REP‘EATED ARTIFICIAL EXPOSURE OF BOVENE UDDERS TO COAGULASE PO$ITWE HEMQLYTIC STAPHYLOCOCC! Thesis {or flu Degree of M. S. MICHIGAN STATE UNIVERSITY Harbhajan Singh Nat 1962 {HESIS LIBRARY Michigan State University .7— .-.—-———h_‘_—'—. ABSTRACT REPEATED ARTIFICIAL EXPOSURE OF BOVINE UDDERS TO COAGULASE POSITIVE HEMOLYTIC STAPHYLOCOCCI by Harbhajan Singh Nat Udders of three young, healthy cows were repeatedly exposed to variable numbers of coagulase positive hemolytic staphylococci (strain 71). During the second series of exposures, mild trauma was applied to the injected quarter. After every exposure, the number of staphylococcic organisms shed in milk were counted. Their pathogenic nature was determined on alternate days. Multiplication of staphylo- coccic organisms was studied in fresh raw milk. Except for the right rear quarter of the first cow, which was exposed four times, all other quarters were ex- posed three times after an interval of 10 days to two months with subsequent increasing doses of staphylococci. Differ- ences appeared in the susceptibility of the quarters to invasion by these organisms. The right rear quarter of cow 1, when exposed to 13.5 x 108 staphylococci developed a mild form of clinical mastitis when the cow was in lacta- tion, but did not show any clinical reaction with nearly the same number of organisms a month later when the cow had become dry. With small numbers of organisms ranging from 16 x 102 to 28 x 102, clinical mastitis was seen in only one quarter (right fore) of cow 1. During the second series of exposures with 38 x 103 to 57 x 104 staphylococci, only two quarters (left fore and left rear) of cow 2 developed a mild form of clinical mastitis which lasted for four days. The re- maining quarters did not show clinical symptoms of mastitis, but in most cases the milk was abnormal for four to six days and contained many flocculi. The third series of ex- posures to a large number of organisms (1 x 1010) created a mild form of clinical mastitis in three quarters of lac- tating cow 191, while the two dry cows did not show any clinical reaction. This mild form of mastitis was over- come without treatment by the fifth day. Rather less vari- able staphylococcic milk counts were observed from cow 1, whereas in the other two cows, counts were greatly variable. Staphylococcic organisms (strain 71) were added to fresh raw milk from cows 191 and 3. During the first three hours of inoculation a sharp decline in the number of staph- ylococcic organisms was observed which was followed by a rapid growth phase. I. REPEATED ARTIFICIAL EXPOSURE OF BOVINE UDDERS TO COAGULASE POSITIVE HEMOLYTIC STAPHYLOCOCCI By HarbhaJan Singh Eat A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1962 ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. A. R. Drury for his advice and encouragement in the prepa- ration of this work. my association with him has been a most memorable and pleasant experience at Michigan State University. Thanks are also extended to Dr. G. H. Conner, my adviser, for his valuable suggestions and advice from time to time. The author deeply appreciates Dr. Drury, Dr. Conner and Dr. Cholvin for their constant help in preparation of this work. Sincere thanks are paid to Dr. W. O. Drinker, Head of Surgery Medicine Department, for his preper guidance and for providing all facilities to carry out this work. I am indebted to the staff of the Department of Surgery and Medicine and particularly the Bovine Mastitis Research Laboratory where this work was carried out. I am thankful to Mrs. Donald Mills and MIs. Carolyn Easter for their help. ii Dedicated to my ever beloved father-- Sardar Basant Singh iii TABLE LIST OF GRAPES. . . . . . . LIST OF TABLES. . . . . . . INTRODUCTION. . . . . . . . REVIEW OF LITERATURE. . . . OF CONTENTS Extramammary reservoir of Staphylococcic organisms in milk and udder. Staphylococci associated with Pathogenic staphylococci. Possible mode of transmission Mode of action. . . . . Changes in milk . . . . Pathology of udder. . . Antitoxins in blood and milk 0 Staphylococcic toxoids. MATERIALS AND METHODS . . . Cultures. . . . . . . . Animals . . . . . . . . O staphylococci. k Plan of the intramammary injections coccic organisms . . . Intramammary injection technique. Collection of milk samples. Agar pour plate method. Culture test. . . . . . Coagulase test. . . . . MicroscOpic test. . . . Test to determine in vitro multiplicatio ylococcic organisms (strain 71). RESULTS 0 0 O O O O O O O 0 DISCUSSION. . . . . . . . . Shedding of staphylococci in the milk . Effect of stress or trauma. Multiplication of staphylococci in fresh raw milk . SUMMARY 0 O O O O O O O O O BIBLIOGRAPHY. . . . . . . . APPENDIX. C O O O O O O O O mastitis. . . I I I . c w 0 O O O O O O O of sta aphylo- n of staph— Page LIST OF GRAPES Page GRAPH SHOWING GROWTH OF STAPHYLOCOCCIC ORGANISMS STRAIN 71 IN FRESHLY DRAWN MILK (10 cc.) FROM COW 3. . . . 39 GRAPH SHOWING GROWTH OF STAPHYLOCOCCIC ORGANISMS STRAIN 71 IN FRESHLY DRAWN MILK (10 cc.) FROM COW 191. . . 4O LIST OF TABLES LEFT FORE QUARTER OF COW 1 . . . . . . . . RIGHT FORE QUARTER OF COW 1. . . . . . . . LEFT REAR QUARTER OF COW 1 . . . . . . . . RIGHT REAR QUARTER OF COW l. . . . . . . . LEFT FORE QUARTER OF COW 2 . . . . . . . . RIGHT FORE QUARTER OF COW 2. . . . . . . . LEFT REAR QUARTER OF COW 2 . . . . . . . . RIGHT REAR QUARTER OF COW 2. . . . . . . . LEFT FORE QUARTER OF COW 191 . . . . . . . RIGHT FORE QUARTER OF COW 191. . . . . . . LEFT REAR QUARTER OF COW 191 . . . . . . . RIGHT REAR QUARTER OF COW 191. . . . . . . SUMMARY OF THE EXPERIMENTAL PROCEDURES FOR INTRAMAMMARY CHALLENGE OF THE THREE BOVINE UDDERS BY STAPHYLOCOCCIC ORGANISMS . . . . GROWTH OF STAPHYLOCOCCIC ORGANISMS (STRAIN IN FRESHLY DRAWN MILK, 10 cc. FROM COW 191 GROWTH OF STAPHYLOCOCCIC ORGANISMS (STRAIN IN FRESHLY DRAWN RAW MILK, 10 cc. FROM COW vi 71) 71) 3 . Page 50 51 53 54 56 57 58 59 60 61 62 63 64 65 66 INTRODUCTION From an economic standpoint, mastitis is one of the most important diseases affecting dairy cattle. It has wide distribution and a high incidence. It lowers the quantity as well as the quality of milk produced by the affected animals. Streptococcus agalactiae has long been recognised as a causative organism in mastitis. Until recently little attention has been paid to staphylococcic organisms as a possible cause of this disease. Most research workers have considered this organism as a normal udder inhabitant or only responsible for a mild udder irritation. When udder flora is dominated by streptococci, the staphylococci do not seem to be a serious problem. Moreover, the media used for the isolation of streptococci has an inhibitory action towards the staphylococcic organisms. Thus, little atten- tion was paid to the staphylococci before World War II. Since then the use of antibiotics has made it possible to eradicate Streptococcus agalactiae; consequently, for vari- ous reasons, staphylococcic organisms have become more prev- alent in the udder flora and have acquired greater signif- icance for the dairy industry and research workers. Jordan (1930) and Litterer and Crabtree (1934) found (I‘ that staphylococci present in milk could cause food poison- ing in man and skin carbuncles in man and various animals. MacDonald and White (1961) expressed doubt that coagulase positive hemolytic staphylococci could cause a mastitis outbreak in a dairy herd. These organisms were found in abundance even after the animals had been rid of the disease. This work has been undertaken to study the possi- bility of causing an acute or gangrenous form of mastitis with coagulase positive hemolytic staphylococcic organisms. An attempt was made to find out the approximate number of staphylococcic organisms that a cow may carry in her udder without showing any clinical manifestation of disease. Factors of stress and trauma, as they occur naturally, were applied to the udders to determine their effect. Multiplication of staphylococcic organisms (strain 71) in freshly drawn milk from two cows was studied. REVIEW OF LITERATURE Burrows (1959) stated that the presence of staphy- lococcic organisms in pus was observed by Pasteur in 1880 and by Ogston the following year. The organisms were ob- tained in pure culture by Becker in 1883 and their relation- ship to suppurative wounds was described by Rosenback in 1884. According to Elek (1959). Garre, in 1885, produced a large orange colored carbuncle in a man by rubbing his skin with a virulent culture of staphylococci. Hucker (1924 B) stated that the term micrococcus was used by Cohn in 1872 prior to the use of staphylococcus by Rosenback in 1884. Hucker (1924 A) further reported that Rosenback originally used trinomial names, such as Staphy- lococcus pyogenes aureus for the golden pigmented pathogenic cocci and Staphylococcus albus pyogenes for nonpigmented pathogenic strains. Extramammary reservoir of staphylococci: Staphylococci are usually found on the skin and mucous membranes of the animal body, especially of the nose and mouth (Burrows, 1959). Evans (1916) found staphylococci in milk which culturally resembled those from bovine skin and other body sources. Mastitis was produced by Little ‘.—‘-I as -..,~_., - and Folly (1935) by injecting staphylococcic organisms iso- lated from skin lesions. McDiarmid (1947) found that Staph- ylococcus aureus was commonly present in milk and on the surface of teats of dairy cows. Hemolytic coagulase positive micrococci were found by Spencer and Lasmanis (1952) on the skin of the teats of dairy cows and on the teat cups of milking machines. Newbould (1960) was of the opinion that Staphylococcus aureus was very widely distributed and could be found in every barn. Hemolytic coagulase positive staphylococci were iso- lated from the ventral surface of the udder and teats by Davidson (1961). He also found these organisms in external orifices such as eyes, nostrils, mouth, vagina and anus. Certain skin areas such as the chest, poll, and caudal folds were also sites of their presence. Staphylococcic organisms in milk and udder: Miller and Heishman (1943 A) has stated that the occurrence of micrococci in the udder has been recognized ever since the inception of bacteriological study of bovine mastitis. He himself noticed a high incidence of staphy- lococcic mastitis in several herds. 