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Studies on the incidence, diagnosis and control of bovine mastitis in British Columbia Tamboline, Florence Rosena 1946

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STUDIES OH THE INCIDENCE, DIAGNOSIS AND CONTROL OF BOVINE MASTITIS IN BRITISH COLUMBIA. - by - Florence Rosena Tamboline A Thesis Submitted In P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the DEPARTMENT OF DAIRYING THE UNIVERSITY OF BRITISH COLUMBIA October, 194-6 • TABLE OF CONTENTS Page I n t r o d u c t i o n . 1 H i s t o r i c a l O u t l i n e . 5 Forms of M a s t i t i s . 6 B a c t e r i o l o g y of M a s t i t i s . 9 1. B a c t e r i a Causing M a s t i t i s . 9 2. Source of I n f e c t i v e Agents. 18 3. Transmission of I n f e c t i v e Agents. 20 4-. Mode of I n f e c t i o n . 22 5• R e l a t i o n of Human Pathogens to M a s t i t i s . 26 Factors Predisposing the Udder to M a s t i t i s I n f e c t i o n . 28 1. Breed. 29 2. I n h e r i t a n c e . 29 3. Stage of L a c t a t i o n . 30 4-. Age of Cow. 31 5. Completeness of M i l k i n g . 32 6. Size of Herd. 33 7. M i l k i n g Machines. 34 8. Season and Weather. 35 9. Feed. 35 10. Trauma. . 36 11. Other I n f e c t i o u s Diseases. 36 Extent of M a s t i t i s I n f e c t i o n . " 37 E f f e c t of M a s t i t i s on M i l k Y i e l d 4.0 E f f e c t of M a s t i t i s on the Composition of M i l k . 43 Experimental. 45 Outline of Research Council Program. 45 General Methods. 46 1. North Okanagan Study. 46 2. General Survey. 47 3. Experimental Herd Study. 48 Methods of C l i n i c a l Diagnosis. 56 Methods of Laboratory Diagnosis. 5*7 1. P h y s i c a l Appearance of the M i l k . 57 2. Modified Whiteside Test. 57 3. Geneva B l o t t e r Test. 53 4. pH Determination with Brom Cresol Purple. 58 5. Hotis Test. 59 6. Percent C h l o r i n e . 59 7. Leucocyte Count. 60 8. Microscopic Study of the Organisms Present i n the M i l k Samples. 6 l 9. C u l t u r a l Study of the.Organisms Present i n the M i l k Samples. 62 Methods f o r C l a s s i f y i n g the Organisms Associated 65 w i t h M a s t i t i s . 1. S t r e p t o c o c c i . 66 2. Staphylococci. 67 3. Rod Forms. 68 Results and Discussion of R e s u l t s . 69 1. Extent of M a s t i t i s I n f e c t i o n i n B r i t i s h Columbia. 70 2. Factors Predisposing the Udder to M a s t i t i s I n f e c t i o n . 76 3. E v a l u a t i o n of the Tests Used f o r the Detection of M a s t i t i s . 80 4-. D e t a i l e d Study of the Organisms As s o c i a t e d with M a s t i t i s . 106 5. Studies on the C o n t r o l of M a s t i t i s . 114 6. Studies on the Treatments Used f o r M a s t i t i s . 118 General D i s c u s s i o n . 127 Summary and Conclusions. 135 B i b l i o g r a p h y . 137 STUDIES ON THE INCIDENCE, DIAGNOSIS AND CONTROL OF BOVINE MASTITIS IN BRITISH COLUMBIA. INTRODUCTION. Bovine m a s t i t i s i s probably responsible f o r a greater economic l o s s to the d a i r y farmer each year than any other s i n g l e f a c t o r . The extent of t h i s l o s s i s v a r i o u s l y r e  ported f o r d i f f e r e n t parts of the world, depending l a r g e l y on the scope of the surveys made and on the methods used f o r the dete c t i o n of the disease. The Bureau of Dairy Industry, United States Depart ment of A g r i c u l t u r e , (20) estimated that the dairymen of that country i n 194-2 l o s t at l e a s t 3% of t h e i r p o t e n t i a l milk production as a r e s u l t of m a s t i t i s . This would amount to a l o s s of 3 1/2 b i l l i o n pounds of m i l k , which at $1.50 per 100 pounds would be worth $52,500,000.00. Hopson i s quoted ( l ) as having stated i n a speech to dairy farmers at Guelph, Ontario, i n October, 194-5, that i n the United States and Canada, cows valued at over $200,000,000.00 are sold every year because they are s u f f e r i n g from m a s t i t i s . Holford (43) i n 1930 reported on a study of 5,000 herds i n New York St a t e , and on the basis of h i s f i n d i n g s c a l c u l a t e d that m a s t i t i s was responsible f o r an annual l o s s i n the United States of $72,011,455.00. This estimate i s very conservative, since i t assumes that only of a l l milk cows are sold each year because of mastitis, that had these animals remained in the herds they would have pro duced only 68% as much milk as i f they had been healthy, and that the average loss in value of the cows discarded is $25.00 per head. Losses due to the reduced milk pro duction of infected cows retained in the herds are not con sidered. If Holford's results are applied to Canada, on the basis of a milk cow population of 3,930,000 and a total milk production of 17,604,823,000 pounds in 1944, and If $1,32 i s taken as the farm value of 100 pounds of milk (22), there i s an annual loss attributable to the removal from herds of mastitic animals of $13,613,509. In order to evaluate f u l l y the financial loss due to mastitis, several factors must be taken into consideration: (1) the decrease in the amount of milk produced by the infected cow, (2) the relatively higher cost of feed per pound of milk produced by the infected cow as compared to that for a normal cow, (3) the decrease in the value of the infected cow, herself, with probable eventual total loss, (4) the loss due to the production of milk unfit for human consumption and of l i t t l e or no commercial value. From the above reports i t can readily be seen that the dairy industry annually sustains a•tremendous loss as a result of mastitis infection. Despite the seriousness of this apparently widespread disease, very l i t t l e work has been done in British Columbia either to study the extent of infection In this province or to try to develop control measures. The f i r s t investigation on mastitis in this province was that reported by Berry and Clark ( 6 ) , who in 1936-37 made a study of 53 cows from several herds in the Lower Fraser Valley. These workers confined their study to speci fied groups of cows, and in their report presented an evalua tion of the relative merits of various f i e l d and laboratory tests for the detection of the disease as applied to the selected groups of animals. Because of the severity of the disease in the dairy herds of British Columbia, with i t s resulting reduction in the revenue of the province, the British Columbia Industrial and Scientific Research Council, through i t s Technical Ad visory Committee on Agriculture, established a Mastitis Sub- Committee to study the c l i n i c a l and laboratory detection of the disease, as well as to devise methods for i t s control and eradication. This Sub-Committee, under the chairmanship of Dr. J.G. Jervis, i s composed of representatives of the Do minion and Provincial Departments of Agriculture, the British Columbia Veterinary Association, milk distributing organiza-- 4 - tions, dairy farmers, and the University of British Columbia Departments of Dairying and Animal Husbandry. The work of the Research Council on mastitis began in May, 1944" The f i r s t undertaking of the Council was a preliminary study of the prevalence of mastitis in the North Okanagan Valley. This survey was carried out in conjunction with work which had been proceeding under a University Re search Fund Grant on problems associated with cheese-making in the North Okanagan Valley. A period of two months was devoted to this study. During the following twelve months, a general survey was made of the incidence of the disease in British Columbia. Milk samples from c l i n i c a l l y infected and suspicious cows were taken by co-operating veterinarians and forwarded to the laboratory for examination. The majority of the samples were obtained from the Lower Fraser Valley. However, a num ber were received from Interior points and from Vancouver Island. In order to acquaint the dairy farmer with the pro ject, and to encourage him to adopt the best possible sani tary methods to prevent the spread of the disease in his herd, a Handbook (43) was prepared by the Committee, and distributed throughout the province by milk processing plants, dairy associations and practising veterinarians. Following the completion of the general survey, an intensive study was made of a few herds, in which a rigid management program had been initiated. This work was under taken with the object of demonstrating that most forms of mastitis can be held in check and eventually eradicated from a herd by careful management practices. A detailed study of the types of organisms present in the milk from the cows of the selected herds was made In order to determine the nature of the flora in specific herds and in certain areas. The classification of the organisms was also used as a basis for experimental treatment studies. Throughout the work, par ticular attention has been paid to the evaluation of the various procedures employed for the detection of mastitis, and of the biochemical and serological techniques used for the classification of the organisms associated with the disease. It is the purpose of this report to review the sig nificant findings of the work undertaken by the Research Council on mastitis together with a summary of the literature pertaining specifically to the program of study of the Coun c i l . HISTORICAL OUTLINE. In the present report, no attempt has been made to enumerate a l l of the papers on mastitis. Reference i s made only to those articles considered pertinent to the work described herein. A complete review of the extensive literature on _ 6 - mastitis up to the end of 1935 may be found in the monograph by Munch-Petersen (75)• A more recent survey of the f i e l d is that of Merchant and Packer in 1944 (68). Although having fewer references to specific articles, the latter publication gives a condensed summary of a l l aspects of the problem. Forms of Mastitis. Mastitis is generally defined as any inflammation of the udder. The disease may be non-infectious or infectious in nature. The non-Infectious form is due to such factors as congestion at freshening, c h i l l i n g , bruising and injury of the teats and udder. The condition usually clears up as soon as the causative factor has been corrected, provided that the remedial measures are not too long delayed. This type of mastitis may, however, leave the udder in a weakened condition, predisposing i t to infection by opportunist bac teria. Infectious bovine mastitis i s caused by various species of bacteria and may be divided into two distinct types: acute and chronic. Bryan (12) further divides the acute form into acute local and acute systemic mastitis. Acute local masti tis is characterized by swelling of the udder accompanied by pain and the production of milk of abnormal physical appear ance. A l l of the symptoms are confined to the udder. In acute systemic m a s t i t i s , the cow shows general signs of i l l  ness i n a d d i t i o n to the symptoms of acute l o c a l m a s t i t i s . The acute systemic type, although r e l a t i v e l y r a r e , i s very o f t e n f a t a l . Acute l o c a l m a s t i t i s may get p r o g r e s s i v e l y more s e r i o u s , becoming acute systemic. Acute systemic m a s t i t i s r e s u l t s when the causative organism metastasizes from the udder i n t o the blood stream. In some cases, only the t o x i c by-products of the organism d i f f u s e i n t o the general c i r c u  l a t i o n . In.some i n s t a n c e s , acute m a s t i t i s may become gan grenous, r e s u l t i n g i n the death of the animal, o r , i f r e  covery takes p l a c e , i n the sloughing o f f of the a f f e c t e d quarter. Acute m a s t i t i s i n v a r i a b l y r e s u l t s i n one of the f o l l o w i n g four conditions: (1) the animal may d i e , (2) the animal may completely recover, e i t h e r spontaneously or i n response to treatment, (3) the a f f e c t e d quarter or quarters may atrophy and become b l i n d , (4) chronic m a s t i t i s may develop. The organism causing the acute form may, i t s e l f , be responsible f o r the chronic c o n d i t i o n , or the acute attack may predispose the udder to i n f e c t i o n by the l e s s v i r u l e n t organisms associated with the chronic type of m a s t i t i s . Chronic m a s t i t i s i s by f a r the most common form of - 8 - the disease. The c o n d i t i o n develops very slowly and may e x i s t i n an animal f o r some time without any v i s i b l e signs of i t s presence. The udder and i t s s e c r e t i o n are normal i n p h y s i c a l appearance. As the disease progresses, the secre t o r y c e l l s of the udder are destroyed and are ev e n t u a l l y r e  placed by connective t i s s u e . Areas of f i b r o s i s or i n d u r a  t i o n can now be detected on p a l p a t i o n of the udder. In the advanced stages of the disease, almost a l l of the secretory t i s s u e i s destroyed, rendering the quarter non-functional f o r milk production. One of the c h a r a c t e r i s t i c s of the chronic form of m a s t i t i s i s the occurrence of p e r i o d i c f l a r e - ups of acute m a s t i t i s . These f l a r e - u p s may be manifested merely by the s e c r e t i o n of f l a k y or c l o t t y m i l k f o r a few m i l k i n g s , or the animal may show symptoms of acute l o c a l or acute systemic m a s t i t i s . Between flare-ups the mil k i s u s u a l l y of normal p h y s i c a l appearance. Because of i t s i n s i d i o u s nature, chronic m a s t i t i s i s l e s s r e a d i l y detected than acute m a s t i t i s . U n t i l the disease has progressed to the point of the formation of f i b r o s i s or in d u r a t i o n i n the udder, or there i s an acute f l a r e - u p , i t i s impossible to detect chronic m a s t i t i s except by a chemical and b a c t e r i o l o g i c a l examination of •the m i l k . Non-infectious m a s t i t i s occurs much l e s s frequently and i s responsible f o r much l e s s serious consequences than the i n f e c t i o u s type. In the studies described h e r e i n , no known cases of non-infectious m a s t i t i s have been encountered. Thus the term " m a s t i t i s " has been used i n the succeeding parts of t h i s report to r e f e r to the i n f e c t i o u s form of the disease o n l y . Ba c t e r i o l o g y of M a s t i t i s . The f a c t that m a s t i t i s i s not a simple disease hut i s manifested hy several d i s t i n c t forms, may he a t t r i b u t e d , i n part at l e a s t , to the great v a r i e t y of organisms which have been shown to be capable of producing an inflammation of the udder. A c e r t a i n set of symptoms may be c o n s i s t e n t l y foTind with a p a r t i c u l a r organism. The a c t i o n of another organism, however, may r e s u l t i n a v a r i e t y of symptoms on d i f f e r e n t occasions depending on the part played by con t r i b u t o r y f a c t o r s . F u r t h e r , a number of d i f f e r e n t organ isms may a l l be associated with the same set of symptoms. These f a c t s e x p l a i n many of the d i f f i c u l t i e s encountered i n the accurate c l i n i c a l and l a b o r a t o r y diagnosis of the d i s  ease, and hence i n the p r e s c r i b i n g of adequate c o n t r o l and treatment measures. 1• B a c t e r i a causing m a s t i t i s . In general, chronic m a s t i t i s i s caused by several species of s t r e p t o c o c c i and l e s s frequently by s t a p h y l o c o c c i . Acute m a s t i t i s i s caused by a number of organisms, i n c l u d i n g s t a p h y l o c o c c i , some species of c o l i f o r m b a c t e r i a , Coryne- bacterium pyogenes and Pseudomonas aeruginosa. Streptococci have been incriminated as the e t i o  l o g i c a l agents of m a s t i t i s i n the great majority of cases. - 10 - Various workers report up to 90% of mastitis infections to be caused by these organisms. There are four species of streptococci which have been definitely proven to be cap able of producing mastitis. Streptococcus agalactiae Lehmann and Neumann, which is also known as Str. mastitidis Migula, has come to be. recognized as the most common cause of chronic mastitis. There is no indication in the literature that this organism ever produces isolated acute cases. An acute attack associa ted with this organism may be a flare-up of an already es tablished chronic condition or i t may mark the beginning of a chronic infection. Str. agalactiae i s characterized by a low invasive power and can apparently live in the udder for considerable lengths of time without producing any demonstrable patho logical changes. This has been shown in work done in Aus trali a (16) where this organism was isolated for varying periods of time from the milk of animals in their f i r s t and second lactation periods. In no case was there any indica tion of mastitis. Evidence suggests that injury to the udder or some other debilitating factor is necessary before In fection by Str. agalactiae can take place. In fact, the ease with which this organism can be isolated from the milk of apparently normal cows, has led some authors (16) to the conclusion that Str. agalactiae should be considered a nor mal inhabitant of the udder, and that the development of - 11 - disease i s due to decreased resistance on the part of the host and not to the activity of the organism. It should be noted at this point, that, because Str. agalactiae had been shown in the early studies on mastitis to be responsible for up to 99$ of the cases, diagnostic procedures have been developed for the sole detection of this organism. As a result, i n many of the more recent studies, cases of sub-clinical mastitis due to organisms other than Str. agalactiae have been overlooked, because the methods used did not detect these organisms. There are two other species of streptococci which are quite similar to Str. agalactiae and which frequently cause mastitis - Str. dysgalactiae (Minett Group II) and Str. uberis (Minett Group III). Merchant and Packer (68) found Str. agalactiae to be responsible for about 9Q%> of cases of streptococcic mastitis in Iowa and Str. dysgalactiae and Str. uberis for about 5% each. Minett et al (74) described the condition produced by Str. dysgalactiae as being severe and more acute than that caused by Str. agalactiae. In some cases speedy destruction of the involved quarter results, while in others recovery takes place. Rarely i s an infection of a more or less per manent nature produced. Mastitis caused by Str. dysgalactiae appears to be less contagious than that produced by Str. agalactiae, and is usually limited to isolated cases in a herd. ! - 12 - Str. uberis infection, on the other hand, is generally mild and may be of a transient or somewhat chronic nature (74)• Only very slight changes are produced in the milk of the affected quarter by this organism. Another streptococcus occasionally incriminated in mastitis is Str. pyogenes Rosenbach, the organism responsible for scarlet fever, septic sore throat, erysipelas and re lated infections in man. Mastitis due to this organism is frequently of the acute type and i s thus readily detected. It may, however, be of a sub-clinical nature and evade de tection until an outbreak of disease in man, caused by the use of raw milk containing this organism, has been traced to the particular infected animal. Merchant and Packer (68) appear to consider Str. zooepidemicus ("animal pyogenes") an etiological agent of mastitis worthy of note. These workers state that this or ganism causes a very severe and often systemic attack which is frequently f a t a l . They report further that many workers believe that Str. zooepidemicus is responsible for most cases of streptococcic mastitis in which the organism metas tasizes from the udder. L i t t l e mention of this organism as an important agent in mastitis infection has been found in the reports of other workers. However, in view of the fact that both Str. dysgalactiae and Str. zooepidemicus belong to Lancefield's serological Group C (102) i t is possible that - 13 - some workers may have reported a l l Group C i n f e c t i o n s to he due to the former organism without f u r t h e r d i f f e r e n t i a t i o n . Various i n v e s t i g a t o r s have reported several other species of s t r e p t o c o c c i to he associated w i t h occasional cases of m a s t i t i s . When the methods of i s o l a t i o n of the organisms and the circumstances r e l a t i n g to the s p e c i f i c cases are considered, i t i s probable that such attacks r e  present chance i n f e c t i o n s . I t may be that the s t r e p t o c o c c i are secondary invaders o n l y , or that f a c t o r s other than b a c t e r i a l i n v a s i o n are of greater importance i n the i n i t i a  t i o n of the disease. The extent to which staphylococci are responsible f o r m a s t i t i s i s v a r i o u s l y reported by d i f f e r e n t i n v e s t i g a t o r s . A complicating f a c t o r i n the diagnosis of staphylococcic m a s t i t i s Is the f a c t that an undefined number of species of staphylococci ("udder micrococci") form part of the normal f l o r a of the udder. Even hemolytic staphylococci are f r e  quently found i n the udder w i t h no evidence of disease (34)• Thus i t i s very d i f f i c u l t to determine whether a staphylococ cus i s o l a t e d from the m i l k of a suspected cow i s responsible f o r the c o n d i t i o n , or i s present merely as a normal i n h a b i  tant of the udder. Fu r t h e r , i t i s d i f f i c u l t to determine whether or not the presence of considerable numbers of s t a  phylococci i n apparently normal udders represents the i n i  t i a l stages of s u b - c l i n i c a l m a s t i t i s . I t i s probable that the a c t u a l i n i t i a t i o n of disease, as i n the case of m a s t i t i s - 14 - caused by S t r . a g a l a c t i a e , i s brought about by other f a c t o r s and that the staphylococci are merely opportunist i n a c t i v i t y . Staphylococci have been most frequently incriminated as the causative agents of an acute m a s t i t i s , e s p e c i a l l y i n r e c e n t l y freshened cows. The disease i s often f a t a l (70). Many workers, however, are of the opinion that more cases of chronic m a s t i t i s are due to staphylococci than have heretofore been considered. Chronic m a s t i t i s caused by staphylococci i s u s u a l l y very m i l l , and i s often l e s s severe than that produced by S t r . a g a l a c t i a e . C e r t a i n members of the c o l i f o r m group have been found to be e t i o l o g i c a l agents of m a s t i t i s . Murphy and Hanson (78) i n a three-year study of 120 cows encountered 79 cases of m a s t i t i s due to c o l i f o r m organisms. Aerobacter aerogenes was i s o l a t e d from 59$ of the cases, "intermediate" types from 26% and E s c h e r i c h i a c o l i from 15$. The c o l i f o r m organisms u s u a l l y cause a very acute att a c k of short d u r a t i o n . There i s generally complete recovery, although Ferguson (35) found a number of cases of m a s t i t i s due to c o l i f o r m organisms to terminate f a t a l l y . On some occasions the attack i s of a milder and more chronic mature. Colifo n n m a s t i t i s i s u s u a l l y sporadic and does not spread e x t e n s i v e l y throughout a herd. Burkhardt et a l (21), however, reported an outbreak of severe acute systemic m a s t i t i s due to A.