma cocci 5 I><>sitive gamma (:occi 6 I’<>sitive gamma (:occi 7 I’<>sitive gamma (:occi 8 I’<>sitive gamma (:occi. 9 I’<>sitive gamma (:occi 10 l’<>sitive gamma <:occi 11 I’<>sitive gamma (:occi 12 I’<>sitive gamma cocci l3 l3<>sitive gamma cocci 14 Positive gamma cocci 15 Positive beta cocci 16 positive gamma cocci 17 Positive gamma cocci 18 Positive gamma TABLE III -- Continued 51 sample Gram Heml sis 33:22“? Growth in Gelatin Mannitol Number Stain y Broth with E.V.A. Lique- Fermenta- 6 5% NaCl Broth faction tion 1 9 Pos it ive gama + + + + cocci 20 Positive gall-a + + + + cocci 21 Positive gamma + + + + cocci 22 l3ositive gamma + + + + cocci 23 l3ositive gamma + + + + cocci 24 Positive gamma + + + + cocci 25 Positive gamma + + + + cocci 25 Posi t ive gamma + + + + cocci 27 Positive ganIna + + + 4 cocci 28 positive gamma + + + + cocci 29 I’ositive gamma + + + + cocci 30 if’ositive gamma + + + * cocci 31 I’ositive gamma + + + + cocci 32 I’ositive gamma + + + + cocci 33 I’ositive gamma + + + + cocci 3 4 lPositive gamma + + i + cocci 3'5 . . l>osit1ve gamma + + + + cocci 36 , I’ositive gamma + + + + cocci 37 Positive beta + + + + cocci r3‘ia. m . 52 TABLE III -- Continued Sample Gram . Gr°"?h 1“ Growth in Gelatin Mannitol . Hemoly51s Nutrient . Number Stain . E.V.A. Lique- Fermenta- Broth ”lth Broth faction tion 6.5% NaCl 51 Positive gamma + + cocci 52 Positive gamma + + cocci 53 Positive gamma + + cocci 54 Positive gamma + + cocci 55 Positive gamma + + cocci 56 Positive gamma + + cocci 58 Positive gamma + + cocci 59 Positive gamma + + cocci 60 Positive gamma + + cocci 61 Positive gamma + + cocci 62 Positive gamma + + cocci 63 Positive gamma + + cocci 64 Positive gamma + + cocci 65 Positive gamma + + cocci 56 Positive beta + i cocci 67 Positive gamma + + cocci 68 Positive gamma + + cocci 69 Positive beta + : cocci 70 {Positive beta + i cocci 53 TABLE III —— Continued Sample Gram . Gr°'?h 1“ Growth in Gelatin Mannitol . HemolySls Nutrient . Number Stain . E.V.A. Lique- Fermenta— Broth WIth Broth faction ti 6.5% NaCl °“ 7O 71 72 72 74 75 76 77 79 85 86 87 89 90 91 92 93 95 96 Positive cocci Positive cocci Positive cocci Positive cocci Positive cocc1 Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci alpha beta gamma alpha gamma gam“a gamma gamma gamma gamma gamma gamma gamma gamma gamma gamma gamma gamma gamma |+ r- 54 TABLE III -- Continued Sample Gram . Growth 1“ Growth in Gelatin Mannitol _ . Hemoly31s Nutrient . Number Stain . E.V.A. Lique- Fermenta- Broth WIth Broth faction tion 6.5% NaCI 97 98 99 100 101 102 103 103 104 105 106 107 108 108 109 109 110 110 111 Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci PositiVe cocci Positive cocci Positive cpcco gamma gamma gamma gamma gamma gamma gamma alpha alpha gamma beta beta alpha beta gamma beta beta gamma beta |+ l+ |+ I+ I+ |+ 55 TABLE III -- Continued Growth in Growth in Gelatin Mannitol Sample Gram . Nutrient . . HemolySls . E.V.A. Lique- Fermenta- Number Staln Broth Wlth Broth factio t' 6.5% NaCl “ 1°“ 112 112 113 113 114 114 114 115 116 117 118 118 119 120 120 121 122 Positive cocci Positive cocci 74-:- Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci Positive cocci gamma beta gamma beta beta gamma ‘alpha gamma gamma gamma gamma beta beta beta gamma gamma gamma |+ l+ |+ l+ |+ I+ 56 TABLE IV SUMMARY OF THE IDENTIFICATION REACTIONS USED FOR E. COLI ISOLATED FROM lOl CANINE ANAL SACS Sample Gram 33:03:10 Indole :2?” v . P. Citrate Number Stain p gy Production . Reaction Utilization on E.M.B. Reaction 1 Negative Typical rods ‘E. coli type - 2 Negative Typical rods E. coli type - 3 Negative Typical rods E. coli type - 4 Negative Typical rods E. coli type - 5 Negative Typical rods E. coli type - 6 Negative Typical rods E. coli type - 7 Negative Typical rods E. coli type - 10 Negative Typical' rods E. coli type - 11 Negative Typical rods E. coli type - 12 Negative Typical rods E. coli type - 14 Negative Typical rods E. coli type - 15 Negative Typical rods E. coli type — 17 Negative Typical rods E. coli type - 18 Negative Typical rods E. coli type - 19 Negative Typical rods ‘E. coli type - 20 Negative Typical rods E. coli type - 23 Negative Typical rods E. ggll_type — 24 Negative Typical rods E. coli type - 57 TABLE IV -- Continued Sample Gram 32?;‘310 Indole It?