0n culture, Ward (1900) isolated micrococci from. 19 apparently normal udders of cows slaughtered as reactors to the tuberculin test. Strippings from 78 cows, when ex— amined by Harding and Wilson (1913). showed micrococci in 70%. Evans (1916) examined 192 milk samples from 161 cows and isolated micrococci from 58% of the samples. Two years later Jones (1918) isolated micrococci from inflamed udders, having acute mastitis. On examination, Bean gp_§1. (1943) found that 21% of the animals were infected with Staphylo- coccus aureus in a herd of dairy cattle. Minett (1937 A), from his study of milk samples from separate quarters of 415 cows, concluded that 145 were shedders of Staphylococcus aureus. In a six-year study, Packer (1952) examined 15,693 samples of milk submitted for a laboratory diagnosis of mastitis and found that 70% of the samples contained micrococci. He revealed that the increase in incidence was associated with the progressive number of lactations. A high infection rate accompanied a low incidence of clinical mastitis. The milk samples with staphylococcic organisms ranged from 37 to 58%, ac- cording to Newberene and Mayer (1960). Their survey in- cluded five major regions of the United States. Spgphylococci associated with mastitis: Rossel and Miller (1933) and Husker and Udall (1933) reported the absence of streptococci in abnormal secretions from abnormal udders, as determined by microscopic examina- tion of milk and physical palpation of udders. Similar conditions were noted by Hastings and Beach (1937), Peter- son and Hastings (1938), and Hastings and Johns (1939). They also observed that the incidence of abnormal milk in- creased from lactation to lactation. They named this con- dition nonspecific mastitis. In the very beginning of the twentieth century, Guillebeau and Svenwall, as described by Minett (1929), were the earliest workers to associate staphylococci with mastitis. They called them galactococci. Among 81 cases of mastitis, Jones (1918) found 24 in which micrococci were the cause. Clinically the cases were mild but occasionally there was acute inflammation which became chronic. Carpenter (1925) examined abnormal udder secretions from 150 cases of mastitis and isolated staphylococci in nine of them. In periodic examinations of approximately 200 cows, Plastridge and Anderson (1934) found that 10% of the cases of abnormal udder secretions were due to staphylococci. Parshall (1934) described an acute gangrenous mastitis in California. Out of 22 cases, mostly in young lactating cows, 15 occurred during the cold, rainy season. The cases occurred sporadically and were often accompanied by sub- cutaneous edema over the abdomen. Nine acute cases of staphylococcic mastitis were described by Little and Folly (1935). Minett (1937 A) reported nine cases of staphylo- coccic mastitis. Studies made on two herds have been re- ported by Schalm (1944) where, in one herd, he found six cases of gangrenous mastitis within a six-weeks period and three cases in another herd of 115 milking cows. I. Pathogenic staphylococci The criteria for Judging pathogenicity of staphy- lococci are the ability to secrete toxins which have the capacity to lyse the red blood cells of certain animal spe- cies and the capacity to coagulate blood plasma of some species. Jones (1918) stated that Savage was the first to describe the biochemical characteristics of micrococci as- sociated with mammitis, but credit went to Glenny and Stevenes (1935) who described two strains on the basis of toxins called alpha and beta for which there are corresponding antibodies. The alpha toxin causes hemolysis in sheep and rabbit blood, while the beta causes hemolysis of sheep blood, but no hemolysis of rabbit blood. The beta toxin is a weak hemolytic agent at 37‘ C but becomes more hemolytic at lower temperatures. This phenomenon is called "hot cold.” The above findings were confirmed by Bryce and Rountree (1936) and Minett (1936). Smith and Price (1938) described a gamma toxin, while a delta toxin was described by William and Harper (1947) in addition to the above toxins. The findings of William and Harper were confirmed by Marks and Vaughan (1950) and Slanets and Bartley (1953). Another hemotoxin called epsilon has been shown by Elek and Levy (1950). It is capable of causing lysis of sheep and rabbit erythrocytes and is secreted by coagulase negative organisms. Bryce and Rountree (1936) and Minett (1936) sug- gested that human strains usually produce alpha hemolysis while the majority of animal strains produce beta hemolysis. Similar findings were reported by Elek and Levy (1950). Chapman and Berens (1934, 1938) and Cruickshank (1937) attributed pathogenicity of staphylococci to the presence of the enzyme coagulase. Pathogenicity of staph- ylococci to the udder, as reported by Plastridge and William (1936), was related to their hemolytic character on ox blood, to the formation of coagulase, and to the fermentation of mannitol. Smith, Hale and Smith (1944) postulated that the pathogenicity of staphylococci for a given species de- pends upon its ability to clot plasma of that species. Pathogenicity of udder micrococci as described by Schalm and Woods (1953 B) was related to the production of beta toxin and coagulation of rabbit plasma. In the same year Slantez and Bartley (1953) found that staphylococcic organ- isms having the capacity to produce alpha, alpha-beta or beta hemolytic toxins were mostly coagulase positive and capable of producing bovine mastitis. The coagulase test is the one most reliable for differentiation between pathogenic and non-pathogenic staph- ylococci as reported by Chapman and Berens (1934, 1938), Cruickshank (1937). and Slantez and Bartley (1945). They further stated that no relationship existed between degree of coagulating power and pathogenicity of a strain. Veilla, Delia and Faber (1947) showed that there was little rela— tionship between blood hemolysis, pigment production and coagulase formation. In spite of the fact that coagulase activity is considered a very important criterion for judging patho— genicity of staphylococci, Dubeler and Cole (1956) claim to have isolated coagulase negative non-hemolytic micro- cocci in eight out of twelve acute cases of bovine mastitis. From this review of literature, it is apparent that the coagulase test is the most practical test to judge pathogenicity of staphylococcic organisms. This, combined with hemolytic ability and the mannitol fermentation test, leave little doubt as to the pathogenicity of a staphylo- coccic strain. Possible mode of transmission There are different theories put forward by various workers, but the exact mode of transmission is not known. According to Jones (1918), the infection was conveyed from animal to animal during the process of grooming and washing before milking. Miller and Heishman (1943 A), on the other hand, thought that the hands of the milker or the teat cups of a milking machine were responsible for infection from an infected animal to a healthy animal. Source of pathogenic 10 staphylococci for invasion of the mammary glands was pri- marily from other infected glands, according to Schalm and Woods (1953 A). Newbould (1960) was of the opinion that the constant use of milking machine inflations laden with bacteria con- tributed to the spread of infection. The most important source of infection was the cow's udder, as revealed by Davidson (1961), who also stated that pathogenic staphylo- cocci commonly multiply outside as well as inside the udder. Mode of action Carpenter (1922), Minett (1936), Schalm (1944), and Miller and Heishman (1943) suggested that the main entrance of infection was through the streak canal. The exact mode of action is not known, but various hypotheses have been put forward. Jones and Little (1922) and Little and Folly (1935) thought the micrococci enter the mammary glands by the way of the streak canal. Jones and Little (1927) hypothesized that after entry into the teat cistern the organisms remain confined there because of the presence of an inhibitory substance in the milk. When the concentration of this in~ hibitory substance in the affected quarter decreased the multiplication of the organisms no longer was confined to the teat canal but extended rapidly to the secretory tissue. Jones further suggested that, after parturition, 11 serum infiltration of the udder may be responsible for the change in character of the colostrum or milk, thus enhanc- ing the establishment of the organisms. Stagnation of blood flow in blood vessels followed by stabilization of staphylococci in the tissues, was re- ported by Cruickshank (1937). The concentration of staph- ylocoagulase formed local thromboses in the small capil- laries, causing infarction and then necrosis or gangrene of the affected part. Spink and John (1940) believed that leucocidin was the important factor present in the products elaborated by staphylococci which prevented the phagocytosis of staph- ylococci by fixed tissues and circulating leukocytes. This factor helped in stabilization of infection. Coagulase prevented phagocytosis of micrococci, thus permitting stabilization of the organisms in the tis- sues was the view expressed by Smith, Hale and Smith (1944). Staphylococcic tissue invasion was followed by a local in- flammatory reaction which served to wall off the infected area. The infiltration of polymorphonuclear leukocytes and microphages prevented further spread of the infection (1212.). Schalm (1959) thought that the production of toxins and the coagulase enzyme favored the tissue invasion and localization of micrococci. Stress might enhance the action of the infective organisms, and in the absence of circulating -." . 