* aerogenes i n 11 cows In a herd of 33. Corynebacteria (diphtheroids) are frequently found i n - 15 - the flora of the normal udder (34)• The only species which has been incriminated in mastitis is Corynebacterium pyogenes. This organism is often encountered in Great Britain and Con tinental Europe as the cause of the so-called "summer mas t i t i s " which is most prevalent in dry cows and maiden heifers on pasture (71). In North America, C. pyogenes has also been found-to cause mastitis, but the condition does not appear to be associated with any particular season of the year nor with any particular stage of the lactation period. C. pyogenes produces a severe acute or chronic con dition accompanied by suppuration and abscessation. The affected quarter frequently sloughs off. Bean et al (4) made a study of 23 quarters of 15 cows infected with C. pyo genes. 22 of these quarters were apparently permanently damaged by the infection, either becoming blind or secreting pus. Pseudomonas aeruginosa has been found to cause spora dic outbreaks of acute purulent mastitis. Permanent injury of the affected quarter usually results. Cone (26) described an outbreak of mastitis in one herd due to this organism. He found that in some cases the acute attack was followed by a mild chronic condition. In Europe, mastitis due to Mycobacterium tuberculosis is f a i r l y common, and plays an important role in the spread of tuberculosis in man in areas where raw milk Is consumed. On this continent, however, bovine tuberculosis has been - 16 - largely eradicated, and so this type of mastitis i s not often encountered. The relationship between Brucella abortus infection and mastitis is not clearly understood. This organism does not appear to produce well defined lesions in the udder. In cases of mastitis where Br. abortus i s shed in the milk, other organisms are also usually present. Thus i t i s d i f f i  cult to determine whether Br. abortus i s the cause of the mastitic condition or whether i t has merely predisposed the udder to infection by other disease-producing bacteria. Most workers attribute gangrenous mastitis to mixed infections of streptococci, staphylococci and colifomv or ganisms. Schalm, however, has reported a number of cases of gangrenous mastitis which he found to be caused by Staphylo coccus aureus (96). Numerous other species of bacteria have been reported in isolated cases of the disease. In most instances i t has not been shown conclusively that the incriminated organism was actually the causative factor, and i t is likely that in many cases the organism found was merely a secondary invader. On other occasions such attacks are probably chance infections due to decreased resistance on the part of the host, and pos sibly to increased virulence on the part of the organism. Hastings and Beach (4-6) and Johns and Hastings (55) and (56) have reported on studies of cows which were not shedding Sc. agalactiae or other recognized pathogen, but - 17 - which repeatedly shewed an abnormal pH, percentage c h l o r i n e and catalase content i n t h e i r m i l k . These workers c a l l e d t h i s c o n d i t i o n " n o n - s p e c i f i c " m a s t i t i s , and found i t to be of a m i l d , p r o g r e s s i v e , chronic nature, i n c r e a s i n g i n i n  cidence from l a c t a t i o n to l a c t a t i o n . Peterson and Hastings (81) concluded from t h e i r studies that t h i s m i l d "non-speci f i c " m a s t i t i s i s the primary i n f e c t i o n i n chronic m a s t i t i s and that the more severe i n f e c t i o n due to S t r . a g a l a c t i a e f o l l o w s a f t e r the udder defence mechanisms have been weakened by the primary i n f e c t i o n . As a r e s u l t of c y t o l o g i c a l s t u d i e s , Peterson et a l (82) concluded that " n o n - s p e c i f i c " m a s t i t i s i s spread from c e l l to c e l l along the m i l k ducts and eventually reaches the udder parenchyma. I t i s never spread through the m i l k . These f i n d i n g s may e x p l a i n why some i n v e s t i g a t o r s have ob served f i b r o s i s i n the udder without any i n d i c a t i o n of S t r . agalactiae i n f e c t i o n . Although they were unable to demon st r a t e i n c l u s i o n bodies i n the udder c e l l s , these authors postulated that "non-specific" m a s t i t i s might be caused by a v i r u s . Broadhurst et a l (8) have reported the I s o l a t i o n of a v i r u s from m a s t i t i c m i l k . They report f u r t h e r the f i n d i n g of i n c l u s i o n bodies i n the leucocytes of the m a s t i t i c m i l k . They claim to have propagated the v i r u s i n t i s s u e c u l t u r e s , to have observed i n c l u s i o n bodies i n the t i s s u e s of mice i n j e c t e d with the v i r u s , and to have r e i s o l a t e d the v i r u s - 13 - from the mouse t i s s u e s . They do not r e p o r t , however, having t r i e d to i n f e c t healthy udders w i t h t h e i r v i r u s p r e p a r a t i o n . Hoerlein (4-7) expresses the opinion that the v i r u s theory of m a s t i t i s i n f e c t i o n has been brought f o r t h i n an attempt to eliminate some of the confusion e x i s t i n g i n the determination of the e t i o l o g i c a l agents of t h i s disease.. The f a c t that no organisms can be i s o l a t e d from many obvious cases of m a s t i t i s has, i n h i s o p i n i o n , done much to strengthen the v i r u s theory. Because of the l a c k of d e f i n i t e evidence of the r e l a t i o n of v i r u s e s to m a s t i t i s , no conclusions can as yet be drawn regarding v i r u s e s as p o s s i b l e e t i o l o g i c a l agents of m a s t i t i s . 2, Source of I n f e c t i v e Agents. There are a number of sources from which the b a c t e r i a causing m a s t i t i s may o r i g i n a t e . Evidence points to the f a c t that S t r . agalactiae l i v e s almost e x c l u s i v e l y i n the udder and i t s s e c r e t i o n . Attempts to i s o l a t e t h i s organism from the t h r o a t s , n o s t r i l s , t o n s i l s and vaginas of cows shedding S t r . agalactiae i n t h e i r m i l k have proven f u t i l e (45) and (39)• Of i n t e r e s t i n t h i s connection i s the report from A u s t r a l i a that t h i s organism has been i s o l a t e d from bovine faeces (16). Because S t r . dysgalacti.ae and S t r . u b e r i s have been l e s s o f t e n incriminated as causative agents of m a s t i t i s than S t r . a g a l a c t i a e , the p o s s i b l e sources of these organisms - 19 " have not been so e x t e n s i v e l y s t u d i e d . I t i s l i k e l y , however, that they, too, are found c h i e f l y i n the udder. S t r . pyogenes i s a human pathogen and i n f e c t i o n of the udder of a cow by t h i s organism can almost i n v a r i a b l y be traced to a human c a r r i e r who has been attending the animal i n question. Merchant and Packer (68) state that S t r . zooepidemicus i s found i n i n f e c t i o n s of the g e n i t a l t r a c t of cows. The staphylococci are much more widely d i s t r i b u t e d i n nature than the m a s t i t i c s t r e p t o c o c c i . They form part of the normal f l o r a of the udder. They are found on the s k i n and mucous membranes of animals. They are also found i n i n f e c  t i o n s of the g e n i t a l t r a c t of cows ( 6 8 ). Because of the ubiquitous nature of the s t a p h y l o c o c c i , m a s t i t i s caused by t h i s group of organisms i s much more d i f f i c u l t to c o n t r o l than that produced by s t r e p t o c o c c i (70). The c o l i f o r m organisms which o c c a s i o n a l l y invade the udder are probably of f a e c a l o r i g i n . A. aerogenes may, how ever, also come from feed and bedding. C. Pyogenes Is one of the most common organisms as sociated with i n t e r n a l suppurative conditions i n c a t t l e (39). I t i s found i n the r e s p i r a t o r y t r a c t and c e r v i c a l lymph nodes (68). I t i s l i k e l y that exudates from such sources provide the inoculum f o r cases of m a s t i t i s due to t h i s o r  ganism. The sources of the other b a c t e r i a which have been - 20 - found capable of causing m a s t i t i s are not w e l l d e f i n e d . Most of these organisms are probably normal i n h a b i t a n t s of the cow's environment. 3. Transmission of I n f e c t i v e Agents. I t has been found t h a t , i n most cases, m a s t i t i c organisms gain access to the udder through the tea t c a n a l . Attempts to produce i n f e c t i o n by the intravenous i n j e c t i o n of m a s t i t i c organisms have f a i l e d . M i l l e r (69) i n j e c t e d animals intravenously w i t h c u l t u r e s of S t r . a g alactiae con t a i n i n g 111 m i l l i o n and 3 b i l l i o n organisms per c.c. w i t h no r e s u l t i n g i n f e c t i o n i n the udder. A report from the United States Department of A g r i c u l t u r e (17) describes the f a i l u r e to induce udder i n f e c t i o n by the intravenous i n j e c  t i o n of 10 c.c. q u a n t i t i e s of 24 hour broth c u l t u r e s of a m a s t i t i c staphylococcus. In considering the spread of m a s t i t i c organisms, i t i s important to remember that each quarter of the cow's udder i s a p h y s i o l o g i c a l e n t i t y , and that i n f e c t i o n of a si n g l e quarter i s quite p o s s i b l e without the other three be coming i n v o l v e d . The transmission of organisms from one quarter to another i n an i n d i v i d u a l animal f o l l o w s the same course as the spread of i n f e c t i o n from one cow to another. Since chronic m a s t i t i s due to S t r . agalactiae Is the only form of the disease which i s a c t i v e l y contagious, and since t h i s organism i s found almost e x c l u s i v e l y In the - 21 - udder, the spread of S t r . a g a l a c t i a e i s brought about l a r g e l y by carelessness during the m i l k i n g procedure. The other o r  ganisms which cause m a s t i t i s do n o t , as a r u l e , produce e p i  z o o t i c outbreaks of the d i s e a s e , and are almost u n i v e r s a l l y present i n the environment of the t e a t . With these organisms, the problem of t r a n s m i s s i o n i s not very important because they are always i n contact with the t e a t . Thus any c o n t r o l measures i n s t i t u t e d i n a herd to prevent the spread of the disease w i l l be of value only f o r S t r . a g a l a c t i a e i n f e c t i o n s . The f o l l o w i n g p r a c t i c e s a l l c o n t r i b u t e to the spread of S t r . a g a l a c t i a e : f a i l u r e to s t e r i l i z e the m i l k i n g machine cups between cows, m i l k e r s n e g l e c t i n g to s t e r i l i z e t h e i r hands between cows, the use of u n s t e r i l e c l o t h s to wipe the udders, m i l k i n g m a s t i t i c cows before healthy ones, m i l k i n g m a s t i t i c m i l k onto the f l o o r or bedding, c a r e l e s s d i s p o s a l of manure and bedding, c a r e l e s s use of te a t p l u g s , d i l a t o r s and ointments. Many workers b e l i e v e t h a t feeding calves on m a s t i t i c m i l k and then p e r m i t t i n g them to suck one another c o n t r i b u t e s to the incidence of S t r . a g a l a c t i a e i n f e c t i o n i n h e i f e r s . S i m i l a r l y , i t i s p o s s i b l e that the p r a c t i c e of l e t t i n g calves nurse both healthy and m a s t i t i c cows may help to spread the organisms to normal animals. F l i e s have come to be suspected as agents i n the spread of m a s t i t i c organisms. Sanders (39) and (90) has - 22 - c a r r i e d out experiments w i t h Musca domestica, the common house f l y , and Hippelates sp., the f r u i t f l y , and has shown that these i n s e c t s are able to carry the organisms i n mas t i t i c m i l k and can implant them i n the teat o r i f i c e i n such a way that i n f e c t i o n f r e q u e n t l y r e s u l t s . He suggests that a drop of mil k i s fr e q u e n t l y l e f t at the bottom of the teat a f t e r m i l k i n g , that the f l i e s feed on t h i s , and that they then f l y to another teat c a r r y i n g w i t h them any organisms which may have been i n the drop of m i l k . I t appears that the i n s e c t s can cause a s l i g h t i n j u r y to the teat o r i f i c e , thus a s s i s t i n g the organisms to gain entrance to the udder. Sanders found that f l i e s were most e f f i c i e n t i n spreading m a s t i t i s among dry cows. This he a t t r i b u t e d to the f a c t that i n such animals, the udder s e c r e t i o n i s not removed and serves as an admirable medium f o r the growth of the m a s t i t i c organisms. These observations help to expl a i n why "summer m a s t i t i s " due to C. pyogenes i s thought to be spread by f l i e s , subsequent to the cows sucking one another and pro bably l e a v i n g a drop of m i l k at the apex of the t e a t . In the case of S t r . pyogenes, the organism i s spread from the i n f e c t e d person to the cow by droplet i n f e c t i o n as a r e s u l t of coughing, or by the handling of the udder and teats w i t h i n f e c t e d hands. -4. Mode of I n f e c t i o n . In the i n i t i a t i o n of any disease, there are three - 23 - factors to be considered: virulence of the invading organism, numbers of the invading organism, and resistance of the host. There is probably no other disease in which the inter-relation ships of these factors i s less understood than i n the case of mastitis. Numerous explanations have been offered as to why on some occasions infection readily takes place, and why on other occasions under apparently identical conditions i t i s impossible to induce infection. None of these theories, however, have been able to account for a l l cases of mastitis. The conflicting results obtained in experiments on the role of the teat in preventing bacterial invasion of the ud der have led to much confusion. There are many reports of intact teats having been dipped in suspensions or cultures of mastitic organisms with no infection resulting. L i t t l e (64) suggests that the teats of heifers act as physical barriers to bacterial invasion, but that in older cows the teat sphinc ter i s more patent and the duct dilated, thus more easily per mitting bacterial invasion. He was unable to induce infection in heifers by applying cultures of a hemolytic Str. agalactiae to the intact teat, but was able to do so in some older cows. He believes, further, that because of the negative pressure created in the udder during the act of milking, the organisms at the tip of the teat may be drawn into the teat by suction. Other workers have found that organisms applied to the teat orifice can invade the udder i f the teat has f i r s t been i n  jured in some way. This explains why mastitis so frequently - 24 - f o l l o w s I n j u r i e s to the tea t s and udder. Ferguson (36) made a study of the milk from 317 cases of i n j u r e d quarters and found 89% to have become i n f e c t e d f o l l o w i n g i n j u r y . However, the f a c t that S t r . agalactiae I n f e c t i o n can spread through a herd at a more r a p i d r a t e than can be accounted f o r by i n  j u r y , suggests that other f a c t o r s , perhaps the v i r u l e n c e of the organism, may play an important r o l e i n such cases. L i t t l e (63) found that when.udders were in o c u l a t e d beyond the teat sphincter w i t h S t r . a g a l a c t i a e , the incidence of i n f e c t i o n was r e l a t e d to the numbers of organisms i n t r o  duced. He suggests that the reason f o r repeated doses of small numbers of organisms being necessary to produce i n f e c  t i o n , i s that i n such cases the udder has to be s e n s i t i z e d to the i n j e c t e d organism, and that f o l l o w i n g s e n s i t i z a t i o n w i t h the f i r s t few doses, i n f e c t i o n can take p l a c e . I t should be pointed out, however, that the reports of many workers regarding the production of acute m a s t i t i s f o l  lowing the i n o c u l a t i o n of the udder w i t h massive doses of s t r e p t o c o c c i , should not be considered evidence that the r e  s u l t i n g disease Is s i m i l a r to what would have been produced by the same organisms under n a t u r a l c o n d i t i o n s . These ex periments show the response of the udder defensive mechanisms to large doses of f o r e i g n p r o t e i n , and do not represent the slow progressive a c t i o n of the b a c t e r i a on the udder t i s s u e as occurs i n chronic m a s t i t i s . G o r i n i (41) b e l i e v e d that the common m a s t i t i c organisms - 25 - were normal inhabitants of the udder, and that they lived in the udder i n a state of equilibrium between the bacteriostatic effect of the udder and the toxins of the organisms. When this equilibrium was" upset in favour of the bacteria, disease resulted. Numerous.workers have reported on the bactericidal effect of fresh cow's milk on various organisms. Jones and Li t t l e ( 6 0 ) studied the bactericidal action of the milk from different cows on a non-hemolytic mastitic streptococcus and found that different milks differed in their action. In some milks the inhibitory action lasted for eight hours, whereas in others i t lasted only four to six hours. They concluded that the inhibitory substance originated in the udder, and suggested that the bactericidal substance or sub stances may be responsible for the limited flora of the udder, since staphylococci and coliform organisms, which live In such close proximity to the teats, do not survive to any extent nor produce disease In the normal udder. If Jones and Little's assumptions are correct, i t is possible that the initiation of mastitis in the udder may be associated with a reduction in the quantity or an alteration in the nature of this bac tericidal substance. It is the writer's opinion that the problem of masti tis control would be much simplified i f the mode of infection were better understood. What Is the efficiency of the teat in keeping bacteria from entering the udder? Is It that on - 26 - c e r t a i n occasions more organisms than usual pass beyond the tea t sphincter and so produce d i s e a s e , or i s i t that the ud der defensive power i s weakened and unable to hold i n check the usual numbers of invading organisms? Does the b a c t e r i  c i d a l substance of the udder have a s e l e c t i v e a c t i o n on bac t e r i a , or does i t have the same e f f e c t on a l l s t r a i n s ? Are the normal udder b a c t e r i a able to increase i n v i r u l e n c e under c e r t a i n circumstances? U n t i l such questions can be answered, i t i s u n l i k e l y t h a t m a s t i t i s i n f e c t i o n w i l l be e f f i c i e n t l y c o n t r o l l e d . 