“ v.13. Citrate Number Stain p w Production . Reaction Utilization on E.M.B. Reaction 26 Negative Typical rods E. coli type 27 Negative Typical rods E. coli type 29 Negative Typical rods E. coli type 31 Negative Typical rods E. coli type 32 Negative Typical rods E. coli type 33 Negative Typical rods E. coli type 34 Negative Typical rods ‘E. coli type 35 Negative Typical :rods E, coli type 36 Negative Typical rods ‘E. coli type 37 Negative Typical rods E. coli type 38 Negative Typical rods ‘E. coli type 39 Negative Typical rods E, goli type 40 Negative Typical rods E. 22}; type 41 Negative Typical rods E. coli type 42 Negative Typical rods E. coli type 43 Negative Typical rods E. coli type 44 Negative Typical rods E. coli type 45 Negative Typical rods ‘E. coli type ‘46 Negative Typical rods E. coli type 58 TABLE IV -- Continued Colony Methyl . Smmfle Gram Morphology P223::tion Red K; :t'o 3:?Iétet' Number Stain on E.M.B. Reaction a 1 n 1 12a 1°“ 47 Negative Typical rods E. coli type - 49 Negative Typical rods E. coli type _ 50 Negative Typical rods E. coli type - 51 Negative Typical rods E. coli type - 56 Negative Typical rods E. coli type - 57 Negative Typical rods E. coli type - 58 Negative Typical rods E. coli type - 62 Negative Typical rods E. coli type — 63 Negative Typical rods E. coli type — 64 Negative Typical rods E. coli type — 65 Negative Typical rods E. coli type - 66 Negative Typical rods E. coli type - 67 Negative Typical rods E. coli type - 68 Negative Typical rods E. coli type - 69 Negative Typical rods E. coli type _ 70 Negative Typical rods E. coli type - 71 Negative Typical rods E, coli type - 72 Negative Typical rods E. coli type - 73 Negative Typical rods E. coli type - 59 TABLE IV -- Continued Sample Gram §°l°2y1 Indole "ethyl v . P. Citrate Number Stain "9 ° °3y Production Red Reaction Utilization on E.M.B. Reaction 74 Negative Typical rods E. coli type 75 Negative Typical rods E. coli type 76 Negative Typical rods E. coli type 77 Negative Typical rods E. coli type 78 Negative Typical rods E. coli type 79 Negative Typical rods ‘E. coli type 81 Negative Typical rods E. ccli type 82 Negative Typical rods E. coli type 84 Negative Typical rods E. coli type 85 Negative Typical rods E. coli type 86 Negative Typical rods E. coli type 87 Negative Typical rods E. coli type 88 Negative Typical rods E. coli type 89 Negative Typical rods E. coli type 90 Negative Typical rods E. coli type 91 .Negative Typical rods E. coli type 92 Negative Typical rods E. coli type 93 Negative Typical rods E. coli type 94 Negative Typical rods E. coli type 60 TABLE IV -- Continued l- Smufle Gram fizioflzlo Indole :zahyl V. P. Citrate Mmmer Stain p 8y Production . Reaction Utilization on E.M.B. Reaction 95 Negative Typical rods ‘E. coli type - 96 Negative Typical rods E. coli type — 97 Negative Typical rods 4E. coli type — 98 Negative Typical rods E, coli type — 99 Negative Typical rods E. coli type — 101 Negative Typical rods E. coli type - 102 Negative Typical rods ‘E. coli type - 103 Negative Typical rods ‘E. coli type - 104 Negative Typical rods E. coli type - 105 Negative Typical rods E, coli type - 106 Negative Typical rods E, coli type - 109 Negative Typical rods