12 antitoxins a fulminating mastitis with gangrene of the udder is more likely to develop. Changes in milk Staphylococcic infected milk as reported by Jones (1918) was yellowish white, containing many large pus floc- culi. 0n standing, many of the large particles coalesced to form a large viscid mass on the bottom of the vial. Sometimes the secretions were purulent in nature. Minett (1936) stated that milk from staphylococcic infected quar- ters was not grossly changed in appearance except that oc- casionally it was watery. Frequently the reaction was a1- kaline and there was an increased amount of sediment follow- ing centrifugation. Leukocyte counts in excess of 500,000 cells per m1. of milk were seen by Plastridge §3_§l. (1939) in milk which has staphylococci present. Gould (1942) reported that fol- lowing infection, the milk secretion was a dirty greyish thin syrup with clots. Murphy (1943) described staphylo- coccic infected milk as being high in pH, chloride concen- tration, and leukocyte counts. Alder and Migaki (1951) found that in staphylococcic infection of udders, there was a marked increase in poly- morphonuclear leukocytes in the milk. Schalm and Woods (1953 B) reported that milk secretion was reduced and turned to straw color in cases of gangrenous mastitis. Edds and *-.- l3 Bieter (1953) reported that the milk secretion became scanty, odorless, and resembled clear serum following staphylococcic infection. Pathology of udder Grossppathology: Jones (1918) stated that in acute cases the affected quarters were swollen, firm and painful to manipulation. After the acute inflammation subsided a more chronic condition continued and the quarters became atrophied and secreted a smaller quantity of milk. Jones was of the opinion that some staphylococcic organisms were able to produce only a mild catarrh while others gave rise to severe parenchymatous inflammation. Actinomycotic-like lesions were described by Albiston (1930), Smith (1934), and Davis (1935) in cases of chronic staphylococcic mastitis. Minett (1957 A) described histo- pathological changes in six udders affected with chronic micrococcic mastitis. In five of the udders he did not find gross pathological changes but in the sixth, one quar- ter was indurated and much of the tissue was brownish in color. Typical induration and cording within the udder tissue were reported by Gould (1942). Parshall (1954) and Schalm (1944) reported postpartum cases of gangrenous mas- titis in dairy cows due to a mixed infection of Clostridium welchi and staphylococci. They found the udders tense and l4 swollen. Within 24 to 48 hours the teats turned black or blue and this coloration extended upwards for a variable distance to involve the udder preper. In some cases there was gas formation and subcutaneous edema anterior to the udder. MicroscOpic pathology: Minett (1937 A) found that in certain areas the acini were filled with solid masses of neutrophils and that these cells also were infiltrating the interaciner septa, which was thickened as a result of increased fibroblastic activity. In other parts where the disease process was advanced, there was a large increase of connective tissue and atrophy of the secreting gland tissue. The histopathology of staphylococcic mastitis was described by Edds and Bieter (1953) as follows: In acute cases there was an active congestion with destruction of glandular tissue epithelium. The lumina of the alveoli were filled with coagulated masses of exu- date and clotted milk. Many of the alveoli contained des- quamated cells and polymorphonuclear leukocytes. The con- gestion was accompanied by lymphocytic infiltration into the area. Interlobular edema developed and the congested areas turned hard and were pink in color. In chronic cases there was an increase in the intralobular connective tissue which led to the ultimate disappearance of most of the al- veoli. l5 Antitoxins in blood and milk of cows Minett (1937 B) found that in cattle and goats there existed a direct relationship between the age of an animal and the antitoxin titer of the blood. He thought that the presence of micrococcic antitoxin in the blood of cowswas the result of previous infection of the udder with M3353- cococcus pyogenes. The incidence of the udder infections also increased with age. In milk whey from normal quarters, the amount of antitoxin found was 1/80th to 1/40th of that in the blood serum. In colostrum the antitoxin titer was higher than in the blood. The increase of antitoxin in the colostrum over that in the milk was due to an increase in the globulin. That staphylococcic antitoxin in blood serum was the result of staphylococcic infection past or present, was reported by Miller and Heishman (1943 B). The normal milk contained one unit or less of antitoxin per cc, whereas the glands with acute mastitis showed 16 to 32 units of antitoxin per co. in the diseased quarters. During arti- ficial exposure of the udder to infection no antitoxin was found in the blood but when in those cases the infection was stabilized, four to eight units of antitoxin developed in the blood within five weeks. Spencer and Lasmanis (1954) found that most cows showing mammary gland infection with Micrococcus pyogenes had two or more units of antitoxin per cc. of serum, whereas l6 noninfected cows had less than two units of antitoxin per cc. Staphylococcic toxoids Boak (1956) demonstrated that rabbits actively or passively immunized against staphylococcic toxins were more resistant than were normal animals to injections of living staphylococci. Immunity lasted for three months when Minett (1939) injected staphylococcic toxoid into the udder of sheep. Derbyshire (1960) vaccinated goats and cows with an adjuvent cell toxoid prepared from Staphylococcus aureus strain 201. A high level of immunity was conferred against experimental challenge with strain 201 and 106 but not against strain BB, N 90 and S 20 T, although the production of gan- grene by these strains was checked in vaccinated goats. Goats showed evidence of immunity to a massive in- tramammary test does of strain BB after they were exposed to living staphylococci of the same strain by Derbyshire (1961). This immunity appeared to be local in nature, since it was not apparent when the other half of the udder was challenged by a similar dose of staphylococcic organisms. Porterfield and Petersen (1959) reported that the bovine udder was a potent source of antibody production and that antibodies appeared in milk within two hours after 1actat1ng cows were infused with an antigen. The antibody contents of the blood serum and milk were higher 215 days following immunization. 17 Derbyshire (1961) has suggested the use of killed or living attenuated staphylococcic organisms in vaccines for use in the field. The use of toxoids for the treatment of subacute staphylococcic mastitis under field conditions was reported favorably by Gould (1942), Wilson (1942) and Roach (1945). Research workers from the University of New Hamp— shire (Anonymous, 1959) reported favorably on the use of a staphylococcic toxoid. Its use decreased the number of acute mastitis flare-ups in infected cows. The vaccine used by Fincher, Hodges, Johnson and Reed (1960) neither produced change in the incidence of mastitis nor reduced the severity and damage to the udder. They also reported the failure of immunity to develop in another herd where the vaccinated animals suffered a severe type of mastitis from hemolytic staphylococci. MATERIALS AND METHODS Cultures A staphylococci,, strain 71, used in this study was recovered from a cow showing acute clinical mastitis. This strain has been maintained by monthly transfers on tryptose agar slants and stored in a refrigerator at 5° C. The subcultures used were always alpha-beta hemolytic on bovine blood agar and did coagulate rabbit plasma. Actively growing 22 to 24 hour broth cultures that previously had been transferred for several consecutive days were used. Transfer of the organism tends to stimulate a constant, uniform, rapid and more vigorous growth. The cultures were incubated for 22 hours at 37° C, then diluted in 0.85% sodium chloride solution and infused immediately. At the time of dilution, poured blood agar plates were made for colony counts and 0.1 ml. of the di- luted culture that was injected was spread on the surface of a blood agar plate to give an approximation of the number of organisms injected. Concurrent with injecting the cul— tures, tests of the hemolytic and rabbit plasma coagulation abilities were made. Samples of milk were obtained for culture after carefully washing the udder with a bacteri- cidal agent. The milk samples were taken to the laboratory 18 l9 and plated for counts in less than five minutes after col- lection. Animals This study was carried out on four clinically healthy cows. All cows were five to six years old. Cows l and 2 were nearing completion of their lactation period, whereas cows 191 and 3 were in the middle of their lactation. Cows 1 and 2 were of the Guernsey breed, while the breed of cows 191 and 3 was Holstein. These animals were obtained from the Michigan State University Dairy Department. A detailed study was made on three cows, 1, 2 and 191. Cow 3 was used only for the study of multiplication of staphylococci in fresh raw milk. Upon palpation the udders of all cows were found clinically normal, without any swelling or hard indurated areas. The milk secretions from all quarters were normal in appearance when examined on a strip cup. Prior to experimentation numerous examina- tions were made for the presence of mastitis-producing or- ganisms from their udders. Two cows (2 and 191) were found shedding coagulase negative nonhemolytic staphylococci in the milk from all quarters. Coagulase negative nonhemolytic staphylococci were also found in milk from the right rear quarter of cow 1. Each individual quarter was considered as a separate unit (Turner, 1948) for artificial exposure to staphylococci. 