5. R e l a t i o n of Human Pathogens to, M a s t i t i s . The organisms most commonly associated w i t h bovine m a s t i t i s are non-pathogenic to man. However, several human pathogens have been found from time to time to cause m a s t i t i s . The most fr e q u e n t l y encountered of these Is S t r . pyogenes. Numerous references can be found i n the l i t e r a t u r e to-epidemics of s c a r l e t fever and s e p t i c sore throat which have been traced to cases of m a s t i t i s due to S t r . pyogenes. The p a t i e n t s had consumed unpasteurized milk or m i l k products containing the s e c r e t i o n from the i n f e c t e d cows. In almost every case i t has been shown that the animal became i n f e c t e d from a human c a r r i e r . Stebbins et a l (104) reported on 7 milk-borne epidemics i n New York State from 1934-1936, i n  v o l v i n g 3 epidemics of s c a r l e t fever w i t h S06 cases and 16 deaths, and 4 epidemics of s e p t i c sore throat with 723 cases - 27 - and 8 deaths. S i x of the seven epidemics were traced to cows s u f f e r i n g from acute m a s t i t i s and harbouring s t r e p t o c o c c i i n  d i s t i n g u i s h a b l e from those i s o l a t e d from the p a t i e n t s . Certain staphylococci are able to produce an entero- t o x i n which causes acute g a s t r o - e n t e r i t i s i n man. Staphylo c o c c i of t h i s type have been i n c r i m i n a t e d i n c e r t a i n cases of m a s t i t i s (24)• These enterotoxin-producing staphylococci may i n f e c t the udder from man or from primary bovine i n f e c  t i o n s . I t should be noted i n t h i s connection that t h i s t o x i n i s heat stable and that i f m i l k , containing toxin-producing s t a p h y l o c o c c i , i s held at temperatures high enough f o r the organisms to m u l t i p l y and produce t o x i n , subsequent p a s t e u r i  z a t i o n of the m i l k w i l l not destroy the t o x i n . Crabtree and L i t t e r e r (27) reported a s e r i e s of 242 attacks of severe gas t r o - e n t e r i t i s i n 97 persons which was traced to two m a s t i t i c cows harbouring toxin-producing staphylococci i n a herd sup p l y i n g raw mil k to the Infected persons. Cone (26) reports that Ps. aeruginosa has been suspec ted of causing a type of dysentery i n man. Although there i s as yet no proof that such Is the case, the source of the cau s a t i v e organism could e a s i l y be m a s t i t i c milk from cows i n  fect e d with Ps. aeruginosa. Man u s u a l l y acquires the human pathogens of m a s t i t i s o r i g i n by dri n k i n g unpasteurized m i l k . I t appears, however, that products made from raw m i l k may also harbour these o r  ganisms. Bryan and Bryan (13) found that a s t r a i n of S t r . - 28 - agalactiae, a strain of Str. pyogenes, and a hemolytic and a non-hemolytic strain of Staph, aureus survived for 6 months in both salted and unsalted butter made from sweet and r i  pened unpasteurized cream. Yale and Marquardt (113) obser ved that Str. pyogenes survived over 18 weeks in Cheddar cheese ripened at 45°F. and 9-11 weeks in cheese cured at 62°F. They concluded that the survival of pathogens i n cheese was dependent on the kind of cheese, pH, moisture content, salt content, and curing temperature. Despite these findings, there are no records of streptococcic epidemics ever having been traced to cheese. The importance of using only pasteurized milk and milk products is to be especially emphasized when i t Is considered that, in many instances, human pathogens cause subclinical mastitis which cannot be readily detected. Factors Predisposing the gdder to Mastitis Infection. Since most of the organisms which commonly produce mastitis are of low invasive power, and since they appear to be present in the environment of the healthy udder without producing disease, i t follows that there must be factors other than bacterial invasion which are essential for the initiation of infection. These other factors have, in fact, come to be considered by many workers to be of greater im portance than the organisms themselves. - 29 - !• Breed. Many surveys have been made to determine whether certain breeds of cattle are more disposed to mastitis i n  fection than others. However, no proof has been forthcoming that mastitis infection i s more prevalent i n any particular breed than in any other. The apparent higher incidence of the disease in certain breeds as reported by different workers may be explained by the fact that the breed in ques tion comprised a greater percentage of the cow population studied. Murphy (76) found that the teat mucous membranes of Holsteins were more pocketed than those of Guernseys. Contrary to what would be expected, however, he did not find that the incidence of mastitis was related to the amount of pocketing. 2. Inheritance. The inheritance of susceptibility to mastitis has been suspected as a contributary factor to Infection. The effect of inheritance is probably of an indirect nature. As a result of the present-day practice of breeding cows for greater milk production, i t i s to be suspected that in the high producing cow the udder tissue is placed mder such an extreme working pressure that i t s normal metabolism is upset, and so the animal is less able to resist bacterial invasion. Also, high producing cows, especially of the Holstein breed, often have large pendulous udders which are readily injured. - 30 - Murphy et al (79) made a study of the mastitis records of two families of cows and concluded that heredity played a part in susceptibility to mastitis in the animals studied. Fincher (37) suggests that bulls improperly selected from dams with weak udders may cause serious amounts of mastitis through genetic influences. Some workers believe that the internal structure of the teat, that i s , the size of the streak canal and the amount of pocketing, which may be an inheritable factor, is related to the lodgement of bacteria and their gaining a foothold i n the udder (59). 3. Stage of Lactation. Acute mastitis due to staphylococci frequently occurs immediately after calving. It is probable that these organisms are opportunists and take advantage of the conges tion in the udder at freshening time to establish themselves. Li t t l e and Foley (66) attribute the greater incidence of staphylococcic mastitis in newly freshened cows to the pre sence of increased amounts of blood serum in the milk at this time. In in vitro studies, they found that the presence of serum overcame the bacteriostatic action of milk on sta phylococci, and concluded that the high serum content of colostrum was conducive to rapid multiplication of staphylo cocci In the udder. "Summer mastitis" caused by C. pyogenes appears to be most prevalent in dry cows. As discussed previously, - 31 - t h i s i s l i k e l y due to a combination of f a c t o r s such as cows sucking one another, and f l i e s feeding on the s e c r e t i o n l e f t on the ends of the teats a f t e r sucking. These conditions apparently favour the development of t h i s bacterium without s t i m u l a t i n g the a c t i v i t y of other m a s t i t i c organisms. With the common chronic form of the disease, there does not appear to be any p a r t i c u l a r stage i n the l a c t a t i o n p eriod when the animal i s most susceptible to i n f e c t i o n . Because the disease i s slow i n i t s development, the f i r s t v i s i b l e signs of i n f e c t i o n are not l i k e l y to appear u n t i l l a t e i n the l a c t a t i o n p e r i o d . The i n i t i a l i n f e c t i o n , how ever, undoubtedly took place much e a r l i e r . 4« Age of Cow. I t i s to be expected, that the older the cow, the greater i s the l i k e l i h o o d of I t s having m a s t i t i s . As the number of l a c t a t i o n s i n c r e a s e s , the udder t i s s u e becomes ex hausted and the teat muscles r e l a x e d , and so the animal i s l e s s able to r e s i s t invading organisms. E s p e c i a l l y i s t h i s so under present-day high production p r a c t i c e s . The chronic nature of the common form of the disease also helps to ex p l a i n why m a s t i t i s i s more prevalent i n older cows. Bryan (11) found that the majority of cows of eight years of age or older were i n f e c t e d with s t r e p t o c o c c i c m a s t i t i s . Many workers have become alarmed at f i n d i n g mas t i t i c s t r e p t o c o c c i i n the milk of f i r s t - c a l f h e i f e r s and i n - 32 - the mammary t i s s u e of v i r g i n h e i f e r s (51)• They do n o t , how ever, report whether there were any signs of i n f e c t i o n other than the presence of m a s t i t i c organisms. The b a c t e r i a p r o  bably reached the udder through a prematurely opened streak canal from calves sucking one another a f t e r d r i n k i n g m a s t i  t i c m i l k . In such instances i t i s l i k e l y t h a t the organisms are l i v i n g i n the udder i n a quiescent s t a t e , not causing any i n f e c t i o n , but ready when s u i t a b l e c o n d i t i o n s a r i s e to produce d i s e a s e . In many cases where S t r . a g a l a c t i a e has been recovered from the milk of f i r s t - c a l f h e i f e r s during the f i r s t few days a f t e r f r e s h e n i n g , the organism has l a t e r d i s  appeared from the m i l k . 5. Completeness of M i l k i n g . The e f f e c t of incomplete m i l k i n g on the develop ment of m a s t i t i s i s not w e l l agreed upon by the v a r i o u s wor kers i n t h i s f i e l d . Some i n v e s t i g a t o r s b e l i e v e that when q u a n t i t i e s of m i l k are l e f t i n the gland a f t e r m i l k i n g , the b a c t e r i a present i n the udder m u l t i p l y r a p i d l y on t h i s ex c e l l e n t food supply, and so are able to i n i t i a t e i n f e c t i o n . When i t i s r e c a l l e d , however, t h a t milk s e c r e t i o n i s a con tinuous p r o c e s s , and that the udder i s never completely f r e e of m i l k , the v a l i d i t y of the above theory may be questioned. Schalm and Mead (97) found t h a t incomplete m i l k i n g had l i t t l e e f f e c t on the p h y s i c a l appearance of milk from h e a l t h y quar t e r s but that i t appeared to aggravate the symptoms of mas-- 33 - t i t i s due to Str. agalactiae. They also found that frequent milking relieved the symptoms of mastitis caused, by Str. agalactiae. In acute cases of mastitis, frequent milking of the affected, quarter i s usually recommended. The purpose of such a procedure i s to eliminate as many organisms as pos sible from the udder and to reduce the intramammary pressure. Some workers advise the cessation of milking as an aid in curing mastitis. They claim that by so doing the secretory cells of the udder are relieved of the duty of milk secre tion, and the udder tissues can then concentrate on over coming the infecting bacteria. When the practice of overstocking is followed to i n  duce the rapid drying up of late lactation cows, the amount of milk l e f t in the udder is probably much greater than that which would remain as a result of careless milking or of not stripping after machine milking. In such cases, the leaving of such large quantities of milk in the udder is undoubtedly conducive to congestion and bacterial multiplication. 6. Size of Herd. In practice, i t has been found that mastitis i s more prevalent in larger herds than in smaller ones. This may be readily explained. In small herds the owner frequent ly looks after the complete herd himself, and consequently readily detects the earliest signs of infection. Although - 34- - there are usually better sanitary f a c i l i t i e s in large herds, because of the increased number of animals, there are more attendants, with the result that less individual attention i s given each cow. Also, milking machines are more frequently used in large herds, and so flakiness in the fore-milk, which Is often one of the f i r s t signs of a flare-up of chronic mas t i t i s , may not be detected. Another important consideration is that in small herds replacement animals are usually raised on the farm, whereas in large herds the number of cows in the herd i s maintained by the purchase of mature cows which are possible sources of mastitis. Bryan has shovm. (11) that un der identical conditions of management, there is no di f f e r  ence in the incidence of mastitis In large and small herds. 7. Milking Machines. The milking machine has frequently been incrimina ted as an important factor in the establishment of mastitis infection in the udder. If properly used, the milking machine should be a definite aid to the farmer In maintaining maxi mum milk production. In view of the fact that the let down of milk i s dependent on the secretion of oxytocin by the pituitary gland, and that the amount of this hormone in the blood i s rapidly depleted, rapid milking as can best be ac complished by the use of the milking machine should increase the amount of milk produced per cow per milking over that obtained by hand milking. - 35 - It is the careless use of the milking machine that is conducive to the development of mastitis. When the teat cups are not properly sterilized between cows, they may spread mastitic organisms from diseased to healthy cows. Too high a vacume, too rapid or uneven pulsation, letting teat cups ride too high on the teats, leaving the machine on too long, a l l produce strain, on the udder tissue, and i f carried on for any length of time may predispose the udder to mastitis infection. Numerous workers have tried to prove that a particu lar brand of milking machine i s more contributory to mas t i t i s infection than another. Because of the complexity of such a problem and of the number of factors to be considered, the results have not been very satisfactory. 8. Season and Weather. The effect of the season of the year and of weather conditions on the incidence of mastitis i s not readi ly studied because of the number of factors involved. It is known, however, that sudden chilling Is conducive to mastitis infection. It i s li k e l y that a non-infectious condition i s f i r s t set up in the udder and that bacterial invasion follows. 9. Feed. The relation of feeding practices to mastitis i n  fection is probably of an indirect nature. In trying to ob tain maximum milk production, the farmer keeps his cows on a - 36 - high protein ration. It i s l i k e l y the effect of the high ' milk production in weakening the udder and not the feed i t  self which contributes to the production of mastitis. Of interest in this connection is the report of Wied- marm (112) that mastitis i s due to a deficiency of carbohy drate in the diet. However, Wesche (110), working on the same problem was unable to duplicate Wiedmann's results. 10. Trauma. It is generally agreed that the most important factor predisposing the udder to mastitis infection is i n  jury. This may entail injury to the udder i t s e l f by bruising from high door s i l l s , stumps, etc., by kicking, horning, and by penetration of the udder tissue by barbed wire. More frequently i t entails injury to the teats especially from being stepped on. Teat injuries allow bacteria to enter the udder more easily. The point of injury acts as a focus per mitting the bacteria to gain a foothold in the udder. Fur ther, the defensive and regenerative mechanisms of the udder are occupied in repairing the injury and are less able to combat the invading organisms. 11. Other Infectious Diseases. As mentioned previously, i t is likely that Br. abortus lesions in the udder predispose to mastitis infection. Vaccinia virus and the virus of foot and mouth disease pro duce lesions on the teats and i t is thought that, on occasion, - 37 - these might a ct as f o c i f o r in v a d i n g b a c t e r i a . An i n t e r e s t i n g o b s e r v a t i o n r e l a t i v e to the e f f e c t of other b a c t e r i a on the development of m a s t i t i c organisms, i s - the r e p o r t of Pounden and. Johnson (86) that an udder s t a p h y l o  coccus enhanced, the a c i d produced i n Hotis t e s t s by m a s t i t i c s t r e p t o c o c c i . I t might be of value i n understanding the r e  l a t i v e a c t i v i t i e s of m a s t i t i c organisms i n the mammary gland to know whether or not a s i m i l a r s t i m u l a t i o n could take place i n the udder. Extent of M a s t i t i s I n f e c t i o n . Because of the m u l t i p l i c i t y of ways i n which the d i s  ease manifests i t s e l f , and because of the v a r i a t i o n s i n the extent of the changes produced i n the udder t i s s u e and i n the a l t e r a t i o n s i n the composition of the m i l k , i t i s very d i f  f i c u l t to define p r e c i s e l y what state of the udder and i t s s e c r e t i o n denotes the d i v i d i n g l i n e between a h e a l t h y and a m a s t i t i c c o n d i t i o n . In e v a l u a t i n g the l i t e r a t u r e , i t i s im portant to note on what b a s i s the var i o u s workers determine whether or not an animal i s m a s t i t i c . In most cases, abnor m a l i t i e s i n the composition of the milk can be detected e a r  l i e r i n the course o f the disease than can changes i n the udder t i s s u e . Organisms capable of causing m a s t i t i s may be i s o l a t e d from milk normal i n chemical composition and from non-indurated udders. On the other hand, i t i s sometimes im p o s s i b l e to i s o l a t e organisms from quarters obviously d i s -- 38 - eased as determined by other t e s t s . Thus i n i n t e r p r e t i n g studies on the extent to which herds are i n f e c t e d with mas t i t i s , i t i s important to take i n t o c onsideration the methods of d i a g n o s i s , s i n c e , i n general, a b a c t e r i o l o g i c a l examina t i o n of the mi l k reveals more p o s i t i v e cases than a chemical examination, and the l a t t e r i n d i c a t e s a greater number of diseased cows than a p h y s i c a l examination of the udder. Gwatkin et a l (42) i n a study of 594 cows i n 28 herds i n Ontario found 39$ of the cows p o s i t i v e f o r m a s t i t i s and 9.6$ su s p i c i o u s . They based t h e i r conclusions on the f o l  lowing t e s t s : p h y s i c a l examination of the udder, appearance of the m i l k , brom thymol blue r e a c t i o n , rennet coagulation t e s t , microscopic and b a c t e r i o l o g i c a l examination of quarter samples of m i l k . The percent of i n f e c t i o n i n i n d i v i d u a l herds ranged from 15-91. In a f u r t h e r study of 265 p o s i t i v e cases, i n c l u d i n g those considered above, they found that 29.8$ of the animals were i n f e c t e d i n one quarter o n l y , 21.9$ i n two quarters, 16.6$ i n three quarters and 31.7$ i n a l l four quarters. When considered e n t i r e l y on a quarter b a s i s , t h i s study revealed that 62.5$ of the t o t a l number of quarters of the 265 p o s i t i v e cows were i n f e c t e d . I f the 362 negative and suspicious animals from t h e i r f i r s t study are added to these 265 p o s i t i v e cows, and the r e s u l t s again considered on a quarter b a s i s , only 26.4$ of the "total number of quarters of the 627 cows are found to be I n f e c t e d . R o s e l l (87) i n a f i v e year study i n Quebec found an - 39 - average of 34-• 6$ of the 1,838 cows studied to he p o s i t i v e f o r m a s t i t i s . In i n d i v i d u a l herds the percent of cows i n f e c t e d v a r i e d from 19-97. On quarter samples of mil k he determined the catalase content, percent c h l o r i n e , pH with brom thymol b l u e , and amount of sediment In the m i l k , and made a micro scopic examination of the sediment. R o s e l l concluded from h i s studies that there were very few herds which, a f t e r ade quate t e s t i n g , were completely free from m a s t i t i s . Bryan (11) made a study of 2,715 cows i n 322 herds i n Michigan, using a leucocyte count of over one m i l l i o n per c.c. together with the presence of st r e p t o c o c c i In incubated com pos i t e m i l k samples as h i s c r i t e r i o n of I n f e c t i o n . 26.2$ of the cows studied were found to have str e p t o c o c c i c m a s t i t i s and only 14$ of the herds were fr e e from i n f e c t i o n . In England, Edwards (33) examined 809 cows i n 18 herds and found 36.4$ of the cows to be i n f e c t e d i n at l e a s t one quarter with s t r e p t o c o c c i c m a s t i t i s , as revealed by a c u l t u r a l examination of the sediment from quarter samples of mil k on Edwards' a e s c u l i n c r y s t a l v i o l e t medium (32). The Incidence i n i n d i v i d u a l herds v a r i e d from 10-84$. In a d e t a i l e d study of 218 i n f e c t e d cows, Edwards found that 36.2$ of the p o s i  t i v e cows were i n f e c t e d i n one quarter only, 27.5$ i n two quarters, 15.6$ i n three quarters and 20.7$ In a l l four quar t e r s . 