E. coli type - 110 Negative Typical . rods E. coli type — 111 Negative Typical rods E. coli type - 113 Negative Typical rods .E‘ coli type - 114 Negative Typical rods E. coli type — 115 Negative Typical rods ‘E. coli type - .116 Negative Typical rods E. coli type - l 17 Negative Typical rods E. coli type - 61 TABLE IV -- Continued Colony Methyl Susie Sign Morphology 1132:3111Ztion Red Re l(:tio Stiflétzt' “" " on E.M.B. Reaction 8 n 1 12 1°" 113 Negative Typical rods ‘E. coli type 119 Negative Typical rods E. coli type 120 Negative Typical rods ‘E. coli type 121 Negative Typical rods ‘E. coli type 122 Negative Typical rods E, coli type 123 Negative Typical rods E, 901i type 124 Negative Typical rods E. coli type 125 Negative Typical rods E, coli type E 87113 ' l b . i TABLE V 62 SUMMARY OF IDENTIFICATION REACTIONS USED FOR AEROBACTER ISOLATED FROM 71 CANINE ANAL SACS ¨e Gram Colony Indole Methyl V. P. Citrate Gelatin . Morphology . Red . Utili- Lique- Mumer Stain Production . Reaction . . on E.M.B. Reaction zation faction 8 Negative Aerobacter rods type - - + + - 9 Negative Aerobacter rods type - — + + _ 10 Negative Aerobacter rods type - - + + - 11 Negative Aerobacter rods type - - + + - 15 Negative Aerobacter rods type - - + + — 16 Negative Aerobacter rods type - - + + — 20 Negative Aerobacter rods type - - + + - 21 Negative Aerobacter rods type - - + + - 22 Negative Aerobacter rods type - - + + - 23 Negative Aerobacter rods type - - + + - 24 Negative Aerobacter rods type - - + + — 25 Negative Aerobacter rods type - - + + — 27 Negative Aerobacter rods type - - + + - 28 Negative Aerobacter rods type — - + + _ 29 Negative Aerobacter rods type - - + + — 30 Negative Aerobacter rods type - - + + - 31 Negative Aerobacter rods type - - + + - 32 Negative Aerobacter rods type - - + + - TABLE V -- Continued 63 Sample Gram Colony Indole Methyl V P. Citrate Gelatin . Morphology R d . Utili- Lique— Mmmer Stain Production . Reaction . . on E.M B Reaction zation faction 33 Negative Aerobacter rods type - — + + - 34 Negative Aerobacter rods type - - + + - 35 Negative Aerobacter rods type - - + + - 36 Negative Aerobacter rods type - - + + 1 37 Negative Aerobacter rods type - — + + - 38 Negative Aerobacter rods type - - + + - 39 Negative Aerobacter rods type - — + + 1 40 Negative Aerobacter rods type - - + + i 41 Negative Aerobacter rods type - - + + 1 42 Negative Aerobacter rods type - - + + 1 43 Negative Aerobacter rods type - — + + i 46 .Negative Aerobacter rods type — - + + i 47 Negative Aerobacter rods type - — + + 1 48 Negative Aerobacter rods type - - + + i 49 Negative Aerobacter rods type - - + + i 50 Negative Aerobacter rods type - — + + l 51 Negative Aerobacter rodS' type - - + + i 56 Negative Aerobacter rods type - - + + i 58 Negative Aerobacter rods type - - + + i .- .ni.-_ L-r_i _ ge—qu—.9_-x TABLE V —- Continued 64 — T -:— fimmle Gram Colony Indole Methyl V. P. Citrate Gelatin . Morphology . Red . Utili— Lique- Number Stain Production . Reaction . . on E.M.B. Reaction zation faction 59 Negative Aerobacter rods type - — + + I 60 Negative Aerobacter rods type - - + + l 64 Negative Aerobacter rods type — - + + i 66 Negative Aerobacter rods type - - + + i 68 Negative Aerobacter rods type — - + + i 70 Negative Aerobacter rods type — - + + i 72 Negative Aerobacter rods type - - + + I 73 Negative Aerobacter rods type - - + + 1 74 Negative Aerobacter rods type - - + + I 75 Negative Aerobacter rods type - - + + I 76 Negative Aerobacter rods type - - + + i 79 Negative Aerobacter rods type - - + + i 80 Negative Aerobacter rods type - - + + I. 82 Negative Aerobacter rods type - - + + i 83 Negative Aerobacter rods type - - + + i 84 Negative Aerobacter rods type - - + + i 85 Negative Aerobacter rods type - - + + 1 89 Negative Aerobacter rods type - - + + l 92 Negative Aerobacter rods type - - + + i TABLE V -— Continued 65 Smufle Gram Colony Indole Methyl V. P. Citrate Gelatin . Morphology . Red . Utili- Lique- Nmflmr Stain Production . Reaction . . on E.M.B. Reaction zation faction 93 Negative Aerobacter rods type - — + + l 94 Negative Aerobacter rods type - - + + i 96 Negative Aerobacter rods type - - + . i 97 Negative Aerobacter rods type - - + + 1 98 Negative Aerobacter rods type - - + + l 99 Negative Aerobacter rods type - - + + l 106 Negative Aerobacter rods type - - + + i 107 Negative Aerobacter rods type - - + + l 110 Negative Aerobacter rods type - - + + i 111 Negative Aerobacter rods type - - + + 1 116 Negative Aerobacter rods type - - + + .1 118 Negative Aerobacter rods type - - + + l 121 Negative Aerobacter rods type - - + + i 122 Negative Aerobacter rods type - - + i 1 124 Negative Aerobacter rods type - - + + 1 I’ '1'“- ..u J.A!~_‘”‘.'Il' Ln 66 TABLE VI SUMMARY OF PROTEUS IDENTIFICATION REACTIONS ISOLATED FROM 41 CANINE ANAL SACS Urea Mannitol Sucrose Maltose Indole Sample Gram Colony . . . Utili- Fermenta- Fermenta— Fermenta- Produc- Number Stain Morphology . . . . . zation tion tion tion tion 66 Negative Swarming rods with odor + _ - _ _ 67 Negative Swarming rods with odor + - - - + 68 Negative Swarming rods with odor + - — — + 69 Negative Swarming rods with odor + - — - + 70 Negative Swarming rods with odor + — — - + 71 Negative Swarming rods with odor + — - - + 73 Negative Swarming rods with odor + - - - + 74 Negative Swarming rods with odor + - - - + 76 Negative Swarming rods with odor + - — - + 77 Negative Swarming rods with odor + - - - + 79 Negative Swarming rods with odor + - - - + 80 Negative Swarming rods with odor + - - — + 81 Negative Swarming rods with odor + — - — + 82 Negative Swarming rods with odor + - - - + 83 Negative Swarming rods with odor + — - - + 84 Negative Swarming rods with odor + - - - + 85 Negative Swarming rods with odor + — - - + 87 Negative Swarming rods with odor TABLE VI -- Continued 67 S 1e Gram Colon Urea Mannitol Sucrose Maltose Indole Nam: Stain Mor hilo Utili- Fermenta- Fermenta- Fermenta- Produc- um er p 8y zation tion tion tion tion 88 Negative Swarming rods with odor + - - _ + 89 Negative Swarming rods with odor + - - - + 90 Negative Swarming rods with odor + - - - + 91 Negative Swarming rods with odor + - — - + 92 Negative Swarming rods with odor + — - — + 93 Negative Swarming rods with odor + - - - + 94 Negative Swarming rods with odor + - — — + 96 Negative Swarming rods with odor + - — - + 97 Negative Swarming rods with odor + - — — + 98 Negative Swarming rods with odor + - - — + 99 Negative Swarming rods with odor + - - — + 107 Negative Swarming rods with odor + - - - + 108 Negative Swarming .rods with odor + — - - + 111i Negative Swarming rods with odor + - - - + 112 Negative Swarming rods with odor + - _ - + 115 Negative Swarming rods with odor + - _ - + 116 Negative Swarming rods with odor + - - — + 117 Negative Swarming rods with odor + - _ - + 121 'Negative Swarming rods with odor 68 TABLE VI -— Continued Urea Mannitol Sucrose Maltose Indole swmflfi' Gram Colony Utili- Fermenta- Fermenta— Fermenta- Produc- Number Stain Morphology zation tion tion tion tion 122 Negative Swarming rods with odor + — - — + 123 Negative Swarming rods with odor + - - - + 124 Negative Swarming rods with odor + - - - + 125 Negative Swarming rods with odor + - - - + 69 a>whw wm Ow Hum HUMZHHWHO>HHOZ wm>neH02m ammo wow vmmccozoz>m Hmor>ewu fiwo: o O>ZHZH >2>h mbnm muscwe mwms neuosw mum Hamowo Zonwwm sosaeanmnwos 2:56am moms: zomcrowomq vsomcoewoz peasanawos Umxafiomo monsoon :mwnomm zmsswnow u Zommewco spedmmoose I + I I I do w ewe: 6 meme: camSman m Zommnw