20 Great care was taken to avoid contamination of other quar- ters, when another one was being exposed to infection. The animals were kept in separate stalls and fed a ration of good quality hay and grain. Infected and noninfected quarters were hand milked twice a day. Plan of the intramammary injections of staphylococcic cr- ganisms Three series of intramammary inoculations of staph— ylococcic organisms were made. During the first series of injections a low number of organisms varying from 16 x 102 to 28 x 102 were used. In the second series (moderate range) the number of organisms varied from 38 x 103 to 57 x 104. High number of staphylococcic organisms (72 x 107 to 27 x 109) were injected in the third series of inoculations. Each quarter was injected three times except the right rear quarter of cow 1 which was infused four times. All the quarters were successively injected with low, mod- erate and high numbers of staphylococcic organisms at inter- vals varying from ten days to two months. The right rear quarter of cow 1 was infused twice with a dose of staphy- lococcic organisms in the high range. The first of the two doses was administered when the cow was lactating and the second injection was repeated one month later when the cow was dry. Details regarding the numbers of the organisms in- jected into each quarter and the interval between the different 21 injections are listed in the appendix, table XIII. Intramammaryinjection technique The technique of intramammary injection was the same in each instance. The external surfaces of the quar- ters were thoroughly washed with warm aqueous chlorine solu- tion containing 200 parts per million of available chlorine and dried completely with muslin cloth. The end of the teat was disinfected with a cotton swab saturated with 70% alcohol. The experimental amount of diluted or undiluted culture was injected through the teat canal, just beyond the sphincter, with a sterile five or ten cc. syringe with a blunt pointed 24 gauge needle. The injections were usually made in the morning and cows were kept under constant ob- servation throughout the day. Before injection, the rectal temperature of the cow was recorded and a milk sample was taken from the quarter. After injection, milk samples were taken at three-hour intervals for the first nine hours and thereafter once daily for 10 to 15 days. Collection of milk samples Before collecting a milk sample the udder and the teats were cleaned as described above. The workers' hands were also washed and then rinsed with 70% alcohol before each milk sample was collected. About 20 m1. of milk was drawn from the desired quarter into a sterile vial. No milk was discarded before the sample was collected. The 1‘ 22 milk samples were promptly taken to the laboratory where the routine tests described below were performed. Agar pour plate method This test was carried out to find the approximate number of organisms infused or being shed in the milk. The basic media for the agar plate consisted of: l Bacto-Tryptose. . . . . . . 10 grams 2 Bacto-Beef Extract. . . . . 3 grams 3 Sodium chloride . . . . . . 5 grams 4 Bacto-Agar. . . . . . . . . 17 grams 5 Distilled water . . . . . . 1000 cc. Autoclaved at 15 lbs. pressure for 15 minutes. Just prior to plating, the agar media were liqui- fied by heating and cooled to 42° C and then poured over a measured quantity of milk on the petri dishes. When the agar became solidified, the petri dishes were kept inverted in the incubator for 24 to 48 hours. At these times the colonies were counted. Serial dilutions were made of cul- tures and milk so as to have count ranges from 30 to 300 colonies per plate. Culture test This test was used to find out the hemolytic char- acter of staphylococci injected in the quarter or shed in milk. One loopful (0.01 ml.) of each milk sample was streaked over one quarter of a blood agar plate. In the blood agar there was bovine blood in the concentration of 5% and agar in the concentration of 2%. Blood was obtained from a cow 23 of known hemolytic susceptibility. The blood plates were dried in the incubator overnight. This test was made by placing a loopful of the milk near the edge of the plate and drawing the 100p over the agar for a distance of two to three centimeters towards the center of the plate. One plate was used for the milk from four quarters from one cow. The inoculated plates were incubated at 37° C for 24 to 48 hours and the results were recorded. Coagulase test Bacto-coagulase plasma purchased from Difco Lab- oratories, Detroit, Michigan, was reconstituted with three m1. of distilled water. In each small sterile vial sup- ported in a rack, 0.5 ml. of the plasma solution was placed. Colonies were transferred with a sterile wire loop from the blood agar plate to the plasma solution. The vials were thoroughly shaken, incubated for 22 to 24 hours at 37° C and then the degree of clotting of the blood plasma was recorded in the following terms: 0 = No clot + = Floccules ++ = 50% clot +++ = Full clot Microscopic test The milk samples were incubated at 37° C for 24 hours before microscopic examination. Then 0.01 ml. of the incubated milk sample was smeared over a glass slide. r. 24 The smear was then dried in the air and stained by Newman's method. The microscopic test was undertaken to provide additional evidence of the presence of staphylococci and to ascertain the degree of leukocyte response. Test to determine in vitro multiplication of staphylococcic organisms (strain 21) This test was performed to find out the rate of multiplication of staphylococcic organisms in freshly drawn milk. Ten cc. of fresh raw milk were drawn from each quar- ter into a separate sterile small tube containing a glass bead. These tubes were inoculated with one loopful of an 18-hour broth culture of staphylococci, strain 71, which was diluted 100 times with 0.85% sodium chloride solution. Each tube was shaken before incubation, and at half-hourly intervals throughout the experiment. Contents of the tubes were withdrawn at various intervals and plated as described for the agar pour plate method. The number of colonies was counted after 24 hours of incubation at 37° C. RESULTS A. Challenge of bovine udders with a low range of staph- ylococcic organisms: » The right rear quarter of cow 1 and all the four quarters of cows 2 and 191 were shedding non-hemolytic co- agulase negative staphylococcus aureus in their milk before they were challenged with staphylococcic organisms, strain 71. Except for the right fore quarter of cow 1, all the other quarters (11 quarters) failed to show any clinical evidence of infection following exposure to staphylococcic organisms. No gross abnormality was observed in the milk from these quarters; however, in every instance the milk did contain non-hemolytic coagulase negative staphylococcic organisms. The injection of approximately 24 x 102 staphylo- coccic organisms resulted in acute clinical infection of the right fore quarter of cow 1 (appendix, table II). The infection later became chronic and lasted for about 10 days. Three hours following inoculation, congestion at the base of the teat was seen. During the next three hours, the quarter was swollen, firm and warm. The swelling of the quarter disappeared by the third day. Nine hours after the time of inoculation, the body temperature was elevated to 105.9° F but returned to 102.5° F within the next two hours. 25 26 Coagulase positive hemolytic staphylococcic organisms were cultured from the milk. No medicinal treatment was given to the cow. Summary All the quarters of three cows were challenged with a low range of staphylococcic organisms (16 x 102 to 28 x 102). These numbers of organisms failed to cause any clin- ical evidence of persistent infection except in the right fore quarter of cow 1 (appendix, table II). B. Moderate range of stapgylococcic organisms: When 38 x 103 to 57 x 104 staphylococcic organisms were infused into the quarters of cows 1 and 191, only the right rear quarter of cow 1 failed to show abnormal milk. At the time the exposures were made, mild trauma was ad- ministered to the udder by a kick. The milk from these quarters (seven quarters) showed shreds and flocculi on the strip cup screen. It was yellowish or dirty grey in color. This abnormality in milk lasted for four to six days. The quarters returned to clinical normality. Hemo- lytic coagulase positive staphylococci were found in the milk from these quarters. Two quarters of cow 2 (right fore and right rear) and one quarter (right rear) of cow 1 failed to show any clinical evidence of infection (appendix, tables IV, VI and VIII). 27 Acute clinical mastitis was seen in the two quar- ters (left fore and left rear) of cow 2 which lasted for four days (appendix, tables V and VII). Three hours after intramammary inoculation, swell- ing was observed in the left fore quarter of cow 2 extend- ing upwards from the base of the teat. Six hours after injection the body temperature was 104.0° F but returned to 102.l° F during the next three hours. On the second day following injection the swelling disappeared from the quarter. The milk continued to appear abnormal for four days and contained coagulase positive hemolytic staphylo- coccic organisms. The left rear quarter of cow 2 developed acute clin— ical mastitis which subsided without treatment after five days (appendix, table VII). The inflamed quarter felt firm and tense until the end of the third day. Six hours after inoculation the body temperature was raised to 105.2° F but returned to normal during the next three hours. Coag- ulase positive hemolytic staphylococci were cultured from the milk of this quarter. Summary All three cows were reexposed to approximately 38 x 103 to 57 x 104 staphylococcic organisms. Mild phys- ical trauma by kicks was applied to the injected quarters. Only two quarters showed clinical response to this moderate range of staphylococcic organisms. 