55.16$ of the t o t a l number-of quarters of the i n f e c t e d animals were p o s i t i v e f o r streptococcic m a s t i t i s . On i n c l u d  ing the 514 negative cows from the f i r s t study with these 218 - 40 - p o s i t i v e cows, and c o n s i d e r i n g the r e s u l t s on a quarter b a s i s , 16.4$ of the t o t a l number of quarters were found to be p o s i  t i v e . The f i n d i n g s of these workers i n d i c a t e that i n any r e p r e s e n t a t i v e group of cows, about 25-40$ of the animals can be expected to be i n f e c t e d with m a s t i t i s , and that about 20$ of the quarters of the t o t a l number of cows w i l l be p o s i t i v e f o r t h i s d i s e a s e . In i n d i v i d u a l herds the disease may be a n t i c i p a t e d to i n v o l v e approximately 10 to over 90$ of the animals. E f f e c t of M a s t i t i s on M i l k Y i e l d . I t i s a w e l l known f a c t that m a s t i t i s reduces the amount of milk produced by I n f e c t e d animals. The extent of t h i s r e d u c t i o n depends' on the number of quarters i n v o l v e d and on the seriousness of the c o n d i t i o n i n each q u a r t e r . I t reaches i t s u l t i m a t e when the a f f e c t e d quarter i s completely atrophied and secretes no m i l k at a l l . Because the amount of milk produced even by normal cows i s such a v a r i a b l e q u a n t i t y and depends on so many f a c t o r s , i t i s very d i f f i c u l t to d e t e r  mine the re d u c t i o n i n milk y i e l d brought about by s u b - c l i n i c a l m a s t i t i s . Among the f i r s t to work on t h i s problem were Shaw and Beam (101), who found that q u a r t e r s , p o s i t i v e to the brom thymol b l u e , percent c h l o r i n e , c e l l count and catalase t e s t s , produced approximately 22$ l e s s m i l k than the opposite non-- 41 - i n f e c t e d quarters, a f t e r the maximum v a r i a t i o n between the y i e l d s from non-infected quarters had been allowed f o r . I t i s important to note, however, that the r e s u l t s were computed by the d i f f e r e n c e i n y i e l d between the i n f e c t e d quarter and the opposite non-infected one. Thus, i f the i n e f f i c i e n c y of one quarter had stimulated the other three to greater than normal milk production, as appears to be the case where a s i n g l e or at the most two quarters are i n f e c t e d , t h i s compen satory a c t i o n would tend to increase the apparent d i f f e r e n c e s between the y i e l d s of Infected and non-Infected quarters. Minett and M a r t i n (73) made a study of two herds of Ayrshire and F r i e s i a n cows over a t o t a l of 373 l a c t a t i o n p e r i o d s . Both herds were i n f e c t e d with s u b - c l i n i c a l m a s t i t i s due to S t r . agalactiae as determined by a b a c t e r i o l o g i c a l examination of the sediment from quarter samples of m i l k . The t o t a l m i l k production of each cow f o r an e n t i r e l a c t a t i o n period was corrected f o r age, length of dry period and month of c a l v i n g , and the corrected r e s u l t s were subject to s t a t i s  t i c a l a n a l y s i s . An average reduction i n milk y i e l d of 954 pounds per l a c t a t i o n was found f o r the i n f e c t e d cows stu d i e d . White et a l ( i l l ) studied 35 cows over a t o t a l of 198 l a c t a t i o n p eriods. On comparing the r e s u l t s obtained from negative cows with those from the same cows a f t e r becoming I n  fected w i t h s u b - c l i n i c a l m a s t i t i s as determined by the brom thymol blue t e s t , sediment t e s t , leucocyte count and bac t e r i o l o g i c a l examination of the m i l k , they found that when - 42 - only one quarter of the udder was a f f e c t e d , there was no de crease i n the milk y i e l d of the cow. A r e d u c t i o n i n y i e l d takes p l a c e , however, when two quarters are inv o l v e d , and t h i s r e d u c t i o n i n c r e a s e s with each a d d i t i o n a l quarter Infected, u n t i l when a l l four quarters are inv o l v e d milk production Is decreased by about 15-20$. Hucker et a l (53) In a study of the milk production records of 35 cows over 3 l a c t a t i o n periods concluded that when the only evidence of m a s t i t i s was a leucocyte count of over 500,000 per c . c , or the presence of s t r e p t o c o c c i , there was no red u c t i o n i n milk y i e l d . The y i e l d was not a f f e c t e d u n t i l the disease had progressed to the point where the f o r e  mil k was a l k a l i n e to brom thymol blue or the p h y s i c a l appear ance of the milk was g r o s s l y a l t e r e d . They a l s o concluded that a m i l d i n f e c t i o n of a l l f o u r quarters had about the same e f f e c t on milk production as a more severe i n f e c t i o n of one quarter only. In the early stages of chronic m a s t i t i s , the milk y i e l d i s undoubtedly a f f e c t e d very l i t t l e i f at a l l . As the disease progresses, more secretory c e l l s are destroyed and the amount of milk produced by the a f f e c t e d quarter diminishes according l y . In cases of s u b - c l i n i c a l m a s t i t i s i t Is not l i k e l y that the r e d u c t i o n i n the milk y i e l d Is very great, but when the p o t e n t i a l milk production of a la r g e number of such i n f e c t e d cows i s considered, the red u c t i o n of y i e l d assumes considerable economic Importance. - 43 - E f f e c t of M a s t i t i s on the., Composition of M i l k . The e f f e c t of m a s t i t i s i n f e c t i o n on the chemical com p o s i t i o n and p h y s i c a l p r o p e r t i e s of milk have been discussed at length by R o s e l l (88). In m a s t i t i s , the f a t content of the m i l k i s u s u a l l y lowered. The s o l i d s - n o t - f a t content i s also decreased. The calcium and potassium contents are d i - m i n i s t e d , but the amounts of sodium and c h l o r i n e are increased. Lactose and casein are reduced but albumin and the non-protein nitrogen f r a c t i o n i n c r e a s e . The pH i s generally more a l k a l i n e than u s u a l . The e l e c t r i c a l c o n d u c t i v i t y i s increased due to the greater s a l t content. The v i s c o s i t y of the m i l k , on the other hand, i s decreased. M i l k from m a s t i t i c animals u s u a l l y shows a reduced s t a b i l i t y to heat. This i s probably due to i t s decreased c a l  cium content and increased albumin content and p o s s i b l y also to i t s high pH. Welch and Doan (109) found that when m a s t i t i c milk was mixed with normal m i l k and the r e s u l t i n g mixture con centrated to a s o l i d s content comparable to that of condensed m i l k , the m a s t i t i c m i l k tended to render the normal m i l k l e s s stable towards heat. I t can thus be seen that i f appreciable q u a n t i t i e s of milk from m a s t i t i c animals are used i n the manu facture of evaporated and condensed m i l k , serious d i f f i c u l t i e s -may be encountered i n the s t e r i l i z a t i o n of these products. As a r u l e , m a s t i t i c milk takes longer to coagulate with rennet than normal m i l k and the r e s u l t i n g curd i s not so f i r m (103)• This i s probably r e l a t e d to the decreased - u - amounts of c a s e i n and calcium and to the hig h pH i n m a s t i t i c m i l k . Johns et a l (57) found that the y i e l d of Cheddar cheese was reduced when milk from cows with s u b - c l i n i c a l m a s t i t i s was used. They d i d n o t , however, f i n d the q u a l i t y of the cheese to be a f f e c t e d . M a s t i t i c m i l k , because of i t s low curd t e n s i o n , tends to produce weak-bodied cheese. I f milk from cows more s e r i o u s l y I n f e c t e d than those studied by Johns et a l were used to make cheese, I t Is very l i k e l y that the f l a v o u r and body of the r e s u l t i n g cheese would be a f f e c t e d (54). The low calcium, c a s e i n and l a c t o s e content of mas t i t i c m i l k renders i t I n f e r i o r to normal m i l k as a source of these substances f o r human n u t r i t i o n . In view of the p r e  valence of m a s t i t i s among the d a i r y cow p o p u l a t i o n , i t i s p r o  bable that these d e f i c i e n c i e s have not r e c e i v e d the a t t e n t i o n warranted. Another c o n s i d e r a t i o n i n t h i s connection i s the report of Schdnberg (98) that milk from m a s t i t i s - i n f e c t e d cows i s d e f i c i e n t i n r i b o f l a v i n . The extent of the chemical a l t e r a t i o n i n the milk of m a s t i t i c animals i s r e l a t e d to the s e v e r i t y o f the disease - the more advanced the case the greater Is the a l t e r a t i o n . Thus the e f f e c t o f the abnormal chemical composition of mas t i t i c milk on processed milk and on human n u t r i t i o n i s de pendent on the s e v e r i t y of the i n f e c t i o n i n the i n d i v i d u a l cow, and a l s o on the amount of abnormal milk i n the t o t a l m i l k supply. - 45 - EXPERIMENTAL. Outline of Research Council,,, Proj^ra^. The work undertaken by the Research Council on mas t i t i s has.as i t s o b j e c t i v e the lessening of the incidence of t h i s disease i n the d a i r y herds of B r i t i s h Columbia. Through out the course of t h i s study, major emphasis has been placed on the f o l l o w i n g aspects of the problem, which are discussed under separate headings: 1. a survey of the extent of m a s t i t i s i n f e c t i o n i n B r i t i s h Columbia. 2. a study of the f a c t o r s predisposing the udder to m a s t i t i s i n f e c t i o n . 3. an evaluation of the t e s t s used f o r the de t e c t i o n of m a s t i t i s . 4- a d e t a i l e d study of the organisms associated with m a s t i t i s and an evaluation of the methods used f o r t h e i r c l a s s i f i c a t i o n . 5. a study of the methods used f o r the c o n t r o l of the spread of m a s t i t i s i n f e c t i o n . 6. a study of the treatments used f o r m a s t i t i s . The experimental data to be discussed under the above headings have been obtained from three sources: 1. a preliminary study of the extent of m a s t i t i s i n f e c t i o n i n the North Okanagan V a l l e y which has been d e s i g  nated as the "North Okanagan Study". - 46 - 2. a general survey of the incidence of the disease in British Columbia which has been called the "General Sur vey" . 3. a detailed study of four experimental herds, es tablished for the purpose of determining the efficiency of a management program for the control of the disease. This project has been referred to as the "Experimental Herd Study". Under some headings, the results from a l l three sources are discussed, under others, only those from one or two. In a l l cases, however, the data from each of the three projects are treated separately. General Methods. 1. North Okanagan Study. During the course of a study of some of the pro blems associated with cheese-making in the North Okanagan Valley, an examination was made of the milk supply of a cheese factory in that area, employing the methylene blue reductase test, the resazurin test, and a semi-quantitative Breed count on the bulk milk from each shipper. Following this study, nine herds were selected on the basis of the rapid partial reduction and slow complete reduction of methylene blue and resazurin, and of the presence of,more than the average num ber of leucocytes and of chaining cocci in the bulk herd milk. These herds were then subjected to analysis for mastitis. - 4? - M i l k samples were taken as a s e p t i c a l l y as p o s s i b l e i n  to s t e r i l e sample b o t t l e s . In each case, about 4° c.c. of f i r s t s t r i p p i n g s from each quarter of the udder were obtained. The samples were taken at the evening m i l k i n g , l e f t overnight at room temperature and t e s t e d the next morning. The f o l  lowing t e s t s f o r m a s t i t i s were performed on the samples: p h y s i c a l appearance of the milk, Geneva b l o t t e r t e s t , modi f i e d Whiteside t e s t , pH determination with brom c r e s o l pur p l e , H otis t e s t , percent c h l o r i n e , leucocyte count and blood agar p l a t e technique. 2. General Survey. The general survey o f the extent of m a s t i t i s i n  f e c t i o n i n B r i t i s h Columbia was made p o s s i b l e by the co-opera t i o n of i n t e r e s t e d p r a c t i s i n g v e t e r i n a r i a n s throughout the prov i n c e . These v e t e r i n a r i a n s undertook the r e s p o n s i b i l i t y o f c o l l e c t i n g m i l k samples from s u s p i c i o u s and p o s i t i v e cows and of o b t a i n i n g f i e l d data r e l e v a n t to the case being studied and to the general problem of m a s t i t i s c o n t r o l . As r e q u i r e d , the v e t e r i n a r i a n was s u p p l i e d with packages c o n t a i n i n g f o u r s t e r i l e screw-cap, 1 1/2 oz., b o t t l e s f o r the c o l l e c t i o n of m i l k samples, to be forwarded to the l a b o r a t o r y f o r examination. He was a l s o s u p p l i e d with a f i e l d note book f o r the c o l l e c t i o n of data regarding the case h i s  t o r y of the cow under study and r e l a t e d information. This data was to be sent to the l a b o r a t o r y with the milk samples. - 48 - A copy of the data sheet from the f i e l d note book i s ap pended hereto as E x h i b i t "A". The f i e l d note book also pro vided f o r the recording of observations concerning the h i s  t o r y of the case subsequent to the taking of the i n i t i a l m i l k samples. A copy of t h i s h i s t o r y sheet i s appended as Ex h i b i t «B«. M i l k samples were taken from c l i n i c a l l y p o s i t i v e and suspicious animals and o c c a s i o n a l l y from negative cows i n h i g h l y i n f e c t e d herds. Quarter samples were to be taken from a l l four quarters of the udder, even though every quarter was not n e c e s s a r i l y i n f e c t e d . In so f a r as was p o s s i b l e , f i r s t s t r i p p i n g s were to be taken. The samples were sent to the labo r a t o r y by m a i l or express. I c i n g of the samples i n t r a n  s i t was not c a r r i e d out. The samples were tested as soon as p o s s i b l e a f t e r reaching the l a b o r a t o r y , and the r e s u l t s of the laboratory diagnosis were sent to the v e t e r i n a r i a n as e x p e d i t i o u s l y as p o s s i b l e , e i t h e r by mail or telephone, i n order to a s s i s t him i n p r e s c r i b i n g treatment measures. The f o l l o w i n g laboratory t e s t s were employed f o r the detection of m a s t i t i s : p h y s i c a l examination of the m i l k , modified White side t e s t , pH determination w i t h brom c r e s o l purple, Hotis t e s t , percent c h l o r i n e , leucocyte count, and blood agar p l a t e technique. 3. Experimental Herd Study. In order to study the e f f e c t of a s t r i c t manage-- 49 - EXHIBIT "A". DATA SHEET FOR THE COLLECTION OF FIELD DATA RELEVANT TO THE CASE UNDER STUDY. MASTITIS RESEARCH PROJECT Doctor. Date... Herd Owner Address /Tattoo or Cow Ident. < E a r T a g B r e e d A g e Date of Calving Daily Milk Production- Duration of Type of Mastitis Attack Case History Herd History- Daily Milkings How Milked ... M P.M.. M P.M.. Machine:- Name of Type of Mfgr Unit Feeding Methods Percent Protein Supplements Used Sanitary Methods , , Treatment Milk Samples for Lab.: R F L F R H LH.... Time First Stripping: Yes.. No.. Please keep record of Treatment and Results on Page 3. - 50 - EXHIBIT "B". HISTORY SHEET FOR THE COLLECTION OF DATA REGARDING THE CASE HISTORY SUBSEQUENT TO THE TAKING OF THE INITIAL MILK SAMPLES. Herd Owner. C o w I d e n t . ( T a t t o o o r [ Ear Tag Breed.... Age Condition of Udder Treatment Used Repeated Clinical Notes Results Obtained Recovered Condition of Udder - 51 - ment program on the c o n t r o l of m a s t i t i s , four herds, known to have a high incidence of m a s t i t i s , were selected i n d i f f e r e n t parts of the Lower Fraser V a l l e y . A l e g a l agreement out l i n i n g the duties and r e s p o n s i b i l i t i e s of the Research Coun c i l , the v e t e r i n a r i a n i n charge of each herd, and the co-op e r a t i n g farmer was drawn up. The Council assumed the r e s  p o n s i b i l i t y f o r the conduct of the research p r o j e c t . The v e t e r i n a r i a n was made responsible f o r the f i e l d , work required f o r the program, and had charge of the supervision of the mi l k i n g p r a c t i c e s to be followed by the co-operating farmer, as o u t l i n e d i n the schedule to the agreement which i s given below. The herd owner undertook to give the v e t e r i n a r i a n access to h i s herd at a l l times, and agreed to carry out the herd management and m i l k i n g procedures as p r e s c r i b e d . The farmer also assumed the r e s p o n s i b i l i t y of rep o r t i n g a l l cases of m a s t i t i s attacks immediately to the v e t e r i n a r i a n , so that there would be the c l o s e s t l i a s o n between the adm i n i s t r a t i o n of treatment by the v e t e r i n a r i a n , and the work of the l a b o r a  tory on milk samples procured from the diseased animals. SCHEDULE.. TO THE AGREEMENT ENTERED INTO. BY THE RESEARCH COUNCIL, THE VETERINARIAN, AND THE HERD OWNER. Outline of Herd Management P r a c t i c e s to be Carried Out By a l l Co-operating Dairy Farm Owners A. GENERAL. SANITATION Barns, paddocks and pasture f i e l d s used by m i l k i n g cows - 52 - to be kept i n good general c o n d i t i o n . Fences i n good r e p a i r - no f r e e wire, hanging l o o s e . F i e l d s f r e e of protuding o b j e c t s , stumps, r o o t s , l o g s , brush and stone upon which udder i n j u r i e s c ould occur. S t a l l s and stan chions to be kept i n good r e p a i r to avoid a l l udder i n  j u r i e s . B. STABLE SANITATION Stable to be kept c l e a n , b r i g h t and a i r y . Walls and c e i l  i ngs to be cleaned once y e a r l y . S t a l l s and g u t t e r s to be washed d a i l y with running water or washed down once each week with l y e s o l u t i o n , I l b . to 20 qts. water. Bedding - a l l s t a b l e s to be kept w e l l bedded with c l e a n , dry straw or other m a t e r i a l . C. FEEDING Feed w e l l balanced r a t i o n s p l u s necessary mineral and v i  tamin supplements when i n d i c a t e d . P r o t e i n i n r a t i o n not to exceed 16$. In case of m a s t i t i s f l a r e - u p or acute a t  tack i n any cow or cov^s, g r a i n r a t i o n s f o r such animal to be reduced at once, and steps taken to i s o l a t e such animals from m i l k i n g l i n e . D. TREATMENT OF MASTITIS CASES Udder i n j u r i e s or t e a t i n j u r i e s to be handled o n l y at and with the advice of the co-operating V e t e r i n a r i a n . Under no circumstances are m i l k i n g tubes, teat bogies or d i l a  t o r s to be used except by and with the advice of the V e t e r i n a r i a n . E. REPLACEMENT COWS Owners are s t r o n g l y advised to r a i s e , i n s o f a r as p o s s i b l e , t h e i r replacement requirements. Cows or i n - c a l f h e i f e r s • purchased f o r replacement are to be i s o l a t e d from the m i l k i n g f o r a p e r i o d of t h i r t y days and to have passed two clean t e s t s f o r M a s t i t i s before being admitted to the m i l k i n g l i n e of cows. F. CALF REARING Owners are requested to r a i s e a l l h e i f e r calves on milk known to be from healthy, M a s t i t i s - f r e e cows. M i l k from M a s t i t i s - i n f e c t e d cows should be p a s t e u r i z e d or b o i l e d f o r one minute before being f e d to any l i v e s t o c k . G. RECOMMENDATIONS FOR MILKING PROGRAMME ( l ) Have a p r e l i m i n a r y t e s t made f o r M a s t i t i s on every cow m i l k i n g on the farm, ( i i ) Arrange cows i n m i l k i n g order i n the d a i r y barn i n such a manner that a l l c l e a n cows are milked i n Group 1, suspicious or questionable cows milked as Group 2, and M a s t i t i c or i n f e c t e d cows as Group 3-- 53 - EQUIPMENT REQUIRED Supplementary (1) One wiping c l o t h f o r each cow i n m i l k i n g herd (un bleached cotton 18" square or 12" x 24" recommended), ( i i ) Four sa n i t a r y p a i l s to supplement m i l k i n g equipment: P a i l No. 1 - Chlorine S o l u t i o n (250 ppm.) to be kept as near 1 3 0° F . as p o s s i b l e . A l l towels f o r udder Trashing to be kept i n t h i s s o l u t i o n . P a i l No. 2 - Empty p a i l to receive used towels as each udder i s washed. P a i l No,. 3 - Clean, c o l d water to r i n s e m i l k i n g cups as removed from each coir. P a i l No. A - Warm ch l o r i n e s o l u t i o n (250 ppm.) to Im merse and hold m i l k i n g cups between each cow being milked. ( i l l ) S t r i p cup and black screen of f i n e mesh to be used as a check t e s t on f i r s t s t r i p p i n g s immediately be for e applying m i l k i n g u n i t , ( i v ) L i g h t weight wheeled truck or suspended c a r r i e r to accommodate above equipment and m i l k i n g p a i l s - c a r r i e d w e l l above spotter l e v e l i n m i l k i n g a l l e y , (v) Dipping Cup - small cup of c h l o r i n e s o l u t i o n to be used to dip each teat of the milked cow as soon as cows have been g i v e n ' f i n a l check s t r i p p i n g . (NOTE: The ch l o r i n e s o l u t i o n i n cup to be discarded a f t e r each cow.) M i l k i n g Equipment X i l I f machine - two u n i t s per operator i s recommended, ( i i ) One extra m i l k i n g p a i l with l i d per u n i t i n opera t i o n s i s e s s e n t i a l so that no pouring of m i l k from p a i l to other containers be required at the m i l k i n g l i n e . ( i i i ) M i l k i n g scales and recording sheets. Routine of M i l k i n g ( i ) Prepare a l l equipment, ch l o r i n e s o l u t i o n s , washing cl o t h s and s t r i p cups i n readiness on movable p l a t  form. ( i i ) Check temperature of ch l o r i n e s o l u t i o n and renew i f temperature drops 130° to 1 0 0° F . unless some arrange ment can be made to maintain heat, ( i i i ) Wash the udder c a r e f u l l y and completely. Ring towel out and wipe dry. Discard towel i n t o empty p a i l . ( i v ) Using s t r i p cup, draw one or two f u l l hand squeezes of milk from each quarter i n r o t a t i o n through screen and note character of s e c r e t i o n . I f c l e a r of a l l f l a k e s , apply machine, (v) Apply m i l k i n g machine f o r three to four minutes. - 54 - Watch c l o s e l y to remove as soon as cow i s milked out. Remove machine promptly, (vi ) Rinse teat cups i n co l d water and then immerse i n ch l o r i n e s o l u t i o n , ( v i i ) S t r i p p i n g cow - This should be done immediately a f t e r machine i s removed. Use only f u l l hand squeezes and avoid excessive s t r i p p i n g , ( v i i i ) Use dipping cup - on a l l four quarters i n r o t a t i o n taking a f r e s h cup of c h l o r i n e s o l u t i o n and d i s  carding i t a f t e r treatment of each i n d i v i d u a l cow. NOTE: I f two men are a v a i l a b l e at m i l k i n g , i t i s advisable to d i v i d e the work as f o l l o w s : One man to wash udders, do f i r s t s t r i p p i n g t e s t and operate m i l k i n g machine. Second man to f i n i s h m i l k i n g by f i n a l s t r i p p i n g , complete the operation by a p p l i c a t i o n of c h l o r i n e s o l u t i o n with dipping cup to each t e a t and weigh and record the mil k f o r each i n d i v i d u a l cow. I . GENERAL Close a p p l i c a t i o n of the above p r i n c i p l e s w i l l m a t e r i a l l y a s s i s t i n preventing M a s t i t i s i n your herd.' I t w i l l as s i s t you i n e l i m i n a t i n g the disease promptly by knowing a l l i n f e c t e d cows; and i n general routine p r a c t i c e i t w i l l speed up the mi l k i n g operation so as to permit i t s com p l e t i o n i n a clea n , s a n i t a r y manner that w i l l pay d i v i  dends . Complete co-operation, exchange of important observations as between the owner, the v e t e r i n a r i a n and the labora t o r y workers w i l l go f a r towards the ultimate objective of the complete c o n t r o l of M a s t i t i s i n B r i t i s h Columbia d a i r y herds. M i l k samples were taken from a l l cows i n the e x p e r i  mental herds at frequent i n t e r v a l s and tested i n the l a b o r a  tory f o r chemical abnormality and f o r the presence of mas t i t i s organisms. P h y s i c a l examinations of the udders of a l l cows were to be made r e g u l a r l y by the v e t e r i n a r i a n i n charge of each herd. In a d d i t i o n , any and a l l treatments used f o r m a s t i t i s i n these herds, and the r e s u l t s of such treatments, - 55 - were to be recorded by the v e t e r i n a r i a n . I t was planned to carry on the experimental herd study f o r at l e a s t two or three years, and so the r e s u l t s reported he r e i n on t h i s p r o j e c t are to be considered as being i n the nature of a progress r e p o r t . The data from t h i s study i n the present report cover the f i r s t two routine t e s t s on the four herds, the second t e s t being performed approximately three months a f t e r the f i r s t . In a d d i t i o n , two of the herds (#1 and #2) were each t e s t e d once before the experimental herd study was i n i t i a t e d . These t e s t s were made about two months before the f i r s t general t e s t on a i l herds. The data from these two examinations are included i n t h i s s e c t i o n of the re p o r t . When an e n t i r e herd was tested at one time, the milk samples were taken by the la b o r a t o r y personnel. The udders and teats were c a r e f u l l y washed w i t h a warm ch l o r i n e s o l u t i o n before the samples were obtained. For the f i r s t part of the work, the samples consisted of the very f i r s t m ilk obtained from a quarter. For the l a s t s e r i e s of t e s t s , however, two streams of milk were discarded from each quarter before the samples were c o l l e c t e d . I t was hoped by t h i s procedure to eliminate most of the contaminating organisms which may have reached the lower part of the udder through the teat canal during the i n t e r v a l between m i l k i n g s . In the f i r s t t e s t i n g of the four herds, quarter samples of milk were taken from a l l cows. In the second t e s t , composite samples were obtained - 56 - from those animals i n herds #1 and #4 which were found f r e e from evidence of m a s t i t i s at the f i r s t t e s t i n g . The samples were taken at the evening m i l k i n g , l e f t at room temperature over night and tested the next day. The same t e s t s f o r m a s t i t i s were employed as i n the general sur vey, namely, p h y s i c a l examination of the m i l k , modified White side t e s t , pH determination w i t h brom c r e s o l p urple, Hotis t e s t , percent c h l o r i n e , leucocyte count and blood agar p l a t e technique. At the f i r s t general t e s t i n g , representative colonies were picked from the plates of a l l samples taken from the cows of herd #1 and the c u l t u r e s so obtained were subjected to d e t a i l e d study. At the second t e s t i n g a s i m i  l a r study was c a r r i e d out on organisms i s o l a t e d from the milk of the animals i n herd #2. Methods of C l i n i c a l Diagnosis. In the case of acute m a s t i t i s , the symptoms of the disease are r e a d i l y observed. With s u b - c l i n i c a l m a s t i t i s , how ever, the f o l l o w i n g procedures must be used i n order to de te c t l e s i o n s i n the udder: the examination of the teat f o r patency and f i b r o s i s , the examination of the udder f o r sym metry and balance, and the p a l p a t i o n of the milked-out udder f o r the extent of i n d u r a t i o n and f i b r o s i s . These methods are described i n d e t a i l and t h e i r usefulness discussed by U d a l l and Johnson (106). - 57 - Methods of Laboratory Diagnosis. The methods used f o r the l a b o r a t o r y diagnosis of mas t i t i s were a l t e r e d slighHy from time to time throughout the study as improvements suggested themselves. An o u t l i n e of the various procedures and the changes made i n them i s given beloYf. 1. P h y s i c a l Appearance of the M i l k . A l l m ilk samples were subjected to a v i s u a l exami n a t i o n f o r the presence of f l a k e s , c l o t s , serum, blood, e t c . 2. Modified Whiteside, Test. The modified Whiteside t e s t was c a r r i e d out on a l l samples according to the method of Murphy and Hanson (77). 5 l o o p f u l s of milk were placed on a microscope s l i d e and 1-2 l o o p f u l s of 1 N sodium hydroxide were mixed with them f o r about 4-0 seconds. The r e s u l t s which are best observed against a dark background, are i n t e r p r e t e d as f OIIOTJS: 0 r e a c t i o n : uniform opaque f l u i d . * r e a c t i o n : s i m i l a r to the 0 r e a c t i o n with the a d d i t i o n of a few very small white f l a k e s . 1* r e a c t i o n : s i m i l a r to * r e a c t i o n but with more and l a r g e r f l a k e s . 2+ r e a c t i o n : c l e a r background with large white f l a k e s . 3* r e a c t i o n : c l e a r background with large f l a k e s t e n ding to clump together. 4--S- r e a c t i o n : v i s c i d mass l i f t i n g o f f the s l i d e with - 58 - the l o o p . 3. Geneva B l o t t e r Test. The Geneva b l o t t e r t e s t i s a t e s t f o r pH which has been adapted f o r use i n the f i e l d . I t consi s t s of a piece of b l o t t i n g paper with four small areas impregnated w i t h brom thymol blue on which a drop or two of milk from each quarter i s placed. Although t h i s t e s t was designed f o r use i n the barn, i t was employed on a l l samples i n the North Okanagan study, 1 or 2 drops of m i l k being applied to each area of dye. 4» pH Determination with_Brpm_Cresol_Purple. The determination of pH was c a r r i e d out i n conjunc t i o n with the Hotis t e s t . 9.5 c.c. of milk were placed i n a s t e r i l e rubber-stoppered t e s t tube. 0.5 c.c. of a 0.5$ aqxie- ous s o l u t i o n of brom c r e s o l purple were added to each tube. The tubes were then shaken and the colour of the mil k observed. The colour reactions are i n t e r p r e t e d as f o l l o w s : normal: greyish mauve colour, very s l i g h t l y a l k a l i n e : ) s l i g h t l y a l k a l i n e : ) i n c r easing shades of purple, a l k a l i n e : J very a l k a l i n e : deep purple colour, very s l i g h t l y acid:) s l i g h t l y acid:) increasing shades of yellow, acid:) very a c i d : b r i g h t canary yellow c o l o u r . During part of the work, a brom c r e s o l purple colour standard was employed. This standard was, however, not com p l e t e l y s a t i s f a c t o r y , owing to the dif f e r e n c e s obtained i n the depth of colour with milks of d i f f e r e n t f a t content. Of ne c e s s i t y , the standard was prepared from skim m i l k . M i l k s from - 59 - Jersey and Guernsey cows tend to give a l i g h t e r , more b l u i s h shade than the standard, whereas milks from Ayrshire and Hol- s t e i n cows give shades of colour more approaching those of the standard. Thus the use of the standard f o r high f a t con tent milks would tend to grade these samples as more normal i n pH than should a c t u a l l y be the case. For most of the sam ples t e s t e d , however, no standard was used, and i t was found r e l a t i v e l y easy, a f t e r a l i t t l e experience, to d i s t i n q u i s h between the various shades of c o l o u r , e s p e c i a l l y i f a f a i r l y l a rge number of samples were examined at one time. 5. Hotis Test. The procedure of Hotis and M i l l e r (49) was used i n c a r r y i n g out the Hotis t e s t . The tubes used f o r the pH determination were incubated at 37°C. and examined a f t e r 24 and 48 hours f o r colour change, presence of f l a k e s along the sides and bottom of the tube, degree of c l o t t i n g and d i g e s  t i o n of the m i l k . Unfortunately, i n the North Okanagan study, the r e s u l t s of t h i s t e s t were very u n s a t i s f a c t o r y . Owing to inexperience at the time i n i n t e r p r e t i n g the data obtained by t h i s t e s t , the r e s u l t s of the Hotis t e s t from the North Okana gan study are not recorded i n the t a b l e s . P o s i t i v e f i n d i n g s , however, were used as confirmatory evidence i n c l a s s i f y i n g the quarters. 6• Percent C h l o r i n e . E o s e l l ' s method (87) f o r determining the c h l o r i n e - 60 - content of mil k was used throughout the study. The t e s t was c a r r i e d out as f o l l o w s : 4-0 c.c. of d i s t i l l e d water, 10'c.c. of m i l k and 8 drops of a 10$ aqueous s o l u t i o n of potassium chrornate were placed i n a f l a s k . N/10 s i l v e r n i t r a t e was t i  t r a t e d i n t o the above s o l u t i o n u n t i l the f i r s t permanent co l o u r change was noted. The percent of ch l o r i n e i n the mil k i s obtained by m u l t i p l y i n g the number of c.c. of AgNO-j r e  quired by 0.0355- Owing to an i n s u f f i c i e n t supply of s i l v e r n i t r a t e , only about h a l f of the samples taken i n the North Okanagan study could be examined by t h i s t e s t . 7. Leucocyte Count. The leucocyte count was c a r r i e d out by a modi f i c a t i o n of the Breed technique f o r counting the b a c t e r i a i n m i l k . Smears were made by spreading a 2 mm. l o o p f u l (i.D.) of m i l k over 1 sq. cm. area on a microscope s l i d e and were stained by the Newman method (80). The number of leucocytes in.30 f i e l d s were counted w i t h a c a l i b r a t e d microscope, and the r e s u l t s c a l c u l a t e d to give the c e l l count per c.c. of m i l k . I t should be noted, however, that the quantity of milk d e l i v e r e d by the 2 mm. loop was s l i g h t l y l e s s than the 0.01 c.c. employed f o r the standard Breed technique. Thus the counts recorded herein may be s l i g h t l y lower than those ob tained by other workers using the standard method. In the North Okanagan study and the general survey the leucocyte count was performed on the f r e s h milk samples. - 61 - I n the experimental herd study, however, the samples, a f t e r the pH, percent c h l o r i n e and Whiteside t e s t s had been p e r f o r  med, were incubated at 37°C. f o r 16-24 hours, and the l e u  cocyte smears made a f t e r i n c u b a t i o n . By t h i s procedure, the b a c t e r i a i n the sample, e s p e c i a l l y the s t r e p t o c o c c i , are given a chance to m u l t i p l y and so should be more r e a d i l y de tected on microscopic examination of the incubated m i l k . Since c a r e f u l l y taken m i l k samples do not u s u a l l y c l o t during t h i s period of i n c u b a t i o n , the procedure does not i n t e r f e r e w ith the preparation of a uniform smear f o r the leucocyte count, and, i n a d d i t i o n , u s e f u l information i s obtained r e  garding the types of organisms present. The samples studied i n the general survey were frequently two or three days o l d on reaching the l a b o r a t o r y , and so i t was deemed inad v i s a b l e to incubate them before making the smears. However, i n some cases i n the general survey, the leucocyte count was p e r f o r  med on the unincubated sample, the sample was then Incubated f o r 16-24 hours, and another smear was made to study the types of organisms which may have developed during i n c u b a t i o n , 8. Microscopic Study of the Organisms Present i n the M i l k Samples. The r e l a t i v e numbers and kinds of b a c t e r i a present i n the smears, made to determine the leucocyte count of the milk samples, were noted. The smears were always c a r e f u l l y examined f o r short and long chaining s t r e p t o c o c c i . - 62 - 9 • Cultural Study of the Organisms, Present i n the M i l k S_amples. The techniques used f o r the preparation of blood agar p l a t e s from the milk samples were a l t e r e d a number of times during the course of the study. Several tryptose agar bases were used i n the e a r l y part of the work f o r making the blood agar, and i t was found that most of these media gave comparable r e s u l t s . The medium f i n a l l y s e l ected and used f o r the greater part of the study was Difco tryptose blood agar base which has the f o l l o w i n g composition: beef e x t r a c t : 0.3$ tryptose: 1.0$ sodium c h l o r i d e : 0.5$ agar: 1.7$ For part of the general survey and f o r a l l of the experimental herd study, the milks were plated on Edwards1 c r y s t a l v i o l e t a e s c u l i n agar (32) as w e l l as on the tryptose blood agar. Edwards' medium i s designed to f a c i l i t a t e the d i f f e r e n t i a t i o n of s t r e p t o c o c c i by suppressing the growth of other organisms while p e r m i t t i n g the s t r e p t o c o c c i to develop normally. At f i r s t , t h i s medium was prepared as d i r e c t e d by Edwards with n u t r i e n t agar, c r y s t a l v i o l e t and a e s c u l i n . I t i s known, however, that some st r e p t o c o c c i do not grow par t i c u l a r l y w e l l on n u t r i e n t agar, and so, i n the l a t t e r part of the study, Difco tryptose blood agar base was s u b s t i t u t e d f o r the n u t r i e n t agar i n t h i s medium. In view of the work of Brown (9) on the use of - 63 - horse blood f o r the d i f f e r e n t i a t i o n of s t r e p t o c o c c i by hemo l y s i s , and i n a n t i c i p a t i o n that s t r e p t o c o c c i would be the p r i n c i p a l organisms encountered, 5$ d e f I b r i n a t e d horse blood was used f o r making the blood agar pl a t e s i n the North Okana- gan study and i n the f i r s t part of the general survey. As the work proceeded, however, appreciable numbers of staphylococci were encountered, and, since horse blood i s unsuitable f o r the study of the hemolytic p r o p e r t i e s of s t a p h y l o c o c c i , i t was de cided to use the more r e a d i l y a c c e s s i b l e cow's blood f o r the blood agar p l a t e s . I t was found that both streptococci and staphylococci grew w e l l on t h i s medium, and that hemolysis by both organisms could be r e a d i l y d i s t i n g u i s h e d . In order to conserve blood i t was found necessary to reduce the concen t r a t i o n of blood, from 5$ to 3% f o r part of the study and the r e s u l t s obtained w i t h t h i s m o d i f i c a t i o n were e n t i r e l y s a t i s  f a c t o r y . Throughout the study, 0.005$ of p-aminobenzoic a c i d was added to the basic medium f o r the blood agar plates In order to counteract the i n h i b i t o r y a c t i o n of sulfanilamide on any s t r e p t o c o c c i which might be present i n the samples, i n the event that the cow had been treated with sulfanilamide p r i o r to sampling and that some of the drug had been shed, i n the m i l k . The technique adopted f o r p l a t i n g was as f o l l o w s : 3-4 drops of the unincubated m i l k sample were used undiluted f o r each pour p l a t e . Approximately 13 c.c. of medium were - 64 - employed to make each p l a t e . The required amount of d e f i b - r i n a t e d blood was added to the melted and cooled basic med ium j u s t p r i o r to pouring the p l a t e s . The pl a t e s were i n  cubated at 37°C. f o r 24 hours. They were examined at the end of t h i s time f o r type of growth, hemolysis, and r e l a t i v e numbers of the d i f f e r e n t types of colonies present. I f there was s u f f i c i e n t growth, they were not incubated f u r t h e r . I f , however, there was only s l i g h t growth I n 1 day, the p l a t e s were incubated another 24 hours. This extended incubation period was u s u a l l y found necessary f o r the p l a t e s made with Edwards' medium. Owing to an i n s u f f i c i e n t supply of media i n the North Okanagan study, only those samples were p l a t e d which appeared suspicious or p o s i t i v e by the other t e s t s employed. A l s o , the l a c k of an incubator f o r t h i s study, rendered the r e s u l t s from t h i s t e s t questionable, and so they are not included i n the t a b l e s . The p o s i t i v e f i n d i n g s were, however, used as confirmatory evidence i n c l a s s i f y i n g the quarters- At f i r s t , i d e n t i f i c a t i o n of the organisms on the p l a t e s was accomplished by p i c k i n g representative colonies i n t o yeast litmus m i l k (Y.L.M.) and by examining smears made from the Y.L.M. c u l t u r e s . Due to pressure of time, i t was l a t e r found necessary to carry out the i d e n t i f i c a t i o n of the o r  ganisms by making gram s t a i n s d i r e c t l y from the colonies on the p l a t e s . I t was found p o s s i b l e by t h i s procedure to d i s  t i n g u i s h between chaining s t r e p t o c o c c i , s t a p h y l o c o c c i , spore-- 65 - forming and non-spore-forming rods and also between gram p o s i  t i v e and gram negative b a c t e r i a . Time was not a v a i l a b l e f o r the f u r t h e r d i f f e r e n t i a t i o n of the organisms. Methods f o r C l a s s i f y i n g the Organisms As.spciat.ed With,...Mastitis. During the course of the experimental herd study, i t was found p o s s i b l e to examine the types of organisms present i n the m i l k of a l l the l a c t a t i n g cows i n herds #1 and #2. Representative colonies from the blood agar and Edwards' me dium pla t e s of a l l samples were picked i n t o D i f c o tryptose phosphate broth containing 0.1$ agar. Gram st a i n s were made from the broth c u l t u r e s , and on the basis of t h e i r morphology, the organisms were di v i d e d i n t o three groups: s t r e p t o c o c c i , staphylococci and rod forms. In cases of doubt In d i f f e r e n  t i a t i n g between staphylococci and s t r e p t o c o c c i , the production of catalase was determined by p l a c i n g a l o o p f u l of growth i n a few drops of 3$ hydrogen peroxide and observing f o r the formation of gas bubbles. Staphylococci produce catalase whereas the s t r e p t o c o c c i do not. The organisms i n each of the three groups were studied f u r t h e r , employing the methods devised f o r the c l a s s i f i c a t i o n of the p a r t i c u l a r group concerned. Before the various d i a g  n o s t i c t e s t s were a p p l i e d , however, the cultures were a l l re- p l a t e d on tryptose blood agar to ensure p u r i t y , and to deter mine the type of hemolysis. Pour pl a t e s were employed f o r the s t r e p t o c o c c i c u l t u r e s and spread plates f o r the staphy l o c o c c i and rod forms. The procedures used f o r the c l a s s i  f i c a t i o n of the three groups of organisms are described below: - 66 - 1. S t r e p t o c o c c i . The c l a s s i f i c a t i o n of the s t r e p t o c o c c i was c a r r i e d out according to the methods of P l a s t r i d g e et a l (83) and Hansen (44)• The t e s t s employed were: (a) the h y d r o l y s i s of sodium hippurate by the method of Coffey and Foley (25). (b) the a b i l i t y to s p l i t a e s c u l i n by a m o d i f i c a t i o n of the method of Diernhofer (29) . The composition of the t e s t medium used i s as f o l l o w s : beef i n f u s i o n : 500 c.c. peptone: 5.0 g. NaCl:• 1.5 g. K2HP0,: 1.0 g. aes c u l i K : 0.5 g. The t e s t , i t s e l f , was performed according to the procedure of P l a s t r i d g e et a l (83). (c) f i n a l pH i n 1% glucose broth a f t e r the method of Avery and C u l l e n (2), except that the basal medium used was n u t r i e n t broth and the pH of the medium was adjusted to 7.2. The pH reached at the end of 7 days incubation was de termined with a Beckman pH meter. (d) r e a c t i o n i n yeast litmus m i l k , using skim m i l k w i t h the a d d i t i o n of 0.1$ yeast e x t r a c t and enough litmus s o l u t i o n to give a d i s t i n c t pale v i o l e t c o l o u r . The cultures were examined at d a i l y I n t e r v a l s up to 7 days f o r r e d u c t i o n , r e - o x i d a t i o n , a c i d production, type of c l o t and p r o t e o l y s i s . (e) reduction of methylene blue m i l k . The t e s t on the s t r e p t o c o c c i from herd #1 was c a r r i e d out with a 1:5000 - 67 - concentration of methylene blue (83). In the case of the organisms from herd #2, the concentration of methylene blue was reduced to 1:1000 (102). (f) p r e c i p i t i n t e s t . Brown's m o d i f i c a t i o n (10) of the L a n c e f i e l d p r e c i p i t i n t e s t was employed on a number of cultures using group A, B , and C sera. (g) carbohydrate fermentation t e s t s using casein peptic digest broth as the b a s a l medium and 1.0$ of the r e  quired carbohydrate. The medium was dispensed i n 5 c.c. q u a n t i t i e s and the various carbohydrates were added to the broth before s t e r i l i z a t i o n . In the study on herd #1, the f o l l o w i n g sugars were employed: trehalose, s o r b i t o l , s a l i c i n , r a f f i n o s e and mannitol. In the study on herd #2, l a c t o s e was used i n a d d i t i o n to the foregoing f i v e carbohydrates. The c u l t u r e s were incubated at 37°C. f o r 14 days and the amount of a c i d produced was determined by t i t r a t i o n w ith N/4 sodium hydroxide using phenolphthalein as i n d i c a t o r . 