28 C. High range of staphylococcic organisms When cow 1 was in lactation, her right rear quarter was infused with 13.5 x 108 staphylococcic organisms. She developed acute clinical mastitis (appendix, table IV). There was swelling of the quarter for two days and the milk was abnormal for five days. Six hours after inoculation, the body temperature was 105.4° F. It returned to normal within the next three hours. Coagulase positive hemolytic staphylococci were cultured from the milk of this quarter. Approximately the same number of staphylococcic organisms did not produce any clinical evidence of infection when injected 25 days previously (appendix, table IV). Except for the right rear quarter of cow 191, all quarters developed an acute clinical mastitis lasting for about five days (appendix, tables IX to XII). The right rear quarter of this cow secreted milk for three days which was yellowish in color, and contained many flocculi. The left fore quarter of cow 191 developed an acute clinical mastitis (appendix, table IX). Milk from this quarter was yellowish in color and contained many pus flocculi. The swollen quarter felt quite firm and warm upon palpation. Four hours after inoculation the body temperature was 104° F but returned to 102.5° F within the next three hours. Hemo- lytic coagulase positive staphylococcic organisms were cul- tured from the milk. 8 Approximately 11.2 x 10 staphylococcic organisms 29 caused an acute mastitis in the right fore quarter of cow 191. Five days following exposure there was no clinical evidence of infection even though treatment was not used (appendix, table X). The left rear quarter of cow 191 developed clinical mastitis which lasted for five days (appendix, table XI). The quarter was hard, swollen and sensitive to the touch. Six hours after inoculation the body temperature was 104.0° F and then raised to 106° F during the next three hours. The swelling of the quarter disappeared next day. The milk was yellowish white and contained many flocculi. The ab- normality lasted for five days. The dry cows (1 and 2) did not show any clinical response to these numbers of staphylococcic organisms (ap- pendix, tables 1 to VIII). Summapy Three cows were challenged with intramammary injec- tions of staphylococcic organisms ranging in number from 72 x 107 to 27 x 109. One quarter of one cow was inocu- lated twice with approximately the same numbers of organ— isms at an interval of 25 days. Four of the 13 quarters gave clinical evidence of acute infection. DISCUSSION Comparatively few references to the artificial in- troduction of small numbers of staphylococcic organisms into the udder have been found in the literature. Most of the work deals principally with the infusion of a rela- tively large number of organisms in contrast to the small number of organisms used in this work. Severe mastitis with systemic involvement followed injection of Staphylo- coccic aureus into the udders of two cows and three heifers by Carpenter (1922). He introduced two cc. of 24-hour broth culture of organisms recovered from a case of clinical mas- titis. Four animals reacted severely showing high tempera- ture with loss of appetite for 36 hours following the inocu- lation. Ten days later, one of the cows died from staphy- lococcic septicemia and at necrOpsy staphylococcic organ- isms were isolated from all the tissues of the body. In the other three animals abscesses appeared in the udders. A two ml. suspension of fresh culture isolated from a nat- ural case of gangrenous mastitis was injected in two cows by Parshall (1934). The quarters became swollen and hot and the milk contained many clots. The reaction was very mild and gradually subsided. Little and Folly (1935) injected from 200 to 2500 30 31 staphylococcic organisms through the teat meatus on the small, beaded end of a glass rod on nine different occas- ions over a period of about five weeks. Acute mastitis was seen on the second day after the last injection. The above staphylococcic organisms for the intramammary infusion were isolated from an abscess found on the skin of the udder. The quarter was swollen, congested and painful to manipula- tion. The secretion was scanty, yellowish and thick. Minett (1936) was able to establish Staphylococcus aureus in one quarter with two exposures nine days apart, using 1 m1. of a 24-hour broth culture for each exposure. The infection persisted for some months and into the next lactation, after which time it disappeared. Twenty-six quarters of seven lactating cows and one dry cow were injected by Miller and Heishman (1943 A) with a small number of organisms varying from 700 to about 30,000 on 55 occasions. Twenty-five attempts out of 55 resulted in infection of 22 quarters. Some variation was found in the ability of the different strains of staphylococci to establish and maintain themselves in the udder. Differences also appeared in the susceptibility of the quarters to in- vasion by these organisms. With some strains repeated ex- posures were necessary to bring about infection and in these cases, a mild type of mastitis of a rather short duration developed. With other strains, infection occurred after one or two exposures and chronic mastitis of a persistent 32 nature was established. Schalm (1944) was able to produce gangrenous mas- titis in two lactating cows following the injection of five ml. of pure broth culture of Staphylococcus aureus, while two dry cows receiving similar amounts developed a transi- tory mastitis. He was of the opinion that lactating udders were more vascular and the gangrenous process probably was the result of injury to the vascular system of the udder. Slantez and Bartley (1955) found that 5 x 107 staph- ylococci were required to set up an acute infection. One cow was able to eliminate the infection after nine days. Drury (1962) injected large numbers of staphylo- coccic organisms of strain 80/81 into the udder and demon- strated the continual shedding of staphylococci in milk for four months following exposure. In this work with small numbers of organisms none of the quarters, except the right fore quarter of cow 1 showed any clinical evidence of mastitis. This particular quarter developed acute mastitis when injected with 24 x 102 staphylococcic organisms. The ability to establish a mas- titis might not be solely due to the organisms but might be due to unknown factors which play an important part in increasing the mastitis-producing potential of the bacteria. This may be further evidence that there are differences in the susceptibility or resistance of an individual quarter to infection with staphylococci. Little and Folly (1935) 35 and Miller and Heishman (1943 A) were also unable to pro- duce clinical mastitis with small numbers of organisms. They maintained that repeated exposures with large numbers of organisms were necessary to produce clinical evidence of mastitis. In this project,the second series of injections (12 quarters) with staphylococci in the range of 38 x 103 to 57 x 104, two quarters (left fore and left rear of cow 2) developed a mild clinical form of mastitis which lasted for four days. In seven quarters the milk secretion was of abnormal color containing many flocculi. The abnormal color of milk lasted four to six days. Stress and trauma might have played some signifi- cant part in causing clinical symptoms of mastitis in the two quarters of cow 2. However, the effect of stress or trauma is difficult to evaluate. During lactation, cow 1 developed acute mastitis following exposure to 13.5 x 108 staphylococcic organisms but failed to do so when injected with nearly the same num- bers of organisms during the dry period. This failure may be explained by increased resistance, or decreased vascu- larity of the udder,as suggested by Schalm (1944). In this work the first exposure, using a small num- ber of organisms, might have produced some immunity against the next higher dose of staphylococci. It is possible that the two cows (2 and 191) which were shedding nonhemolytic 34 coagulase negative staphylococci before inoculation might have developed some immunity. From udders considered clinically normal, staphy- lococci may be shed without showing clinical infection. Coagulase positive hemolytic staphylococci may be respon- sible for subclinical infection in these udders. Due to the antigenic nature of staphylococci, they may possibly cause formations of antibodies which protect against further attacks of staphylococci. Whenever the resistance of the udder is lowered by unknown factors or if overwhelming num- bers of staphylococcic organisms are introduced, clinical mastitis may develop. Shedding of staphylococci in the milk Staphylococci may constitute an important inhabitant of the apparently normal udder and may be shed in the milk from these udders. The numbers of staphylococcic organisms in each quarter may vary from the fore milk to the strip- pings and there may also be a variation in the daily counts. Minett (1937 A) reported that there was an enormous variation in the count from time to time even when the milk samples were obtained at short intervals. On occasion he found less than ten colonies of staphylococci on the first plate, while from the same cow two days later there were innumerable colonies. Little and Folly (1935) counted staphylococci from a clinically sound udder for a period of 31 days. They 35 found that staphylococcic counts varied between 10,000 to 20,000 per ml. of milk. In this work higher and more constant counts were observed in cow 1 than in cows 2 and 191. Minimum and max- imum staphylococcic counts were in the range of 1 x 103 to 1.7 x 106 per ml. of fore milk. The counts varied from 1 x 103 to 5 x 104 per ml. of fore milk. The number of staphylococcic colonies from the milk of cow 1 varied slightly from each quarter each day. In the other two cows (2 and 191) more variable counts were found from each quarter each day. This may be explained as biological phenomena. There appeared to be no definite pattern to the daily variations in numbers of staphylococcic organisms shed in the milk. Effect of stress or trauma According to McDonald and White (1961), hemolytic coagulase positive staphylococcic organisms were found in abundance even after the animals had recovered from clin- ical mastitis. Many workers doubt whether staphylococci are really the cause of infection or whether some unknown factor is responsible. Factors such as stress and trauma were suggested by Schalm (1959) as having the effect of increasing the susceptibility of the udder to infection. With this in mind, mild kicks were given to the quarters when they were injected for the second time. Only two quarters (left fore and left rear) of cow 2 developed a mild form of clinical mastitis. It is not clear whether ”*1.