2. Staphylococci. The procedures of P l a s t r i d g e et a l (84) were em ployed to c l a s s i f y the s t a p h y l o c o c c i . They are as f o l l o w s : (a) reduction of n i t r a t e according to the method of P l a s t r i d g e et a l (84) except that the concentration of potassium n i t r a t e i n the medium was reduced to 0.1$. (b) l i q u e f a c t i o n of n u t r i e n t g e l a t i n , observations being made at i n t e r v a l s up to 14 days. - 68 - (c) r e a c t i o n i n yeast litmus milk as o u t l i n e d above f o r the s t r e p t o c o c c i . (d) pigment production on n u t r i e n t agar s l a n t s . (e) u t i l i z a t i o n of ammonia nitrogen a f t e r the method of P l a s t r i d g e et a l (84) except that 0.0018$ phenol red was used as i n d i c a t o r i n the medium. This t e s t was per formed only on the cu l t u r e s obtained from herd #2. (f) coagulase t e s t employing approximately 3-4- drops of a 1:2 d i l u t i o n of human plasma In a s t e r i l e 3/8" by 2 7/8" (O.D.) corked t e s t tube. The tubes were examined f o r coagulation a f t e r 1, 4- and 24- hours incubation at 37°C. This t e s t , a l s o , was performed only on the organisms i s o l a t e d from herd #2. (g) carbohydrate fermentation t e s t s , using a nu t r i e n t broth base, with 1.0$ of the required carbohydrate and 0.0018$ phenol red as i n d i c a t o r . The sugars employed were l a c t o s e , sucrose, mannitol, r a f f i n o s e and g l y c e r o l . Aqueous so l u t i o n s of the various carbohydrates were prepared and s t e r i l i z e d by S e i t z f i l t r a t i o n . The basic b r o t h , containing the i n d i c a t o r , was s t e r i l i z e d by au t o c l a v i n g . A f t e r s t e r i  l i z a t i o n the broth and sugar s o l u t i o n s were mixed and d i s  pensed a s e p t i c a l l y Into s t e r i l e tubes. 3. Rod Forms. Unfortunately time d i d not permit the c l a s s i f i c a  t i o n of the rod forms from herd #1. Those i s o l a t e d from - 69 - herd #2 were studied i n d e t a i l using the f o l l o w i n g procedures: (a) r e a c t i o n i n yeast litmus milk as described ab ove f o r the s t r e p t o c o c c i and s t a p h y l o c o c c i . (b) reduction of n i t r a t e s as o u t l i n e d f o r the s t a p h y l o c o c c i . (c) l i q u e f a c t i o n of g e l a t i n as o u t l i n e d f o r staphy l o c o c c i . (d) i n d o l production from 1% tryptone b r o t h . (e) carbohydrate fermentation t e s t s using the fo l l o w i n g sugars: l a c t o s e , f r u c t o s e , galactose, mannitol, d e x t r i n , glucose, g l y c e r o l , maltose and sucrose. The pro cedure used was i d e n t i c a l with that used f o r the staphylo c o c c i except that 0.002$ brom thymol blue was used i n place of phenol red as the i n d i c a t o r . Results and Discussion of R e s u l t s . The c l a s s i f i c a t i o n of the quarters as negative, sus p i c i o u s or p o s i t i v e was based on the i n t e r p r e t a t i o n of the fi n d i n g s obtained by the use of the various t e s t s applied to each quarter sample, employing the sum of the c h a r a c t e r i s t i c s as a guiding p r i n c i p l e . In general, the standards established f o r each t e s t were those accepted by other workers i n the f i e l d of m a s t i t i s . The r e s u l t s from a l l the quarters of each cov/ were considered as a u n i t , and a l l the a v a i l a b l e informa t i o n was taken i n t o consideration i n c l a s s i f y i n g the respec t i v e quarters. I t was thought that t h i s procedure would give - 70 - a b e t t e r p i c t u r e of the m a s t i t i s c o n d i t i o n of a given cow than would be the case i f the standards of c l a s s i f i c a t i o n were r i g i d l y set down. The data are discussed under the general headings de noted at the beginning of the Experimental s e c t i o n . In the d i s c u s s i o n of the r e s u l t s , the f i n d i n g s from each of the North Okanagan study, general survey, and experimental herd study have been considered separately. For the sake of con venience i n the t a b l e s , the three pr o j e c t s have been de signated by the f o l l o w i n g numbers: I: North Okanagan study. I I : general survey. I l l : experimental herd study. The data from the three studies are recorded i n every case i n the above order. 1• Extent of M a s t i t i s I n f e c t i o n i n B r i t i s h Columbia. The nine herds i n the North Okanagan study together contained 104 cows. A number of these animals were tested more than once, g i v i n g a t o t a l of 121 cows studied and 4#4 samples t e s t e d . In the general survey, 435 d i f f e r e n t animals were examined, and these together with the number tested more than once gives a t o t a l of 510 animals and 1628 milk samples t e s t e d . There were 120 cows involved i n the experimental herd study. Most of them were tested at l e a s t t w i c e , g i v i n g a t o t a l of 279 animals and 1034 samples t e s t e d . Thus 3146 milk samples have been tested f o r m a s t i t i s during the course - 71 - of the e n t i r e study. In compiling the data on the incidence of m a s t i t i s , the f o l l o w i n g procedure was adopted: i n the case of cows tested t w i c e , the r e s u l t s of each t e s t were considered as 1/2, f o r those animals tested three times they were considered as 1/3, f o r animals tested more than three times the r e s u l t s were treated i n a l i k e manner. Thus the r e s u l t s from a s i n g  l e animal, regardless of the number of times t e s t e d , have the e f f e c t of a s i n g l e t e s t on the c a l c u l a t i o n s . For the deter mination of the number of quarters i n f e c t e d i n the general survey, a l l cows i n which samples were taken from a l l four quarters were i n c l u d e d , no adjustment being made f o r the num ber of times the animal was t e s t e d . This m o d i f i c a t i o n was found necessary because, i n the case of a great many of the cows studied i n the general survey, samples were submitted from only one or two quarters. In the North Okanagan study, two cows were i n advanced stages of l a c t a t i o n , and so the r e  s u l t s from these animals were not included i n the c a l c u l a t i o n s . In Table 1 i s found a summary of the f i n d i n g s on the extent of m a s t i t i s i n f e c t i o n i n the province as revealed by the present study. I t can be seen that approximately 35$ of the cows examined i n the North Okanagan study and the e x p e r i  mental herd study were found, p o s i t i v e f o r m a s t i t i s . This f i g u r e compares favorably with the r e s u l t s obtained by the other workers, whose f i n d i n g s have already been reviewed. TABLE 1. EXTENT OF MASTITIS INFECTION IN BRITISH COLUMBIA. Source of Data Number of cows Studied Percent of cows classified as Number of Quarter Studied Percent of quarters classified as Negative Suspicious Positive Negative Suspicious Positive I 102 ^ 8 . 5 3 1 3 . 7 6 37.7k 7 5 . 2 1 8 . 7 ^ 1 6 . 0 5 II >*35 7 . 8 5 2 0 . 7 5 71.^0 1^32 2 7 . 3 7 3 0 . 5 2 ^2.11 III 120 3 8 . 5 0 27 .H8 3^.02 h6S 5 9 . 0 8 2 3 . 8 1 1 7 . 1 1 - 73 - A summary of the f i n d i n g s of these authors together with the r e s u l t s from pr o j e c t s I and I I I of the present study i s con tained i n Table 2. These r e s u l t s i n d i c a t e that m a s t i t i s i n  f e c t i o n i n B r i t i s h Columbia i s of approximately the same magnitude as i n other parts of the world. I t i s also observed from Tables 1 and 2 t h a t , whereas approximately 35$ of the cows studied were p o s i t i v e f o r mas t i t i s , only about 17$ of the t o t a l number of quarters examined were considered i n f e c t e d . These r e s u l t s are explained i n Table 3 where i t i s seen that most of the p o s i t i v e cows studied were i n f e c t e d i n one quarter only and that the per centage of cows i n f e c t e d i n more than one quarter decreased i n v e r s e l y to the increase i n the number of quarters i n v o l v e d . These f i n d i n g s are i n keeping with the conception that chro n i c m a s t i t i s i s a slow, progressive disease, which begins i n one quarter of the udder and which, i f not checked, gradually spreads to the other three quarters. TABLE 3. RELATIVE NUMBERS^ OF QUARTERS INFECTED IN INDIVIDUAL COWS. Source of Data Number of P o s i t i v e Cows Studied Percent of p o s i t i v e cows Infected i n I Quarter 2 Quarters 3 Quarters 4- Quarters I 38.5 56.71 •21.65 16.45 5.19 I I 232 27.16 21.55 15.09 36.20 I I I 41 4-9.55 19.44 16.44 14.57 TABLE 2 . SUMMARY OF FINDINGS RELATIVE TO THE INCIDENCE OF MASTITIS IN VARIOUS PARTS OF THE WORLD. Author Locality Number of Herds Studied Number of Cows Studied Percent of Cows Positive for Mastitis Percent of Quarters Positive for Mastitis Percent of Positive cows in individual Herds. Rosell ( 8 7 ) Quebec mm* I838 3^.6 •»«* 19 - 97 Bryan ( 1 1 ) Michigan 322 2715 2 6 . 2 — 0 - 100 Gwatkin et a l (*f2) Ontario 28 3 9 . 0 2 6 A 15 - 9 1 Edwards ( 3 3 ) England 18 809 3 6 A 16 A 10 - 81+ North Okanagan Study North Okanagan Valley 9 102 37.7h 1 6 . 0 5 8 - 6 0 Experimental Herd Study Lower Fraser Valley h 120 3^.02 1 7 . 1 1 10 - k2 - 75 - I t i s seen from Table 2 that the incidence of m a s t i t i i n i n d i v i d u a l herds v a r i e s w i d e l y . This i s probably due i n part to the great v a r i a t i o n i n the s i z e of the herds studied I t was found, f o r example, that the most s e r i o u s l y i n f e c t e d herd i n the North Okanagan study consisted of only f i v e cows and that three of these had m a s t i t i s . A s i m i l a r number of i n f e c t e d animals i n a l a r g e r herd would not have such a s t r i k i n g e f f e c t on the c a l c u l a t i o n of the percentage of i n  f e c t i o n i n the herd. These data do, however, i n d i c a t e that a great many herds are h e a v i l y i n f e c t e d with t h i s d isease. The f i n d i n g s from the general survey, as recorded i n Tables 1 and 2 are to be i n t e r p r e t e d w i t h c a u t i o n . The sam ples studied i n the course of t h i s survey were taken, f o r the most p a r t , from c l i n i c a l l y p o s i t i v e cows and so do not c o n s t i t u t e a representative sampling of the cow popu l a t i o n . This accounts f o r the very high percentage of p o s i t i v e cows found i n t h i s study and also f o r the large number of infeete< quarters. Nevertheless, these r e s u l t s demonstrate that mas t i t i s i s a widespread and serious disease among the d a i r y cows of t h i s province. In the North Okanagan study, there were no a c t i v e cases of m a s t i t i s at the time of sampling. The various owners, however, reported that a number of the cows studied had p r e v i o u s l y had attacks of the common chronic form of the disease. A l l forms of m a s t i t i s were encountered i n the gen e r a l survey - chronic, fl a r e - u p of chronic, sub-acute, acute. - 76 - and gangrenous. The h i s t o r y of the various eases at the time of sampling extended from a few hours i n some instances to three years i n others. The c l i n i c a l h i s t o r y of the cows ex amined i n the experimental herd study i n d i c a t e s that most of the m a s t i t i s i n these herds was of a s u b - c l i n i c a l nature. Only four cases of acute f l a r e - u p s have been encountered i n t h i s study to date. 2. Factors Predisposing the Udder to M a s t i t i s I n f e c  t i o n . During the course of the work reported upon her e i n , i t was found p o s s i b l e to study a number of the f a c t o r s which have been suspected as predisposing agents to udder i n f e c t i o n . These are discussed below. (a) Breed. As can be seen from Table 4> there does not appear to be any r e l a t i o n s h i p between the breed of the cow and the l i k e l i h o o d of i t s being i n f e c t e d w i t h m a s t i t i s . The r e l a t i v e numbers of animals of the d i f f e r e n t breeds examined i n the North Okanagan study were too few to permit the draw ing of conclusions. The data from the other two s t u d i e s , however, i n d i c a t e that the four common breeds of d a i r y c a t t l e are i n f e c t e d to approximately the same degree. In the general survey, the incidence of i n f e c t i o n among the four breeds i s seen to be of the order of 60-70$, whereas i n the experimen t a l herd study the l e v e l of i n f e c t i o n i s found to be approx imately 30-40$ f o r the four breeds. This di f f e r e n c e i n l e v e l - 77 - i s due to the high proportion of p o s i t i v e cows included i n the general survey. These f i n d i n g s are i n exact agreement wit h the generally accepted idea that the incidence of mas t i t i s i s not influenced by the breed of the animal. TABLE L. RELATION OF THE BREED OF COW TO THE INCIDENCE OF MASTITIS. Source of Data Jersey Holstein. Guernsey Ayrs h i r e I No. of Cows Studied % Infected 60 44 • 17 13 23.08 5 00.00 8 37.50 I I No. of Cows Studied % Infected 139 68.35 125 72.80 47 59.58 24 75.00 I I I No. of Cows Studied % Infected 56 34-* 82 22 4,0.91 42 29.33 — (b) Age of 0 O W• The r e s u l t s from the North Okanagan study and the experimental herd study (Table 5) demonstrate very c l e a r l y that the l i k e l i h o o d of a cow being i n f e c t e d with m a s t i t i s i n  creases with increasing age of the animal. Again, the d i s  t r i b u t i o n of the cows studied i n the general survey renders - 78 - the data from t h i s study as u n s u i t a b l e f o r determining the e f f e c t of the age of the cow on the incid e n c e of m a s t i t i s , other than to show t h a t even young animals are subject to considerable m a s t i t i s i n f e c t i o n . In general, then, the r e  s u l t s recorded h e r e i n agree with the f i n d i n g s of other work ers that the chances of a cow being i n f e c t e d with m a s t i t i s Increase as the number of l a c t a t i o n s i n c r e a s e s , and that very few cows of over e i g h t years of age are completely f r e e from evidence of i n f e c t i o n (11). TABLE 5. RELATION OF THE AGE OF COW TO THE INCIDENCE OF MASTITIS. Source of Data 2 — 4 Years 5 - 6 Years 7-9 Years Over 9 Years I No. of Cows Studied % I n f e c t e d 25 28.00 7 28.57 11 40.91 6 83.33 I I No. of Cows Studied % I n f e c t e d 143 66.43 112 77.68 79 73.42 26 76.92 I I I No. of Cows Studied % I n f e c t e d 73 25.68 23 35.22 13 66.69 8 60.00 - 79 - ( c) Method, .of ..mjLXklnjg; and sa n i t a r y c o n d i t i o n s . Owing to the l a c k of s u f f i c i e n t data relevant to the numbers of p o s i t i v e cows as compared to the t o t a l num ber of animals i n i n d i v i d u a l herds, I t i s impossible to give any exact r e s u l t s on the e f f e c t of s a n i t a r y methods and me thod of m i l k i n g on the incidence of m a s t i t i s i n the herds s t u d i e d . Prom the l i t t l e data a v a i l a b l e , i t would appear that these f a c t o r s do not g r e a t l y i n f l u e n c e the extent of m a s t i t i s i n f e c t i o n i n i n d i v i d u a l herds. I t was observed th a t herds, i n which the general s a n i t a r y p r a c t i c e s were ex c e l l e n t , were as h e a v i l y i n f e c t e d with m a s t i t i s as those i n which the sa n i t a r y conditions were poor. Some herds i n which the animals were hand, milked were s e r i o u s l y i n f e c t e d , where as, some machine-milked herds were l i g h t l y i n f e c t e d . (d) Trauma. A number of the samples submitted during the general survey were obtained from c l i n i c a l cases of m a s t i t i s , r e l a t i v e to which i t was reported that the m a s t i t i s attack had been preceded by an i n j u r y to the udder or t e a t s . These r e s u l t s i n d i c a t e that i n j u r y i s an important predisposing agent to udder i n f e c t i o n . However, the small number of cases recorded does not permit the drawing of general conclusions. In general, then, the fi n d i n g s from the study reported h e r e i n , agree with the accepted concepts that the breed of the animal and the method of mil k i n g have l i t t l e - 80 - effect on the incidence of m a s t i t i s , but that advancing age and injury predispose to udder i n f e c t i o n . 3• Evaluation of...the Tests Used for the Detection of M a s t i t i s . In order to render the evaluation of the tests used for the detection of mastitis as r e l i a b l e as possible, no samples were included i n which factors other than mastitis may have contributed to a positive reaction. For this reason, the results from two cows, i n the North Okanagan study, which were i n an advanced stage of l a c t a t i o n , were omitted. Simi l a r l y , the data obtained i n the general survey from cows which had freshened less than a week before the samples were taken, and from those which were almost dry at the time of sampling, were not used. Also, because the indirect tests used for the diagnosis of mastitis are not required to de tect abnormalities i n milk samples of grossly altered physi cal appearance, and because the r e l i a b i l i t y of the reactions of these tests i s influenced by the presence of c l o t s , serum, blood, etc., only those samples from the general survey which were normal i n physical appearance were included for study on the tests for mastitis. For the same reason, nine samples from the experimental herd study which were grossly abnormal In physical appearance were withheld from the present study. (a) Physical Examination of the Milk. A l l of the milk samples examined i n the North Okanagan study were of normal physical appearance. In the experimental herd study, only 50 out of a t o t a l of 1034- sam ples tested were abnormal with respect to p h y s i c a l appearance. In most cases, the degree of a l t e r a t i o n was very s l i g h t , and consisted merely of the presence of a few f l a k e s or of a watery appearance. When these f i n d i n g s are considered i n the l i g h t of the incidence of m a s t i t i s among the cows i n these s t u d i e s , i t i s r e a d i l y observed that the predominant type of m a s t i t i s i n these animals i s of a s u b - c l i n i c a l nature, such that the p h y s i c a l appearance of the milk i s not a f f e c  t e d . I t has also been found that i t i s only i n the acute or advanced stages of the disease that the p h y s i c a l appearance of the milk i s a l t e r e d . Hence, i t i s obvious that t e s t s other than the examination of the m i l k f o r p h y s i c a l abnormality must be resorted t o , i n order to detect the s u b - c l i n i c a l forms of m a s t i t i s i n f e c t i o n . However, the presence of f l a k e s or c l o t s i n the foremilk i s often the f i r s t i n d i c a t i o n to the farmer of a fl a r e - u p of chronic m a s t i t i s , and so from t h i s point of view, the p h y s i c a l examination of samples of f o r e  m i l k i s of considerable p r a c t i c a l value. In the general survey, the milk samples received f o r a n a l y s i s presented a great v a r i e t y of appearances - normal, f l a k y , c l o t t y , serumy, e t c . Some of the samples contained varying amounts of blood, others gave the appearance of co agulated serum, and s t i l l others 'were of a b r i g h t yellow co l o u r . The se c r e t i o n of such grossly abnormal m i l k s , de monstrates the tremendous e f f e c t which advanced m a s t i t i s i n f e c t i o n lias on the secretory c e l l s of the udder. (b) Modified tJhiteside Test. Murphy and Hanson (77), who developed the modi f i e d Whiteside t e s t f o r m a s t i t i s , observed that i t s a b i l i t y to detect udder i n f e c t i o n p a r a l l e l e d c l o s e l y that of the l e u  cocyte count. Dunn et a l (30) found that the Whiteside r e  a c t i o n was due to the presence of leucocytes i n the m i l k , and that the i n t e n s i t y of the r e a c t i o n increased with i n c r e a s i n g leucocyte count. They postulated that the r e a c t i o n i s s i m i l a r to that which takes place between n u c l e i c a c i d and sodium hy droxide . They suggested, f u r t h e r , that the sodium hydroxide breaks down the leucocyte s t r u c t u r e and forms a gelatinous mass w i t h the c e l l n u c l e i c a c i d , p o s s i b l y w i t h the formation of the sodium s a l t of the a c i d . I t i s thought that f a t g l o  bules and serum s o l i d s are adsorbed on t h i s gelatinous mass g i v i n g the appearance of white f l o c c u l e s . The f i n d i n g s obtained with the modified White side t e s t i n the present study are shown i n Table 6 . These data i n d i c a t e considerable v a r i a t i o n between the r e s u l t s of the three s t u d i e s . I f a l l reactions of t or higher are taken to i n d i c a t e udder disturbance, then, i n the North Okanagan study, 92.44$ of the negative samples and 96.20$ of the p o s i  t i v e samples would be c o r r e c t l y c l a s s i f i e d by t h i s t e s t . The r e s u l t s a r e , of course, not so d e f i n i t e wi th the suspicious samples. In the case of the general survey and the experimen-- 83 - t a l herd study, however, there are found to he quite a number of f a l s e p o s i t i v e and f a l s e negative r e a c t i o n s . TABLE 6. THE USE OF THE MODIFIED WHITESIDE TEST IN THE DETECTION OF MASTITIS SOURCE OF DATA REACTION PERCENT OF TOTAL NEGATIVE QUARTERS PERCENT OF TOTAL SUSPICIOUS QUARTERS PERCENT OF TOTAL POSITIVE QUARTERS No. of Samples 357 40 79 0 92.44 32.50 3.80 I 6.16 17.50 11.39 1* I.40 32.50 2*, 3+, 4* — 17.50 67.09 No. of Samples 304 207 196 0 61,18 44 • 45 10.71 I I z 22.04 24*. 