— 36 trauma made the udder more susceptible to infection or whether the infection was due to the greater number of staphylococci injected. Multiplication of staphylococci in fresh raw milk Many workers have commented on the bactericidal property of raw milk. It is well known that when certain bacteria are introduced into fresh raw milk their number declines for a time and subsequently increases. Hunziker (1901) was apparently the first investigator who showed a marked decrease in numbers of bacteria when they were intro- duced into the milk from some cows, whereas in others, the bactericidal effect was not noted. He observed only the action of bovine milk on its own milk flora and used no pure culture. It was demonstrated by Chambers (1920) that milk contained a definite bactericidal constituent which is destroyed by heating at 80° C or 90° C for two minutes. He found that the inhibitory action wee specific and de- pended on both the cow and species of the bacteria employed. He also pointed out that lactic acid type of organisms were not inhibited in milk. Hassen (1924) found that fresh raw milk would inhibit the growth of Bacillus typhosus and Bacillus paratyphosus for one to four hours at 37° C. He further showed that there were periodic variations in the ability of a cow's milk to inhibit bacterial growth. He attributed this variation to the ration, since he thought 57 the bactericidal agent was an oxidizing enzyme which sup- posedly originated in the food and that its concentration in the milk was dependent upon the concentration in the ration. According to Jones and Little (1927), the concen- tration of the inhibitory substance varied in the secretions of different animals. It might be as concentrated in a young cow early in her first lactation period as in an older or more resistant animal which has been repeatedly exposed to infection with streptococci. The bactericidal activity of fresh raw milk from a number of cows was tested with nonhemolytic mastitis streptococci. It was reported that there was a sharp decline in the number of organisms in the first four hours of incubation. After this, there was a rapid multiplication particularly in cases of young cows and cows susceptible to mastitis. In cases of resistant cows, there was no multiplication during the first eight hours of incubation. They added that this substance did not originate in food, but was in the udder, and that it did not increase when the cows were artificially immunized or repeatedly exposed to natural infection. In this work the multiplication of hemolytic coag- ulase positive staphylococcic organisms (strain 71) was studied in the fresh raw milk from cows 191 and 3. It was observed that in milk from cow 191 there was a sharp decline in the numbers of staphylococcic organisms after the first 58 three hours of incubation. After this, there was a slight increase for two hours, followed by rapid multiplication at 24 hours. In cow 3 there was a definite decrease in the numbers of staphylococcic organisms for the first three hours, followed by a phase of rapid growth (see graphs on pages 39 and 40). These results are quite similar to those that have been reported by Jones and Little (1927) for strep- tooocci. I'll se III II III. -II' III-II'IIIIII lllel II eel Staphl [Colonies in Thousands .an _ A40 GRAPH SHOWING GROWTH OF STAPHYLOCOCCIC ORGANISMS STRAIN NO: 71 IN FRESHLY DRAWN MILK (10 CC.) FROM COW NO. 191 i , T" 70- m c l g: I ~ 2; l ’1 need * at Time Hours SUMMARY Coagulase positive hemolytic staphylococci of strain 71, recovered from a clinical case of mastitis, were injected into all quarters of three cows at various intervals. Ex- cept for one quarter, which was injected four times, all the otherwquarters were infused three times with increas- ingly greater numbers of organisms at intervals of ten days to two months. For the first series of injections, small numbers 2 to 28 x 102) were infused into each of organisms (16 x 10 quarter. Only one of the 12 quarters developed an acute clinical mastitis. This subsided in 12 days without treat- ment. For the second series of intramammary injections, 38 x 103 to 57 x 104 staphylococcic organisms were intro- duced into each quarter. In addition, each quarter was mildly traumatized by physical force at the time of the injection. Two of the 12 quarters developed acute clinical mastitis. From seven additional quarters abnormal milk was obtained over periods ranging from four to five days. The remaining three quarters did not show any evidence of clinical mastitis. For the third series of intramammary injections, 41 o. I, 42 approximately 1 x 1010 staphylococcic organisms were used. Four of the 13 quarters gave clinical evidence of acute infection. Staphylococci counts from milk were made for 10 to 15 days after each intramammary injection of staphylo- coccic organisms. In one cow the counts were high (5 x 104) and less variable,while in other cows the counts varied from 1 x 103 to 5 x 103. The staphylococcic strain 71 used in this work was incubated in fresh raw milk. Milk from all four quarters of one cow inhibited growth of the organisms for eight hours, after which time they multiplied rapidly. Fresh raw milk from another cow inhibited multiplication for three hours only. Conclusion 1. The response of bovine udders to the injections of ap- proximately the same numbers of coagulase positive staphylococci will vary from quarter to quarter and from cow to cow. Clinically, the response ranged from no evidence of mastitis to an acute mastitis of short duration. 2. Coagulase positive hemolytic staphylococci may be ap- parently harmless inhabitants of bovine udders and may be shed in milk which grossly appears to be normal. 3. There appears to be no definite pattern to the daily variations in numbers of staphylococcic organisms shed in the milk. BIBLIOGRAPHY Adler, H. E., and Migaki, H. 1951: Cell Type in Milk of Cows in a Chronic Staphylococcal Mastitis Herd. Vet. Med., 46,:89. Albiston, H. E. 1930: Actinomycosis of the Mammary Gland of Cow in Victoria. Austral. Vet. J., 6,:2. Anonymous. 1959: Mastitis Treatment. Vet. Med., 54,:20. Bean, 0. W., Miller, W. T., and Heishman, J. 0. 1943: Chemotherapy of Streptococcal Bovine Mastitis. Am. J. Vet. Res., 14,:544. Boak, W. C. 1956: Antistaphylocoagulase in Experimental Staphylococcal Infection. J. Immunol., 76,:89. Bryce, L. M., and Rountree, P. M. 1936: The Production of Beta Toxin by Staphylococci. J. Path. and Bact., 45.8175. Burrows, William. 1959: Text Book of Microbiology. 17th ed. W. B. Saunders Co., Philadelphia and London,: 598- Carpenter, C. M. 1922: Experimental Production of Bovine Mastitis with Streptococci and Other Bacteria. J. Infect. Dis., 31,:1. Carpenter, C. W. 1925: The Bacterial Count of Milk or Inflammatory Exudates from Bovine Mastitis. J.A. V.M.A., 67,:317. Chambers, H. W. 1920: (Bacterial Inhibition) Germicidal Action in Milk. J. Bact., 5,:527. Chapman, G. H., and Berens, C. 1934: Coagulase and Hemoly- sine Test as a Measure of the Pathogenicity of Staph— ylococci. J. Bact., 28,:343. Chapman, G. H., and Berens, C. 1938: The Differentiation of Pathogenic Staphylococci from Nonpathogenic Types. J. Bact., 35,:311. 45 44 Cruickshank, Robert. 1937: Staphylocoagulase. J. Path. and Bact., 45,:295. Davidson, Ian. 1961: Observation on the Pathogenic Staph- ylococci in a Dairy Herd During a Period of Six Years. Res. Vet. Sci., 2,:22. Davies, G. O. 1935: Actinomycotic Mastitis. Vet. Rec., 15,:15. Derbyshire, J. B. 1960: Immunological Studies in Experi- mental Staphylococcus Mastitis. J. Comp. Path. and Therap., 70,:222. Derbyshire, J. B. 1961: Further Immunization of Goats Against Staphylococcus Mastitis by Means of Experi- mental Infection of the Skin and Udder. Res. Vet. Sci., 2,:112. Dubeler, M. J., and Cole, E. J. 1956: Studies on Micro- coccal Mastitis in an Individual Herd. Vet. Med., 51,:113. Drury, A. R. 1962: The Response from Artificial Introduc- tion of Staphylococcus aureus, Phage Type 80/81 into the Bovine Udder Followed by Treatment with Various Antibiotics. Am. J. Vet. Res., 23,:262. Edds, G. T., and Bieter, R. N. 1953: Effects of Certain Drugs Alone on Micrococci Isolated from the Bovine Udder. Am. J. Vet. Res., 14,:355. Elek, D. Stephens. 1959: Sta h lococcus o enes and its Relation to Disease. E. and S. Livingstone, Ltd., Edinburgh and London. Elek, S. D., and Levy, E. 1950: Distribution of Hemoly- sine in Pathogenic and Nonpathogenic Staphylococci. J. Path. and Bact., 62,:541. Evans, A. C. 1916: The Bacteria of Milk Freshly Drawn from Normal Udders. J. Inf. Dis., 18,:437. Fincher, M. G., Hodge, H. D., and Johnson, S. D. 1960: Staphylococcal Toxoid Bacterin in the Control of Bovine mastitis. Report of N.Y. State Vet. College. Cornell University, 1959-1960. Glenny, A. T., and Stevenes, M. F. 1935: Staphylococcal Toxin and Antitoxin. J. Path. and Bact., 40,:2017. 45 Gould, G. N. 1942: Use of Toxoid in the Field. Vet. Rec., 54,:106. _ Harding, H. A., and Wilson, J. K. 1913: Study of Udder Flora of Cows. N.Y. State Agric. Exper. Sta. Tech. Bull. No. 27. Hassen, F. S. 1924: Bactericidal Property of Milk. Brit. J. Exper. Path., 6,:271. Hastings, E. G., and Beach, B. A. 1937: The Production of Milk of Abnormal Composition by Animals Free from Udder Streptococci. J. Agric. Res., 54,:191. Hastings, E. G., and Johns, C. K. 1939: The Relation Be- tween Bacterial Numbers and Biochemical Values in Milk from Streptococci Free Udders. J. Agric. Res., 53’ :543e Hucker, G. J. 1924 A: Studies on Coccaceae. Previous Taxonomic Studies Concerning the Genre of Coccaceae. N.Y. State Agric. Sta. Tech. Bull. No. 99. Hucker, G. J. 1924 B: Studies on Coccaceae. Classifica- tion of the Genus Micrococcus. N.Y. State Agric. Sta. Tech. Bull. No. 102. Hucker, G. J., and Uddal, D. H. 1933: Relation Between the Presence of the Fibrotic Tissues in the Udder and Streptococci or Cells in Freshly Drawn Milk. Cornell Vet., 23,:32. Hunziker, O. F. 1901: Investigations Concerning Germicidal Action in Cows' Milk. N.Y. Cornell Sta. Tech. Bull. No. 197. Jones, F. S. 1918: Studies on Bovine Mastitis. Infection of the Udder with Micrococci and Other Organisms. J. Expt. Med., 28,:721. Jones, F. S., and Little, R. B. 1927: The Bactericidal Property of Cows' Milk. J. Expt. Med., 45.:319. Jordan, E. 0. 1930: The Production by Staphylococci of a Substance Causing Food Poisoning. Amer. J. Med. Assoc., 94,:1648. Litterer, W., and Crabtree, J. A. 1934: Outbreak of Milk Poisoning Due to a Toxin Producing Staphylococci Found in the Udders of Two Cows. Am. J. Pub. Health, 24,:1116. 46 Little, R. B., and Folly, E. J. 1935: Staphylococci As- sociated with Mastitis. J.A.V.M.A., 87,:637. Marks, J., and Vaughan, A. C. T. 1935: Staphylococcus Delta Hemolysine. J. Path. and Bact., 62,:597. McDiarmid, Archibald. 1947: The Carriage of Sta h lococcus aureus in the Milk and on the Tests of NormaI Cows. Vet. Rec., 59,311. McDonald, 1., and White, F. 1961: Some Observations on an Outbreak of Staphylococcic Mastitis in a Herd. J. Com. Path. and Therap., 71,:159. Miller, W. T. 1936: Bovine Mastitis. North. Am. Vet., :32. Miller, W. T., and Heishman, J. O. 1943 A: Staphylococcus Bovine Mastitis Artificial Exposure of Bovine Udder with Staphylococcus aureus. Am. J. Vet. Res., 4,:318. Miller, W. T., and Heishman, J. O. 1943 B: Staphylococcal Antitoxin in the Blood, Milk and Colostrum of Cows. Am. J. Vet. Res., 4,:265. Minett, F. C., and Edwards, S. J. 1929: Studies on Bovine Mastitis. The Bacteriology of Mastitis. J. Comp. Path. Therap., 42,:213. Minett, F. C. 1936: Toxin Production with Particular Ref- erence to Staphylococci from Animals. J. Comp. Path. and Therap., 49,:247. Minett, F. C. 1937 A: Mastitis Due to Staphylococci. Studies on Bovine Mastitis. J. Comp. Path. and Therap., 50,:101. Minett, F. C. 1937 B: Staphylococcal Antitoxin in Blood and Milk of Cows and Other Animals. J. Comp. Path. and Therap., 50,:173. Minett, F. C. 1939: Prevention of Bovine Mastitis by the Use of Staphylococcal Toxoid. J. Comp. Path. and Therap., 52,:168. Mhrphy, J. M. 1943: The General Effect of Staphylococcal Infection on the Composition of Strict Fore Milk. Cornell Vet., 33,:52. Newberene, J. W., and Mayer, K. 1960: Bacteriological Survey of Milk Samples from 582 Problem Mastitis Herds. The Allied Vet. (Nov.-Dec.). l- 47 Newbould, F. H. S. 1960: Mastitis a Problem for Research and Education. Biochem. Rev., 30,:3. Packer, R. A. 1952: Six Years of Laboratory Diagnosis of Mastitis in Iowa. North. Am. Vet., 33,:777. Parshall, J. G. 1934: Natural and Experimentally Produced Gangrenous Mastitis in Cows. Cornell Vet., 24,:146. Peterson, E. G., and Hastings, E. G. 1938: The Pathology of Nonspecific Mastitis and Consideration of Pos- sible Etiological Agent. Cornell Vet., 28,:307. Plastridge, W. N., and Anderson, E. O. 1934: The Strep- tococci of Chronic Bovine Mastitis. Storrs. Agric. Expt. Sta. Bull. No. 195. Plastridge, W. N., and William, L. F. 1936: Observation on Staphylococci Associated with Bovine Mastitis. J. Bact., 31,:88. Plastridge, W. N., Anderson, E. 0., and Weirether, F. J. 1939: Infected Bovine Mastitis. Characteristics of Udder Staphylococci. Storrs. Agric. Expt. Sta. Tech. Bull. No. 251. Porterfield, I. D., Petersen, W. E., and Campbell, B. 1959: Antibody Response of the Udder. Vet. Med., 54,:1. Roach, R. W. 1945: Mastitis and the Practitioner. Vet. Rec., 57,:25. Rossel, J. M., and Miller, W. T. 1933: A Biological and Bacteriological Study of Mastitis. J.A.V.M.A., 82,:587. Schalm, O. W. 1944: Gangrenous Mastitis in Dairy Cows. Vet. Med., 39,:279. Schalm, O. W. 1952: Effect of Massive Doses of Penicillin or Dehydrostreptomycin Employed Singly or in Com- bination on Staphylococcic Infection in Mammary Glandse Me Je Vets Rees, 13,:26e Schalm, O. W., and Woods, M. G. 1953 A: The Mastitis Com- plex. J.A.V.M.A., 122,:462. Schalm, O. W., and Woods, M. G. 1953 B: Micrococcus pyo- genes in Bovine Milk. Identification. Am. J. Vet. Res., 149 3530. 48 Schalm, O. W. 1959: Mammary Glands in Health and Disease. Students' Book Store, California: 393. Slantez, L. W., and Howe, A. F. 1945: Characters of Staph- ylococci and Staphylococcal Toxins. Uni. New Hamp- shire Agri. Expt. Sta. Tech. Bull. No. 84. Slantez, L. W., and Bartley, C. H. 1953: The Diagnosis of Staphylococcus Mastitis with Special Reference to the Characteristics of Mastitis Staphylococci. J. Infect. Dis., 55,:139. Smith, H. 1934: Two Cases of Actinomycosis Mastitis. J.A.V.M.A., 84,8635. Smith, W., Hale, J. N., and Smith, M. N. 1944: The Role of Coagulase in Staphylococcus Infection. J. Expt. Paths, 28,5570 Smith, M. L., and Price, A. 1938: Staphylococcus Gamma Hemolysine. J. Bact., 47,:379. Spencer, G. R., and Lasmanis, J. 1952: Reservoir of In- fection of Micrococcusppyogenes. Am. J. Vet. Res., 13,:500. Spencer, G. R., and Lasmanis, J. 1954: Antibodies for Hemolysine of Mgcrococcus pyogenes in Whey and Serum of Dairy Cattle. Am. J. Vet. Res., 15,:517. Spink, W. W., and John, R. P. 1940: The Bactericidal Power of Blood from Patients and Normal Controls for Staph- ylococci. J. Immunol., 38,:383. Turner, C. W. 1948: Machine Milking Efficiency as Influ- enced by Normal Formation of Cow's Udder. Babson Bros. Co., Chicago, 1,:1. Veilla, D. M., Delia, M., and Faber, E. J. 1947: A Tech- nique for the Isolation of Coagulase Producing Staph- ylococci from Milk in Bovine Mastitis. Am. J. Vet. Res., 8,:275. Ward, A. R. 1900: The Invasion of the Udder by Bacteria. N.Y. Cornell Agric. Expt. Sta. Tech. Bull. No. 178. Wilson, H. M. 1942: Use of Staphylococcal Toxoid in the Field. Vet. Rec., 54,:137. William, R. E. 0., and Harper, J. G. 1947: Staphylococcus Hemolysine on Sheep Blood Agar with Evidence of a Fourth Hemolysine. J. Path. and Bact., 59,:69. 0‘ APPENDIX 50 TABLE I LEFT FORE QUARTER OF COW 1 After intramammary infusion of 1600 staphylococcic (strain 71) organisms Date Time Rectal N0. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /ml. Milk Quarter Milk 7/10 0 102.5 -- 0 - Normal Normal 3 101e6 le5* O " II N 6 101.3 0.8 " " 9 100.9 __ n H 7/11 101.0 1.7 O - " " 7/12 101.3 4.8 " " 7/13 100.6 20 " " 7/14 101.4 5.6 0 - " " 7/15 101.7 60 " " 7/17 101.2 165 " " 7/18 100.0 80 O - " " 7/19 101.6 830 " " 7/20 101.3 80 " " 7/22 100.4 72.5 0 - " " 7/24 101.0 43 " " 7/25 101.5 62 O - " " 7/26 101.3 67 " " 2(28 100.9 75 " " 40 x 10 staph. and trauma 8/25 0 100.5 560 +++ + Flocculi Normal 3 101.4 480 ' 6 101.6 720 " " 9 100.9 1760** ++ + " " 8/26 101.4 612 " " 8/27 101.3 830 n " 8/29 100.8 240 ++ + Normal 3 8/30 101.1 580 u n 9/1 100.6 120 n n 9/2 101.2 720 + + n " 9/3 101.5 450 n n 9/4 101.4 700 n n 9/6 100.7_ 440 ++ + 27.6 x 105 staph. 10/15 0 101.5 N0 count*** Normal Nonmal 6 100.7 N n 9 101.4 *Recorded in thousands **Highest count ***Dry cow .- “a..- -1 o- -. 4. e. *4- 51 TABLE RIGHT FORE QUARTER OF COW 1 After infusion of 2400 staphylococcic (strainll) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /ml. Milk Quarter Milk 7/15 0 101.6 -- O - Normal Normal 3 101.5 4.8* + + Yellow Congestion, 6 103.5 6.5 white swollen, 9 105.9 10 with firm and 11 102.5 8 flocculi.adematous. 7/16 102.0 5.4 + + Yellow Slightly 7/17 101.4 25 serum- swollen. 7/18 110.9 35 like Normal 7/19 101.7 4 + + with " 7/20 100.6 32 flocculi. " 7/21 101.4 150 + + " " 7/22 102.3 61 ” " 7/25 100.9 71 " " 7/25 100.5 60 " " 7/27 100.6 52 + + Normal " 7/28 101.5 130 " " 7/29 101.3 145 " " 7/30 100.7 240 O + " " 8/1 101.5 880 " " 8/2 101.1 120 " " 8/4 101.4 105 + + " " s/5 101.6 96 " " 8/7 100.8 147 + + " " 8/10 101.4 165 " " 8/12 101.2 88 fl " 8 1 100.6 105 ' " 55 x 103 staph. and trauma 9/10 0 100.5 50 ++ + Dirty Normal 3 100.8 640 grey n 6 101.3 80 with n 9 101.9 720 ++ + flocculi. " 9/11 100.9 150 " " 9/12 101.2 56 + + 3 a 9/14 100.7 176 9/15 101.5 140 + + n H 9/17 101.3 191 " " 9/19 101.7 192 + + Normal " 9/20 101.1 112 ” " 9/22 100.8 173 + + " " 9/23 101.4 115 " " 9/25 101.2 450 + + " " c 4.... ”a- “— .. -u- 52 TABLE II--Continued 12 x 107 staph. organisms 10/28 3 101.3 Cow was dry Normal 6 100.9 " ” 9 101.2 ” " *Recorded in thousands 53 TABLE III LEFT REAR QUARTER OF COW 1 After infusion of 2600 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /ml. Milk Quarter Milk 7/25 0 101.2 -- - - Normal Normal 3 101.2 3.4* + + ' ' 6 102.2 1.4 9 101.2 13 7/27 100.8 80 0 + " " 7/28 101.2 9.6 7/29 101.5 12 O - " " 7/50 100.9 58 7/31 101.5 56 O - " " 8/2 100.7 87 8/4 101.4 4.8 O - " " s/5 101.1 19 8/6 101.2 7.8 O - ” " 8/8 100.9 180 8410 101.3, 67. 0 - " " 50 x 103 staph.§nd mild trauma 9/20 0 101.5 3.2 0 - Normal Normal 3 101.4 46 ++ + Straw color 6 101.7 80 Flakes and ” 9 101.2 425 ++ + clots. " 9/21 100.6 120 9/22 101.3 456 ++ + " " 9/24 100.9 396 9/25 101.2 126 + + " " 9/26 100.7 422 9/27 101.6 675 + + Normal ” 9/28 101.2 322 9/30 101. 3 13 + + I! u lO/l 101.2 590 10/2 100.9 386 + + " " 10/5 101.2 425 10/5 100.9 130 + + " " 17.5 x 108 staph. organisms 11/2 0 101.5 - No c0unts** " 3 100.9 " 6 101.2 " 9, 101.4 " *Recorded in thousands **Cow was dry 54 TABLE IV RIGHT REAR QUARTER OF COW 1 After infusion of 2600 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 7/30 0 101.3 2.8* O — Normal Normal 3 101.2 7.8 6 101.5 12 + + ” " 9 100.9 9.6 7/31 101.4 14 + + " " 8/1 100.8 52 8/2 101.3 180 O — " " 8/3 101.4 70 8/5 100.6 12 0 — " " 8/6 100.9 24 8/7 101.2 9.4 O - " " 8/8 101.5 12 8/10 101.2 8.9 8/12 101.3 72.7 0 - " " 8/13 101.2 44 8/15 100.9 22.1 0 - " " 40 x 103 staph. organisms and mild trauma 10/1 0 101.2 - 3 101.5 44 ++ + Normal Normal 6 100.6 68 9 100.9 120 10/2 101.5 86 ++ + " " 10/3 100.5 240 10/4 101.4 58 10/5 101.3 146 ++ + " " 10/6 100.7 88 10/8 101.5 57 +4 + n n lO/lO 101.4 54 13.5 x 10 staph. organisms 10/11 0 101.4 97 + + Normal Normal 3 101.2 184 +++ + Yellowish Swollen, 6 105.4 780 white with firm. 9 102.5 1130 +++ + flocculi. " 10/12 101.8 540 10/13 100.9 772 +++ + " Normal 10/15 101.2 1270 10/16 102.1 662 ++ + " " 10/17 100.5 780 + + Normal " 10/18 100.9 449 10/20 101.5 768 ++ + " " 55 TABLE IV--Continued 16 x 10é staph. organisms 11/5 0 100.6 - ** Normal 3 100.9 6 100.8 9 101.5 *Recorded in thousands **The cow was dry 56 TABLE V LEFT FORE QUARTER OF COW 2 After infusion of 2800 staphylococcic organisms (strain 71) Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /ml. Milk Quarter Milk 8/22 0 101.5 1.8* 0 - Normal Normal 3 101.2 6.0 6 101.3 4.2 O - ” " 9 101.1 10.0 8/23 100.9 2.8 O - " " 8/26 101.3 0.9 8/28 101.5 2.0 O — " " 8/29 100.9 6.1 8/30 101.2 5.5 0 - n w 911 101.5 0.8 0 - " " 240 x 103 staph. organisms and mild trauma 9/20 0 101.2 - Normal Normal 3 101.2 160 ++ + Yellowish Inflam— 6 104.5 80 white mation 9 . 