64 10.20 1* 13.16 24.15 23.47 2*, 3-s-, 4* 3.62 6.76 55.62 No. of Samples 558 276 191 0 73.4S 44.20 9.43 I I I A, 16.13 22.10 13.09 1* 9.50 25.00 41.36 0.89 8.70 36.12 The reason f o r t h i s v a r i a b i l i t y i n the r e s u l t s from the three studies appears to be connected with the age of the - H - m i l k samples at the time of t e s t i n g . In the North Okanagan study, the samples were examined f o r Whiteside r e a c t i o n about 16-20 hours a f t e r being taken. In the experimental herd study, however, i t was f r e q u e n t l y found impossible to perform the Whiteside t e s t u n t i l the milks were considerably over 20 hours o l d , the samples having stood at room temperature i n the mean time. In the general survey the samples were often two or three days o l d on a r r i v a l at the l a b o r a t o r y . I t was u s u a l l y found that the r e a c t i o n was l e s s d i s t i n c t w ith the older sam ples . This may have been due i n part to the leucocytes having r i s e n w i t h the cream, and formed a dense mass which had pro tected the leucocytes from the a c t i o n of the sodium hydroxide. In other cases, the concentration of the leucocytes i n the cream l a y e r may have r e s u l t e d i n a f a l s e p o s i t i v e r e a c t i o n , i f the sample had not been w e l l shaken before the t e s t was performed. The r e s u l t s i n d i c a t e that when the modified Whiteside t e s t i s applied to s t r i c t l y f r e s h quarter samples of m i l k , i t i s a very accurate means of detecting the presence of abnormal conditions i n the udder. Hence, the r e l i a b i l i t y of t h i s t e s t , together w i t h the small amount of equipment required and the ease with which i t can be performed, should render the modi fied. Whiteside t e s t of considerable value as a f i e l d t e s t f o r m a s t i t i s . (c) Geneva B l o t t e r Test. The Geneva b l o t t e r t e s t i s an adaptation of - 85 - the brom thymol blue tube t e s t f o r the determination of the pH of m i l k , and was developed e s p e c i a l l y f o r use i n the f i e l d as an a i d i n the detection of m a s t i t i s . The r e s u l t s obtained w i t h t h i s t e s t are found i n Table 7. These f i n d i n g s i n d i c a t e t h a t , while the t e s t appears to have c l a s s i f i e d the negative samples f a i r l y a c c u r a t e l y , only 29.12$ of the p o s i t i v e sam ples were c o r r e c t l y c l a s s i f i e d by t h i s method. A l s o , 11.39$ c f the p o s i t i v e samples would have been considered negative had t h i s t e s t alone been employed to detect m a s t i t i s . I t would appear, then, that the Geneva b l o t t e r t e s t i s not par t i c u l a r l y s e n s i t i v e to minor changes In the udder and that i t i s l i k e l y to c l a s s i f y a considerable number of p o s i t i v e quar t e r s as fre e from i n f e c t i o n . P o s s i b l e explanations f o r these observations are discussed i n the f o l l o w i n g s e c t i o n on the determination of pH using brom c r e s o l p u r p l e . TABLE 7. THEJJSE OF THE GENEVA BLOTTER TEST IN THE DETECTION OF MASTITIS SOURCE OF DATA REACTION* PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 357 40 79 N 93.84 45.00 11.39 I 6.16 55.00 59-49 G 29 • 12 *N: normal - yellow green colour. ?: suspicious - green colour. . G: garget ( P o s i t i v e ) - blue green colour. - 86 - I t should be emphasized that when the Geneva b l o t t e r t e s t i s used i n the f i e l d , i t should be employed only under w e l l v e n t i l a t e d c o n d i t i o n s , f o r any quantity of ammonia i n the atmosphere from an accumulation of manure, w i l l tend to give f a l s e p o s i t i v e r e a c t i o n s . (d) p_H_JQe_termination_with_Br^m_Cre^l_£urpj^. I t i s generally found t h a t , i n cases of udder disturbance, the pH of the milk tends to become more a l k a l i n e than u s u a l , the degree of the a l k a l i n i t y depending on the s e v e r i t y of the upset. I t i s thought t h a t , i n mild cases of m a s t i t i s , t h i s a l k a l i n i t y i s due to the increased permeability of the c e l l membranes to bicarbonates from the blood. In more advanced cases, i t i s l i k e l y that blood serum and pos s i b l y lymph f l u i d s pass almost unchanged i n t o the m i l k . Blood serum has a pH of about 7-4, whereas the pH of normal milk i s about 6.4 - 6.8. Hence, i t i s reasonable to conclude t h a t , i n the milder forms of the disease where only a small part of the udder t i s s u e i s i n v o l v e d , not enough bicarbonate i s se creted to have very much e f f e c t on the f i n a l pH of the m i l k . This would e x p l a i n , then, why the pH t e s t i s not very s e n s i  t i v e to s l i g h t changes i n the udder. The d r a s t i c changes which take place i n cases of acute m a s t i t i s and advanced chronic m a s t i t i s vrtiere the udder metabolism i s grossly a f f e c  t e d , can r e a d i l y be detected by t h i s t e s t . The r e s u l t s obtained by the determination of the pH of the m i l k samples, which are found i n Table 8, sub s t a n t i a t e the above statements and agree i n general with the f i n d i n g s obtained w i t h the Geneva b l o t t e r t e s t , namely, that a great many p o s i t i v e samples are c l a s s i f i e d as negative by these t e s t s . Hence t h i s t e s t i s of value i n d i f f e r e n t i a t i n g the most s e r i o u s l y i n f e c t e d animals i n a herd, but i s not so r e l i a b l e as the modified Whiteside t e s t i n detecting cases of m i l d or i n s i p i e n t m a s t i t i s . The rather large proportion of negative samples i n the North Okanagan study which appeared s l i g h t l y a l k a l i n e to brom c r e s o l purple, might i n d i c a t e that the pH of the m i l k from normal cows i n t h i s area i s s l i g h t l y higher than that found elsewhere. The small number of samples s t u d i e d , however, pre  vents the drawing of d e f i n i t e conclusions on t h i s matter. - A few samples were encountered which were acid to brom c r e s o l p urple. These f i n d i n g s were always associated with very l a r g e numbers of s t r e p t o c o c c i , and i t i s thought that these organisms had formed enough a c i d i n the m i l k to lower the pH s u f f i c i e n t l y to change the colour of the brom c r e s o l purple. Whether the milk was a c i d on leaving the udder or whether the change had been brought about during the i n t e r v a l between taking the samples and t e s t i n g them i s not known, but the l a t t e r explanation appears more tenable. - 88 - TABLE 8. THE USE OF THE DETERMINATION OF PH WITH BROM CRESQL PURPLE IN THE DETECTION OF MASTITIS SOURCE OF DATA REACTION PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 357 40 79 N. 72.55 32.50 8.86 I V.S1.A1. 2.24- S1.A1. 24,. 09 42.50 30.38 A1.,V.A1.,V.S1. Ac.,81.Ac.,Ac. 1 « 12 25.00 60.76 No. of Samples 304, 207 196 N. 68.75 39.13 18.37 I I V.S1.A1. 13.16 19.81 12.76 S1.A1. 9.21 23.19 19.39 A1.,V.A1.,V.S1. A c , S I . A c , Ac. 8.88 17.87 49.48 No. of Samples 557 272 188 N. 71»1*4" 27.21 11 • 17 I I I V.S1.A1. 17.32 17.28 11.17 S1.A1. 10.24 27.21 25.00 A1.,V.A1.,V.S1. A c ,SI.Ac,Ac. 1.30 28.30 52.66 - 89 - TABLE 8 Cont'd. KEY_T0„RlMC2I0iai, N.: normal (pH approx. 6.4 - 6.8) V. S I . A l . : very s l i g h t l y a l k a l i n e (pH approx. 6.9) S I . A l . : s l i g h t l y a l k a l i n e (pH approx. 7.0 - 7.1) A l . : a l k a l i n e (pH approx. 7.2 - 7.3) V. A l . : very a l k a l i n e (pH approx. 7.4 or higher) V. S I . Ac.: very s l i g h t l y a c i d (pH approx. 6.3) S I . Ac.: s l i g h t l y a c i d (pH approx. 6.2 - 6.1) A c : a c i d (pH approx. 6.0 - 5.6) I t should be emphasized at t h i s point that colostrum has an a l k a l i n e pH, and that the m i l k from animals l a t e i n t h e i r l a c t a t i o n period also tends to show a high pH. The extent of t h i s r i s e i n pH during l a t e l a c t a t i o n v a r i e s with the i n d i v i d u a l cow, some animals showing a much greater change than others. Hence, the pH t e s t i s of no value i n the det e c t i o n of m a s t i t i s i n animals i n e i t h e r e a r l y or l a t e l a c  t a t i o n . A l s o , since the a l t e r a t i o n i n the pH of the milk i n i n s i p i e n t cases of m a s t i t i s i s so s l i g h t , the t e s t should be used only on quarter samples of m i l k , as a mi l d i n f e c t i o n i n one quarter would not be detected i f the pooled milk of a l l four quarters of the udder were examined. Another f a c t o r to be considered i n t h i s connection i s the report by Kleckner (6l) that the pH of milk tends to r i s e s l i g h t l y during estrum. The p o s s i b l e e f f e c t of t h i s r i s e i n pH on the v a l i d i t y of the pH t e s t f o r the detection of m a s t i t i s has not as yet received a t t e n t i o n . The problem, however, i s worthy of study. During the course of t h i s study, i t was observed that - 90 - the d i f f e r e n t shades of colour associated w i t h the various pH's of the milk could be more r e a d i l y d i s t i n g u i s h e d w i t h brom c r e s o l purple than w i t h brom thymol blue as was used i n the Geneva b l o t t e r t e s t . The former i n d i c a t o r , then, i s suggested as being more s u i t a b l e i n determining the pH of m i l k as an a i d i n detecting m a s t i t i s . (e) Hotis Test.. The Hotis t e s t as o r i g i n a l l y proposed by Hotis and M i l l e r (49), was claimed to be 95$ accurate i n detecting the presence of S t r . agalactiae i n milk samples, as compared to the blood agar p l a t e method. M i l l e r (70) l a t e r reported that Staph, aureus could also be detected by t h i s t e s t . S t r . a g a l a c t i a e forms canary yellow f l a k e s at the bottom and along the sides of the tubes, whereas Staph, aureus tends to form white f l a k e s w i t h rust-coloured margins. M i l l e r stated f u r  ther that other m a s t i t i c organisms do not produce such f l a k e s i n incubated milk samples. He b e l i e v e s , however, that t h i s does not detract from the value of the t e s t , since S t r . aga l a c t i a e i s the only organism of importance i n a c o n t r o l pro gram f o r the e r a d i c a t i o n of m a s t i t i s , and that i f t h i s o r  ganism can be accurately detected then the t e s t has served i t s purpose. The r e s u l t s obtained from the Hotis t e s t i n the present study are shown i n Table 9. I t w i l l be noted that a l l of the samples examined i n the general survey were - 91 - used, i n the preparation of t h i s t a b l e . As can r e a d i l y be seen, very l i t t l e information i s revealed by these f i g u r e s . There are several reasons f o r t h i s . The large percentage of p o s i t i v e samples which were graded negative by t h i s t e s t may have been due to the f a c t that f o r most of the cases s t u d i e d , the causative organism was probably not S t r . a g a l actiae nor Staph, aureus. I t i s thought, a l s o , that i n the general sur vey, the udder staphylococci may have outgrown the S t r . aga l a c t i a e present, when the samples were held at f a i r l y low temperatures during t r a n s i t , such that the S t r . a g a l a c t i a e were unable to form t h e i r c h a r a c t e r i s t i c f l a k e s when the milks were subsequently incubated at 37°C. In a great many inst a n c e s , doubtful reactions were en countered i n which there was u s u a l l y a s l i g h t amount of yellow sediment i n the tubes even a f t e r 4-8 hours i n c u b a t i o n . I t was d i f f i c u l t to determine whether such rea c t i o n s were p o s i t i v e t e s t s f o r S t r . a g a l a c t i a e , or whether they merely represented the beginning of the formation of a c l o t i n the tube. Un f o r t u n a t e l y , the organisms i s o l a t e d on the blood agar plates from the milk samples could not be i d e n t i f i e d , and so the num ber of f a l s e p o s i t i v e and f a l s e negative reactions of the Ho t i s t e s t are not known. Bryan and Devereux (14-) and Berry and Clark ( 6 ) also found d i f f i c u l t y i n i n t e r p r e t i n g suspicious r e  actions obtained with t h i s t e s t . Bryan and Devereux, i n f a c t , observed many f a l s e p o s i t i v e reactions which they found to be caused by udder m i c r o c o c c i . These workers also found that the - 92 - r e s u l t s of the Hotis t e s t were not constant on repeated t e s t  ing of the milk from negative and p o s i t i v e cows. In contrast to these f i n d i n g s , consistent r e s u l t s were obtainted w i t h the microscopic and blood agar p l a t e methods when applied to the same mil k samples. TABLE 9. THE USE OF THE HOTIS TEST IN THE DETECTION OF MASTITIS DUE TO STREPTOCOCCUS AGALACTIAE SOURCE OF DATA REACTION PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 381 369 606 Negative 90.03 81.57 84- .32 I I Pos. f o r S t r . agalactiae 7.09 13.28 13.86 Pos. f o r Staph. aureus 2.88 5.15 1.32 No. of Samples 557 272 188 Negative 70.56 50.37 55.32 I I I Pos. f o r S t r . agal a c t i a e 8.62 32.72 29.25 Pos. f o r Staph, aureus 20.82 16.91 15-43 McGulloch and F u l l e r (67) determined that the Hotis r e a c t i o n was due to an a g g l u t i n a t i o n phenomenon, and that the a g g l u t i n a t i n g f a c t o r was present i n the milk of most cows even - 93 - though the milk was negative to the Hotis t e s t . They found a l s o , that s t r e p t o c o c c i other than S t r . agalactiae occasion a l l y gave p o s i t i v e r eactions w i t h t h i s t e s t . The r e s u l t s obtained i n the present study, together w i t h those reported by the above authors, i n d i c a t e that the Hoti s t e s t i s not of very great value In the detection of m a s t i t i s . This t e s t may be u s e f u l among animals badly i n f e c  ted w i th S t r . a g a l a c t i a e , but where organisms other than S t r . a g a l a c t i a e are found to any extent to be responsible f o r mas t i t i s , the t e s t i s of l i t t l e v a l u e . A l s o , the number of f a l s e p o s i t i v e reactions which are frequently obtained, ren der i t s use questionable. Hence, i n view of the f a c t that almost as much information can be obtained by a microscopic examination of incubated m i l k samples, the use of the Hotis t e s t f o r the det e c t i o n of m a s t i t i s i s not warranted. (f) P e t e m i n a t i o n . of. .Percent C h l o r i n e . The r e s u l t s obtained by the determination of the percent c h l o r i n e i n the milk samples i s shown i n Table 10. Various workers disagree as to what percent c h l o r i n e should be taken as the d i v i d i n g point between normal and mas t i t i c samples. R o s e l l (87), whose method was followed i n the work reported h e r e i n , stated that a chlori d e content i n the milk of over 0.142$ should be considered an i n d i c a t i o n of m a s t i t i s i n f e c t i o n . Blood and Rowlands (7) on the other hand, from a study of several methods f o r the determination of the - 94 - c h l o r i n e content of m i l k , concluded that R o s e l l ' s method gave r e s u l t s approximately 25$ higher than methods i n which the pr o t e i n i s p r e c i p i t a t e d or otherwise removed before the c h l o r  ine t i t r a t i o n i s made. Despite these disagreements, however, i t should he pos s i b l e to set a standard f o r each method em ployed. The r e s u l t s reported i n the present paper r e v e a l , t h a t , whereas almost a l l of the p o s i t i v e samples had a c h l o r  ine content of over 0.142$, an appreciable number of negative samples had a chlo r i n e content between 0.142$ and 0.160$, and a few negative samples even had a ch l o r i n e content of over 0.l60$. These f i n d i n g s may i n d i c a t e that 0.160$ Is a b e t t e r f i g u r e than 0.142$ to be taken as the d i v i d i n g l i n e between negative and p o s i t i v e samples w i t h t h i s procedure. They may also i n d i c a t e that the ch l o r i n e content of the milk from nor mal cows i n the areas from which these samples were obtained i s higher than i n the l o c a l i t i e s where R o s e l l made h i s studies. Davies (23) found that Ayrshire' milk was s l i g h t l y higher i n ch l o r i n e content than Shorthorn and Guernsey m i l k . Sharp and Struble (100) observed that milk from H o l s t e i n cows had a higher chlorine content than that from Jerseys and Guer nseys. They a t t r i b u t e d t h i s , i n p a r t , to the f a c t that there are more i n t e r f e r i n g proteins i n H o l s t e i n milk which might a f f e c t the end-point of the t i t r a t i o n such that higher read ings might r e s u l t . These workers'also found that the chlorine content of colostrum i s h i g h , and that the chlorine content of the milk drops r a p i d l y during the f i r s t few days a f t e r calving, - 95 - reaches a minimum, slowly r i s e s during the f i r s t 60$ of the l a c t a t i o n p e r i o d , then increases more r a p i d l y , and f i n a l l y r i s e s very r a p i d l y during the l a s t 10$ of the l a c t a t i o n period. I t i s thought that the c h l o r i n e content of milk changes as occasion demands i n order to maintain i s o t o n i c conditions between the milk and the secretory c e l l protoplasm. I f f o r any reason the s o l i d s - n o t - f a t content of the milk should be decreased, the c h l o r i n e content r i s e s a c c o r d i n g l y . I t i s known that the s o l i d s - n o t - f a t content of milk i s a f f e c t e d by such f a c t o r s as type of feed, drought, e t c . , and that the ch l o r i n e content of the milk i s thus i n d i r e c t l y a f f e c t e d by the same f a c t o r s . I t has been found that one molecule of sodium c h l o r i d e i s i s o t o n i c w i t h two molecules of l a c t o s e and i t i s thought that the high c h l o r i d e content of m a s t i t i c milk may be due to a disturbance i n the synthesis of l a c t o s e , so that the amount of l a c t o s e secreted i s reduced and hence the ch l o r i n e content of the milk i s increased. In the present study, the value of the chl o r i n e t e s t i n the det e c t i o n of m a s t i t i s was found to be g r e a t l y reduced In the case of l a t e l a c t a t i o n animals. A considerable num ber of samples, from cows l a t e In l a c t a t i o n , which appeared normal by the pH t e s t and leucocyte count were decidedly abnormal with respect to percent c h l o r i n e . - 96 - TABLE 10. THE USE OF THE CHLORIDE TEST IN THE DETECTION. OF MASTITIS SOURCE OF DATA CHLORINE CONTENT ' OF MILK IN PERCENT PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 153 18 49 < 0.124 12.4-2 2.04 I O.I24.