102.1 95 with extending flocculi. upwards. 9/21 101.4 50 +++ + ” Swelling disappeared. 9/22 100.9 70 " Normal 9/23 101.2 4.0 ++ + " " 9/25 102.1 3.0 Normal 9/27 101.4 1.0 ++ + " " 9/50 101.2 0.09 10/1 100.9 10 + + " " 10/2 101.5 11 10/5 100.7 23 + + " " 10/8 101.3 2.6 " " lO/9 101.2 1.4 O + " " 10/11 100.6 5.0 + + " " 2711 109 staph. organisms . 10/19 0 100.9 No count Cow was dry Normal 3 101.0 6 101.3 9 p100.7 *Recorded in thousands - - , .- - 4 I , - - - .4 . p-.. . . . . A - . , fl - .. u . -. . — ,_ . . - g u p- . - 4 . . - - - .1--- --u» . -e -. - e 4 r. . .- -A‘- 57 TABLE VI RIGHT FORE QUARTER OF COW 2 After infusion of 2400 staphylococcic organisms (strain 71) Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 8/27 0 100.9 2.2* - 0 Normal Normal 3 101.2 305 I I! 6 101.5 2.7 O + ” " 9 101.5 2.9 8/28 100.7 8.8 0 + " " 8/29 101.3 12 8/30 100.9 4 o - " n 9/1 101.5 96 9/2 100e9 le2 O - H II 9/4 101.4 12 9/5 102.0 1.3 0 - " " 100.9. 3.7 44 x 103 staph. organisms and mild trauma 9/27 0 101.3 1.2 0 - Normal Normal 3 101.2 96.0 ++ + n " 6 102.2 56 9 101.5 112 ++ + " " 9/28 101.2 86 9/29 101.1 65 ++ + " " 9/30 100.9 7.6 10/1 101.4 3.7 + + " " 10/2 100.6 9.6 10/4 101.5 52 + + " " 10/5 101.7 4.5 10/6 100.9 18 10/8 101.3 7.6 + + " " 10(10 102.2 11.7 18 x 10 staph. organisms 10/25 0 101.3 No counts ** Normal Normal 3 101.2 6 101.5 19 100a9 *Recorded in thousands **Cow was dry -“ - *fl‘. - . I . ._ 58 TABLE VII LEFT REAR QUARTER OF COW 2 After infusion of 2100 staphxlococcic (strain 7ll_organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1 Milk Quarter Milk 9/4 0 100.9 1.4* 0 - Normal Normal 3 101.3 2.7 6 101.2 1.8 O + " " 9 101.5 3.1 9/5 100.9 1.6 0 + " " 9/6 102.0 2.9 9/8 101.2 0.7 0 - " " 9/9 100.9 0.9 9/10 100.6 7.6 O - " " 9/12 101.5 5.6 9/14 101.4 1.1 0 - ” " 9/15 101.1 0.8 9/17 g! 101.4 8.7, 0 - " " 545 x 102 staph. organisms and mild trauma 10/5 0 101.4 9.6 0 - Normal Normal 3 101.4 12 Yellowish Congestion, 6 105.2 4.7 ++ + white hard and 9 101.6 120 with edematous 10/6 100.9 76 + + " swelling. 10/7 101.3 6.7 Swelling 10/8 101.2 9.7 + + ” disappeared. 10/9 101.6 15.4 " " 10/10 100.8 4.5 + + Normal " 10/12 101.4 7.6 10/13 101.3 18 + + " " 10/15 100.6 28.5 10(16 101.2 95.7 + + " " 5.1 108 staph. 11/7 0 102.0 No count ** Normal 3 101.4 6 101.3 9 100.9 *Recorded in thousands **Cow was dry 59 TABLE VIII RIGHT REAR QUARTER 0F 00' 2 After infusion of 1800 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. or F. /ml. Milk Quarter Milk 9/10 0 101.3 0.9* 0 - Normal Normal 3 101.2 1.1 6 101.2 3.6 + + " " 9 101.5 0.3 9/10 100.9 3.9 9/11 101.3 2.4 0 " " 9/12 102.0 5.6 9/14 101.1 1.4 0 ' " 9/15 100.8 4.4 9/18 101.3 0.9 0 ” " 9/19 101.1 8.9 9/20 100.6 0.8 0 " ” 7.8 2(21 101.2 5191 10 staph. and mild trauma 10/15 0 100.7 2.4 0 — Normal Normal 3 101.1 86.0 ” ” 6 101.5 5.4 ++ + " " 9 100.9 9.6 " " 10/17 101.3 17.5 10/18 101.4 8.1 ++ + ” " 10/19 100.9 71.0 10/20 101.3 18.5 + " " 10/21 100.6 7.5 10/22 101.4 19.5 + " " 10/24 101.3 8.8 10/25 101.5 12.0 + " " 10/26 100.9 9.6 10(28 101.2 14 + + ” ” 5 x 10 staph. 11/12 0 101.2 No count ** Normal Normal 3 101.5 6 101.4 9 101.2 *Recorded in thousands **Cow was dry . .— . ‘ —-- ... 7-7 - . -o o .- C 60 TABLE IX LEFT FORE QUARTER OF COW 191 After infusion of 1800 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 10/3 0 101.4 1.2* 0 - Normal Normal 3 101.4 2.0 + + ” ” 6 101.8 6.2 9 101.2 3.5 10/4 100.5 9.0 0 + " " 10/5 101.2 5.5 10/6 101.2 0.8 0 - " " 10/7 100.9 1.4 1.0/8 101s2 300 O - I. " 10/10 101.4 96.0 10/12 101.2 2.0 0 - " " 10/14 100.7 0.7 10116 101.6 3.9 0 - ” " 44 x 103 staph. organisms and mild trauma 10/17 0 101.2 4.4 0 - Normal Normal 3 101.2 7.6 6 101.3 15.0 ++ + Yellowish " 9 101.4 8.7 white with 10/18 101.1 1.2 + + flocculi. " 10/19 100.9 14.5 10/20 101.2 2.6 + + Normal " 10/22 101.2 96 10/24 100.9 5.8 + + 9 " 10/26 101.3 0.9 10/28 101.4 1.2 + + " " 10.2 x 108 staph. organisms 10/29 0 101.2 3 100.5 TNC** ++ + Yellowish Hard and 6 104.0 24.0 serum diffused 102.5 7.8 like with swelling. 10/30 101.4 5.6 ++ + pus floc- Normal 10/31 100 . 9 4 . 2 culi . 11/2 100.9 18.4 ++ + ' ' 11/4 101.2 76 11/5 101.4 3.3 ++ + ” " ll/6 101.5 1.6 Normal " 11/8 100.9 2.6 + + " " 1119 101.2 3.5 *Recorded in thousands **Too numerous to count -.—~. .- _. .— 'V :-::-. I.- - . -_ ,' 61 TABLE I RIGHT FORE QUARTER OF COW 191 After infusion of 2400 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 10/15 0 101.7 2.2% o - Normal Normal 3 101.5 4.5 6 101.3 6.6 O + " " 9 101.1 4.2 10/16 100.7 5.0 0 — " ” 10/18 101.2 2.1 10/19 100.5 3.3 0 + " " 10/22 101.2 1.7 10(24 101.1 2.3 0 - “ " 38 x 10 staph. organisms and mild trauma 10/25 0 100.9 19.5 Normal Normal 3 100.9 19.5 ++ + Yellowish " 6 101.2 7.2 with 9 101.1 12.5 ++ + flocculi. " 10/26 100.5 9.8 10/27 100.9 4.5 + + . . 10/28 101.2 17.5 10/30 101.2 5.0 + + Normal " 11/2 100.7 94.0 11/4 100.6 4.8 + + " " 11/5 101.3 4.2 11.2 x 10 staph. 11/7 0 101.2 39.0 + + Normal Normal 3 101.5 225.0 Yellowish Hard and 6 102.0 144.0 ++ white diffused 9 103.5 280.0 with swelling. 11/8 101.2 25.0 ++ + flocculi. " 11/10 101.3 186.0 Swelling 11/11 101.3 TNC** ++ + " disappeared. 11/12 100.9 TNC Normal " 11/13 101.2 15.0 + + " ” 11/15 101.5 9.6 11/16 100.7 7.6 + + " " 11/18 101.2 140.0 11/20 101.3 544 ++ + " " *Recorded in thousands **Too numerous to count o . n..- 7 I a > - ‘ -c-A- - - O l . on. v o no—- 0 + - ‘ .. A . - --..--. 4.. 62 TABLE XI LEFT REAR QUARTER OF COW 191 After infusion of 1800 staphylococcic (strain 7llorganisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 10/10 0 101.2 2.2* o - Normal Normal 3 101.4 3.5 6 101.4 4.2 0 + " " 9 101.5 2.0 10/11 101.2 2.5 10/12 101.3 8.5 0 - " " 10/13 101.4 4.2 10/15 100.7 2.4 0 - " ” 10/16 101.3 4.0 10/17 100.8 2.1 10(18 101.3 0.9 0 - " " 444 x 103 staph. organisms and mild trauma 10/20 0 100.5 1.2 0 - Normal Normal 3 100.5 12.0 6 100.9 7.0 ++ + Yellowish " 9 101.0 9.5 serum like. 10/21 100.6 13.0 10/22 101.4 3.5 ++ + " " 10/24 100.5 9.5 10/26 100.5 150.0 ++ + Normal ' 10/27 101.4 75.0 10/29 100.8 5.4 ++ + " " 10/31 100.5 2.8 11/1 100.2 40.0 + + " " 29 x 108 staph. organisms 11/2 0 101.8 5.6 + + Normal Normal 3 102.5 95.0 6 104.0 5.6 ++ + Yellowish Hard and 9 106.0 125.0 white tense with swelling. 11/3 101.2 50.0 ++ + flocculi. Swelling 11/4 101.2 4.9 disappeared. 11/5 100.9 5.6 + + " " 11/6 101.4 156.0 11/7 101.5 37.0 + + Normal " 11/8 100.9 3.4 11/10 101.4 148.0 + + " " 11/12 102.1 38.0 11/14 101.3 7.6 + + " " 11/15 100.9 4.5 ++ + " " *Recorded in thousands 63 TABLE XII RIGHT REAR QUARTER OF COW 191 After infusion of 2300 staphylococcic (strain 71) organisms Date Time Rectal No. of Coag. Hemo. Gross appearance hrs. Temp. Staph. of F. /m1. Milk Quarter Milk 10/21 0 100.8 1.2* - - Normal Normal 3 100.8 1.5 0 + " ” 6 100.9 6.4 9 101.1 4.3 10/23 101.2 9.0 0 + " " 10/24 101.4 8.5 10/25 100.6 0.9 0 - ” " 10/26 101.3 1.1 10/27 101.1 2.5 0 - " " 10/29 102.1 1.8 1.0/30 lOOs6 008 O " N " 57_x lo4 staph. organisms and mild trauma 10/31 0 101.3 1.7 0 - Normal Normal 3 101.2 68.0 ++ + Yellowish " 6 100.9 12.0 white 11/1 101.2 7.8 ++ + " " 11/2 100.9 7.7 11/4 101.2 26.0 + + " " 11/6 101.4 51.2 11/7 101.3 7.2 + + Normal " 11/8 101.3 9.6 11/10 100.9 6.7 + + " " 11111 101.1 4.5 88 x 10 staph. organisms 11/15 0 101.4 5.7 + + Normal Normal 3 101.2 166.0 Yellowish " 6 100.9 34.0 ++ + white with 9 100.8 9.0 flocculi. 11/16 100.7 14.0 11/17 101.3 5.6 ++ + " " 11/18 101.5 1.6 Normal 11/20 100.5 89.0 11/21 101.5 36.0 + + ” " 11/22 100.9 9.8 11/23 101.2 54.0 + + " " 11/24 101.1 2.6 11/26 100.9 87.0 + + " " 11/27 101.3 62.0 11/30 101.1 9.7 + + n n *Recorded in thousands v”— u— .- - . e --.—a— 64 TABLE XIII SUMMARY OF THE EXPERIMENTAL PROCEDURES FOR THE INTRAMAMMARY CHALLENGE OF THE THREE BOVINE UDDERS BY STAPHYLOCOCCIC OR- GANISMS Cow Quarter Staphylococci Date of injec- Interval in injected tion of staph- days in between ylococcic two consecutive organisms inlections 1 LF* 16 x 10; 7/10 -— 1 RF** 24 x 102 7/15 -- 1 LR*** 26 x 102 7/25 -- 1 RR**** 26 x 102 7/30 -- 2 LP 28 x 103 8/22 -- 1 LE 40 x 102 8/25 46 2 RE 24 x 102 8/27 -- 2 LR 21 x 102 9/4 - 2 RR 18 x 103 9/10 -- 1 RP 54 x 103 9/10 56 1 LR 50 x 104 9/20 56 2 LP 24 x 103 9/20 28 2 RP 44 x 104 9/27 30 1 RR 40 x 102 10/1 63 191 LE 18 x 10 3 10/3 -- 2 LR 54.5 x 20 10/5 30 19% it i? 6 1° 03 i822 15 s I 2 RR 67 x 10 3 10/15 34 1 LF 27.6 x 10 10/15 50 191 RF 24 x 103 10/15 -- 191 LP 44 x 109 10/17 14 2 LE 27 x 10 4 10/19 28 191 LR 44.4 x 10 10/20 10 191 RR 23 x 103 10/21 -- 191 RF 38 x 108 10/25 10 i 2 2:187 8 1863 i; 191 LP 10.2 x 0 10/29 12 191 RR 54 x 108 10/31 10 191 LR 24 x 10 8 11/2 11 1 LR 17.5 x $0 11/2 42 1 RR 16 x 10 8 11/5 25 191 RP 11.2 x810 11;? 12 2 LR 5 x 10 11 7 32 2 RR 5 x 1097 11/12 27 191 RR 88 x 10 11/15, 14 * Left fore ** Right fore Left rear Right rear c- *w .- 65 TABLE XIV GROWTH OF STAPHYLOCOCCIC ORGANISMS (STRAIN 71) IN FRESHLY DRAWN MILK; 10 cc. FROM COW 191 Quarter 0 Hrs. 3 Hrs. 6 Hrs. 8 Hrs. 24 Hrs. Date: 9/25 L.F. 66* 57* 69* 140* TNC** R.F. 78 72 86 125 TNC L.R. 94 87 99 148 TNC R.R. 4O 29; 42 98 TNC Date: 9/27 L.R. 3O 27 32 75 TNC R.R. 16 10 19 51 TNC L.R. 15 14 9 56 TNC R.R. 15 9 12 64 TNC*** Date 10/1 L.F. 75 62 78 198 ‘ TNC R.F. 82 67 96 215 TNC L.R. 68 52 72 140 TNC R.R. 96 8O 95 186 TNC *Staph. colonies counted in thousands in milk **Too numerous to count ***For this reading see graph on page 40 66 TABLE XV GROWTH OF STAPHYLOCOCCIC ORGANISMS (STRAIN 71) IN FRESHLY DRAWN RAW MILK, 10 cc. FROM COW 3 Quarter 0 Hrs. 3 Hrs. 6 Hrs. 8 Hrs. 24 Hrs. Date: 8/25 L.R. 840* 620* 1120* TNC** TNC** R.R. 1420 1100 1490 TNC TNC L.R. 620 410 1240 TNC TNC R.R. 910 740 1550 TNC TNC Date 8/30 ' L.R. 157 144 296 TNC TNC 3.2. 143 139 260 TNC TNC L.R. 81 62 126 TNC TNC*** R.R. 72 42 210 TNC TNC *Staph. colonies counted in thousands in milk **Too numerous to count ***For this reading see graph on page 39 M 3:? 3 o '53 OCT 1 4 '63 OCT 2 8 '83 42:1! 11 ’53 440V 25 ’63 JUL'2 '84 2.31; i "('14. oc'r24’62