-O.14.2 41.83 5.56 0.14.2-0.160 30.72 16.66 2.04 0.160-0.178 9.15 27.77 14.29 >0.178 5.88 50.01 81.63 No. of Samples 304 207 195 <0.124 20.40 7.24 1.03 I I 0.124-0.142 39.80 10.63 3.59 0.14-2-0.160 22.37 20.29 7.69 0.160-0.178 9.54 22.71 13.33 >0.178 7.89 39.13 74.36 No. of Samples 492 270 188 <0.124 31.50 8.52 3.19 I I I 0.124-0.142 35.98 14.07 1.59 0.142-0.160 25.81 16.67 3.19 0.160-0.178 5.69 19.63 16.49 >0.173 1.02' 4i . l l 75.54 - 97 - I t was also observed that the ch l o r i n e content of milk drops r a p i d l y during the m i l k i n g process. In a pre l i m i n a r y experiment with 8 cows, i t was found t h a t , whereas i n a num ber of cases samples of foremilk were p o s i t i v e f o r m a s t i t i s as judged.by the percent c h l o r i n e and pH determinations, samples taken a f t e r the m i l k i n g machine had been removed, appeared normal. The greater the i n i t i a l reading, the great er appeared to be the reduction as m i l k i n g proceeded. I t i s thus very evident that when use i s made of the c h l o r i d e and pH t e s t s f o r the detection of m a s t i t i s , samples of f o r e  milk must be used. The removal of two or three streams of milk before the samples are taken i n order to eliminate con taminating b a c t e r i a should not a l t e r the r e s u l t s from these t e s t s to any extent. I t appears that the other t e s t s em ployed i n the detection of m a s t i t i s are not so g r e a t l y a f f e c  ted by the use of f i r s t or l a s t s t r i p p i n g s as the ch l o r i d e and pH t e s t s . No data i s a v a i l a b l e from the present study regarding the e f f e c t of the breed of the animal on the c h l o  r i d e content of normal m i l k . I t may be concluded, then, that the chloride t e s t i s too s e n s i t i v e to minor changes i n the udder c e l l metabolism to be of value as a f i e l d t e s t i n m a s t i t i s c o n t r o l work. This conclusion was also a r r i v e d at by Frayer (4-0) . In view of the f a c t that the ch l o r i d e t e s t i s so r e a d i l y a f f e c t e d by the stage of the l a c t a t i o n p e r i o d , and considering that cows vary g r e a t l y i n the length of t h e i r l a c t a t i o n periods, i t i s - 98 - l i k e l y that t h i s t e s t , i f used as the sole means of detec t i n g m a s t i t i s i n f e c t i o n i n the udder, may report considerable numbers of f a l s e p o s i t i v e r e a c t i o n s , (g) Leucocyte Count. The f i n d i n g s obtained with the leucocyte count are shown i n Table 11. These r e s u l t s i n d i c a t e that most of the negative samples had a leucocyte count below 500,000 per c . c , but they r e v e a l , f u r t h e r , that appreciable numbers of the p o s i t i v e samples also had a leucocyte count below 500,000. These f i n d i n g s , then, suggest that t h i s t e s t Is of about the same value i n detecting evidence of m a s t i t i s as the pH t e s t , i n that both of these t e s t s tend to give a considerable num ber of f a l s e negative r e a c t i o n s . However, the f a c t that only a 2 mm. l o o p f u l of milk was spread over a 1 sq. cm. area i n place of the 0.01 c.c. employed under Standard Methods, may account f o r some of these I r r e g u l a r r e s u l t s obtained with the leucocyte count. I t should be remembered i n i n t e r p r e t i n g the r e  s u l t s of the leucocyte count, that the number of leucocytes i n colostrum i s very h i g h , and that the leucocyte count of the milk of normal animals tends to increase i n l a t e l a c t a  t i o n . I t Is probable that i f a leucocyte count of over 100,000 rather than one of over 500,000, as i s u s u a l l y accepted, be taken to i n d i c a t e a m a s t i t i c c o n d i t i o n , l e s s posi t i v e samples would be graded as negative by t h i s t e s t . A l s o , - 99 - i f the leucocyte count Is combined with the examination of the smear f o r the types of organisms present, 'the use of the two t e s t s should he of considerable value i n the detection of ab normal conditions i n the udder and of the organisms respon s i b l e f o r such disturbances. IABLE_11. THE USE OF THE LEUCOCYTE COUNT IN THE DETECTION OF MASTITIS SOURCE OF DATA LEUCOCYTE COUNT IN THOUSANDS PER c.c. OF MILK PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 40 79 <100 80.11 27.50 13.93 I 100-500 19.33 52.50 22.78 500-1,000 0.56 15.00 13.92 >1,000 5.00 49.37 No. of Samples 301 203 191 < 100 75.08 44 * 83 6.80 I I 100-500 . 21.93 38.42 9,43 500-1,000 1.99 12.81 24.09 >1,000 1.00 3.94 59.68 No. of Samples 517 254 176 < 100 85.69 65.36 13.63 I I I 100-500 13.73 20.87 24.43 500-1,000 0.58" 12.20 22.16 >1,000 1.57 39.78 - 100 - (h) Microscopic, Study of the_J3rgani,sms_P^ i n the M i l k Samples. A number of i n t e r e s t i n g f a c t s are revealed by the microscopic examination of the smears made from the milk samples, the r e s u l t s of which are found i n Table 12. I t can be r e a d i l y seen that a considerable number of p o s i t i v e samples showed no organisms at a l l on the smear. In the case of the experimental herd study, t h i s i s e s p e c i a l l y noteworthy, since a l l of the milks had been Incubated f o r 16 to 18 hours before the smears were made. I t would thus appear t h a t , i n many cases of obvious udder disturbance, no detectable number of organ isms were being shed In the m i l k . Whether "non-specific" mas t i t i s was present i n such i n s t a n c e s , or whether i t so happened that the causative organism was not being shed i n the milk at the time the samples were taken, i s not known. I t can also be seen that a greater proportion of the p o s i t i v e samples harboured s t r e p t o c o c c i than d i d the negative samples. The reverse r e l a t i o n s h i p was found to e x i s t f o r staphylococci and rod forms. Hence, i t would appear t h a t , i n the diseased udders, the m a s t i t i c s t r e p t o c o c c i had replaced, the normal udder m i c r o c o c c i . On comparing the r e s u l t s of t h i s t e s t w i t h those obtained w i t h the blood agar plate technique, the f i n d i n g s from which are shown i n Table 13, i t can be seen that a greater number of samples harbouring st r e p t o c o c c i was revealed by the l a t t e r t e s t . - 101 - TABLE 12. THE USE OF THE MICROSCOPIC EXAMINATION .Off MILK SAIl/IPLES IN THE DETECTION OF MASTITIS SOURCE OF DATA PREDOMINANT TYPES OF ORGMISMS • IN EACH SAMPLE PERCMT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 383 376 623 No Organisms 42.30 46.28 47.35 I I Chaining Strep t o c o c c i , alone & with Staph. & Rods 15.40 19.15 30.34 Staphylococci alone & with Rods 24.28 25.26 15.25 Rods & D i p l o c o c c i 18.02 9.31 7.06 No. of Samples 558 276 191 No Organisms 31.18 22.10 27.75 I I I Chaining Strep t o c o c c i , alone & with Staph. & Rods 3.23 25.00 39.79 Staphylococci alone & with Rods 37.45 32.97 21.99 Rods & D i p l o c o c c i 28.14 19.93 IO.47 The microscopic examination of the smears made from the milk samples appears to "be of considerable value i n the rapi d detection of the types of organisms responsible f o r cases of m a s t i t i s . The t e s t requires l e s s time and fewer materials than the blood agar plat e method, and, i f applied to incubated samples of m i l k , should reveal the great majority of - 102 - the types of organisms present i n the milk samples. I t should he noted at t h i s point t h a t , i n compiling the data f o r both the microscopic and c u l t u r a l studies of the m i l k samples f o r Tables 12 and 13, the r e s u l t s from a l l of the samples studied i n the general survey were employed. ( i ) C u l t u r a l . Study of the Organisms,L Pre sent i n the. M i l k .Samples. The r e s u l t s obtained from the c u l t u r a l study of the types of organisms present i n the milk samples using blood agar p l a t e s are found i n Table 13. The data obtained by the use of t h i s t e s t are s i m i l a r to those found with the microscopic examination of the milk samples. A few p o s i t i v e samples were again found which di d not harbour any d e f i n i t e organisms. Also the proportion of s t r e p t o c o c c i was again ob served to be much greater i n the p o s i t i v e than i n the negative samples. However, the percentage of s t r e p t o c o c c i found i n the p o s i t i v e samples by t h i s method was considerably higher than that found by the microscopic method. I t i s of s p e c i a l i n  t e r e s t to note the d i f f e r e n c e i n the proportion of non-hemo- l y t i c staphylococci to hemolytic staphylococci i n the negative and p o s i t i v e samples. In the negative samples the two types of staphylococci were present i n about equal numbers. In the p o s i t i v e samples, on the other hand, there were approximately three times as many hemolytic as'non-hemolytic types. These f i n d i n g s would suggest, then, that hemolytic staphylococci are more d e f i n i t e l y associated with m a s t i t i s than are the non hemolytic types. - 103 - TABLE 13. THE USE OF BLOOD AGAR PLATES IK THE DETECTION OF ORGANISMS ASSOCIATED WITH. MASTITIS SOURCE OF DATA PREDOMINANT TYPES OF ORGANISMS IN EACH SAMPLE PERCENT OF TOTAL NEGATIVE SAMPLES PERCENT OF TOTAL SUSPICIOUS SAMPLES PERCENT OF TOTAL POSITIVE SAMPLES No. of Samples 383 376 623 No S i g n i f i c a n t Growth 6.00 4.52 I I N.H. Staph, alone & w i t h Rods H. Staph, alone & with N.H. Staph. & Rods 43.34 31.86 25.80 32.71 11.08 35.31 Streps, alone & with Staph. & Rods 6.00 28.20 45.59 Rods 12.80 8.77 6.10 No. of Samples 558 276 191 No S i g n i f i c a n t Growth 16.31 7.61 6.81 I I I N.H. Staph, alone & with Rods H. Staph, alone & with N.H. Staph. & Rods 27.06 36.38 17.39 23.91 9.42 26.18 Streps. alone or with Staph. & Rods 7.88 40.22 50.26 Rods 12.37 10.87 7.33 N.H. Staph: non-hemolytic sta p h y l o c o c c i . H. Staph: hemolytic s t a p h y l o c o c c i . Streps: s t r e p t o c o c c i (both non-hemolytic and hemolytic) - 104 - I t i s generally agreed that the hemolytic pro p e r t i e s of the various s t r e p t o c o c c i associated with m a s t i t i s hear no r e  l a t i o n s h i p to the a b i l i t y of the organisms to produce udder i n f e c t i o n . In f a c t , d i f f e r e n t s t r a i n s of S t r . a galactiae have been found to e x h i b i t alpha, b e t a , weak beta and gamma haemo l y s i s as described by Brown ( 9 ). With the s t a p h y l o c o c c i , how ever, the non-hemolytic types are considered to be non-patho genic, and the hemolytic types, e s p e c i a l l y I f t h i s property i s associated with the power to coagulate human or r a b b i t plasma., are thought to be probable pathogens. The hemolytic staphy l o c o c c i encountered i n the present study u s u a l l y formed c l e a r zones around, the colonies on blood agar. Although hemolytic staphylococci can be i s o l a t e d from the healthy udder, the f i n d i n g of considerable numbers of these organisms, combined T/ith other i n d i c a t i o n s of disease, may be considered evidence that the causative organism i n such cases i s the staphylo coccus i s o l a t e d . Regarding the use of Edwards' medium as a rap i d means of detecting s t r e p t o c o c c i i n mi l k samples, i t was observed i n the present study that very r a r e l y were st r e p t o c o c c i found on the Edwards' medium pl a t e s and not on duplicate plates made with ordinary blood agar. Admittedly, the d i f f e r e n t i a t i o n of S t r . u b e r i s , by the property of t h i s organism to form dark areas around the colonies on Edwards1 medium, cannot be ac complished by the use of ordinary blood agar. Otherwise, since Edwards' medium reveals only s t r e p t o c o c c i , and since i t - 105 - has been found i n the present study that organisms other than s t r e p t o c o c c i are l i k e l y to be present i n a f a i r l y l a r g e pro p o r t i o n of the milk samples, i t appears that tryptose blood agar i s a s a t i s f a c t o r y general p l a t i n g medium f o r routine studies on the types of organisms associated with m a s t i t i s . I t may be concluded that the blood agar p l a t e tech- nique i s u s e f u l i n the detection of the types of organisms present i n the milk from animals suspected of having m a s t i t i s . This technique i s required f o r the I s o l a t i o n of organisms from the milk samples i n order to d i f f e r e n t i a t e i n greater de t a i l between the various types of m a s t i t i c organisms. This d e t a i l e d study i s necessary i n order to determine whether a case of streptococcic m a s t i t i s i s of the common contagious form due to S t r . agalactiae or whether i t i s of the l e s s con tagious types caused by S t r . dysgalactiae and S t r . u b e r i s . The use of blood agar pl a t e s i s also of value i n d i f f e r e n t i a  t i n g between hemolytic and non-hemolytic s t a p h y l o c o c c i , a l  though some i n d i c a t i o n of whether the staphylococci present i n a milk sample are normal udder b a c t e r i a or are probable pathogens may be obtained by noting the r e l a t i v e numbers of staphylococci observed i n d i f f e r e n t milk samples on the micro scopic examination of the m i l k s . I t i s probable t h a t , i f the predominant types of organisms responsible f o r the m a s t i t i s i n a given herd, were once determined by c u l t u r a l methods, then, on subsequent t e s t i n g s of t h i s herd, the blood agar plate method need not be employed. - 106 - 4* D e t a i l e d Study of the Organisms A s s o c i a t e d with M a s t i t i s . The c u l t u r e s obtained by p i c k i n g r e p r e s e n t a t i v e c o l o n i e s from the blood agar p l a t e s of the milk samples from a l l of the cows i n herds #1 and #2 of the experimental herd study, were subjected to a v a r i e t y of t e s t s , which were se l e c t e d to detect the organisms most commonly associated "with m a s t i t i s . In Table 14 are found the d i f f e r e n t i a t i n g charac t e r i s t i c s employed to d i s t i n g u i s h between the species of strep t o c o c c i which have been found capable of producing m a s t i t i s i n f e c t i o n . The i n f o r m a t i o n contained i n t h i s t a b l e has been compiled from the data of Hansen (44)> Sherman (102), Merchant and Packer (68) and P l a s t r i d g e et a l (83). On the b a s i s of the f i n d i n g s of Merchant and Packer . (68) and P l a s t r i d g e et a l (83) that none of the pathogenic s t r e p t o c o c c i ferment r a f f i n o s e , whereas many of the sapro p h y t i c types do ferment t h i s carbohydrate, a l l of the r a f f i - nose-fermenting s t r a i n s i s o l a t e d were considered as being saprophytic, and were not c l a s s i f i e d f u r t h e r . Over h a l f of the streptococcus c u l t u r e s obtained were found to be S t r , a g a l a c t i a e , S t r . d y s g a l a c t i a e or S t r . u b e r i s . Some of the s t r a i n s i s o l a t e d were found to be a t y p i c a l with respect to one or two p r o p e r t i e s . These organisms were c l a s s i f i e d as be longing to the species they most resembled. S t r . zooepidemi cus and S t r . pyogenes were not encountered. There were a num-w ro *< «+ o C9 • (!) P © CO cc ro t J ct- H* H pi« CP 3 N H - O O O £ ! - 01 O ro CD • w <K) CO $» <+ j» • O ct- P $» ca CD I H ro o ^  <l> (TO P5 I $0 <l C $» CO H * « I <D H - • I O O o o vnvn -F" -P" • * • * ro o \o ON i i -r-r -r-r • • • » \0-vJ ON -f I t I + ro i i I I H I Organism Hippurate Hydrolysis Aesculin Splitting Hemolysis Lancefield Group Trehalose Sorbitol Salicin Mannitol Raffinose •*j o CD P B 0 4 cr o SB £b <+ H p- CO O ct £3 CD Final pH in Glucose Broth Reduction of Methylene Blue Milk Reduction Before Clotting Reduction After Clotting Days to Clot tr* Sri CD I o H * O P e 1-3 M H3 H ro ro •-3. h3 O O o o o M Sri {> o *-3 H O ro s te) 1-3 O to | M N 1-3 M |H3 - LOT ~ - 103 - ber of s t r a i n s which di d not f i t i n t o any of the recognized species; these c u l t u r e s have been designated as " u n c l a s s i f i e d 1 1 . For the r a p i d d i f f e r e n t i a t i o n of the common m a s t i t i c s t r e p t o c o c c i , Brown's m o d i f i c a t i o n of the L a n c e f i e l d p r e c i p i  t i n t e s t .(10), together with the determination of the a b i l i t y of the organisms to bydrolyze sodium hippurate and s p l i t aescu l i n appear to be s a t i s f a c t o r y t e s t procedures, I f more pre c i s e work i s d e s i r e d , the r e a c t i o n i n m i l k , the f i n a l pH i n glucose broth and the fermentation of trehalose, s o r b i t o l , s a l i c i n and mannltol may be employed. In the c l a s s i f i c a t i o n of the s t a p h y l o c o c c i , the des c r i p t i o n s of the p r o p e r t i e s of Staph, aureus given by Bergey (5) and Minett (72) were employed as a guide. The other staphylococci encountered were d i f f e r e n t i a t e d according to the system proposed by Hucker (50). About one t h i r d of the staphy lococcus cultures studied were Staph, aureus. Another quarter of the cultures could not be f i t t e d i n t o any of the recognized species. The other staphylococci encountered appeared to be of the types u s u a l l y found i n the normal udder. The f o l l o w i n g species were found In the milks of both herds: Micrococcus albus, M. candidus, and M. epidermidis. M. c a s e o l y t i c u s , M. c i t r e u s , M. f l a v u s , and M. luteus were also i s o l a t e d from the samples from herd #2. In evaluating the methods used to c l a s s i f y the staphy l o c o c c i , i t would appear from the present study that Staph, aureus can be r e a d i l y i d e n t i f i e d by i t s a b i l i t y to hemolyze - 109 - cow's blood agar, coagulate human blood plasma, ferment man- n i t o l , reduce n i t r a t e to n i t r i t e and l i q u i f y g e l a t i n . I t seems evident, however, that the methods employed to d i f f e r e n  t i a t e the udder micrococci should be r e v i s e d . In Hucker's system of c l a s s i f i c a t i o n , the primary d i v i s i o n s are determined on the basi s of the type of pigment produced by the various organisms. Since i t i s w e l l known that a given species may show considerable v a r i a t i o n i n the type of pigment formed, depending on environmental c o n d i t i o n s , I t i s obvious that such a system of c l a s s i f i c a t i o n i s not very sound. In any attempt to rev i s e the c l a s s i f i c a t i o n of these organisms, a study would f i r s t have to be made using recog n i z e d species of m i c r o c o c c i . I t i s probable that such pro p e r t i e s as g e l a t i n l i q u e f a c t i o n , n i t r a t e reduction, a c t i o n on milk and fermentation of carbohydrates could be u t i l i z e d i n such a scheme. Another complicating f a c t o r i n the c l a s s i  f i c a t i o n of these organisms i s that a number of workers, i n  cluding Bergey (5), d i v i d e the staphylococci from the micro c o c c i merely on the b a s i s of the si z e of the i n d i v i d u a l bac t e r i a l c e l l s , and the way i n which they form, groups. I t has been found i n the present study that such morphological c h a r a c t e r i s t i c s vary g r e a t l y depending on the medium the o r  ganism i s grown i n . There i s also the question as to whether the micrococci and staphylococci"should be considered as separate genera or whether these organisms, which have many c h a r a c t e r i s t i c s i n common, should be grouped under one genus. - 110 - I t may be concluded, then, that the c l a s s i f i c a t i o n of the staphylococci and micrococci i s f a r from s a t i s f a c t o r y , and that considerable work on t h i s group of organisms Is urgently needed. The rod forms i s o l a t e d from the various samples were found d i f f i c u l t to c l a s s i f y . Most of them appeared to be types which are widely d i s t r i b u t e d i n nature, and which are freq u e n t l y found i n the mi l k from normal animals, but which, because of t h e i r I n a c t i v i t y i n various t e s t media, have been studied very l i t t l e . They were c l a s s i f i e d as f a r as was pos s i b l e by the d e s c r i p t i o n s given i n Bergey ( 5 ) . The rods I s o  l a t e d from the milks of herd #1 were not very ex t e n s i v e l y s t u d i e d , but i t was determined that approximately h a l f were species of corynebacteria, the rest being other gram p o s i t i v e types and gram negative b a c t e r i a . The rod forms i s o l a t e d from herd #2 were studied In greater d e t a i l . . About h a l f of these were found to be of the fo l l o w i n g types: Microbacterium flavum, Microb. l i q u e i a c i e n s , Microb. l a c t i c u m , C. flavidum, C. bovis (Bacterium l i p o l y t i c u m ) , and C. xerose. Approxi mately one t h i r d of the rods i s o l a t e d from herd #2 were gram p o s i t i v e forms which apparently do not f i t i n t o any of the groups described by Bergey. A few gram negative types were encountered: Proteus sp., S e r r a t i a sp., and E c o l i , but i t appears l i k e l y that these organisms were contaminants or chance inhabitants of the udder, and were not r e l a t e d i n any way to m a s t i t i s . - I l l - Because most of these rod forms appear so i n a c t i v e , i t i s probable that they do not play a very s i g n i f i c a n t r o l e i n the i n i t i a t i o n of m a s t i t i s i n f e c t i o n i n the udder. The only rods which commonly produce m a s t i t i s are E. c a l l , Aer. aero genes and C. pyogenes, and these forms can be r e a d i l y i d e n t i  f i e d . I t i s not known whether or not the other rods, which may be considered normal Inhabitants of the udder, play a part i n predisposing the udder to i n f e c t i o n by other invading bac t e r i a . I t may be noted i n t h i s connection that the majority of the Corynebacterium and Microbacterium types were i s o l a t e d from negative quarters i n which no other types of b a c t e r i a were found. I t would appear, from the evidence at hand, that the rods commonly incriminated i n m a s t i t i s can be f a i r l y e a s i l y I d e n t i f i e d by recognized procedures, and t h a t , i n a l a r g e - s c a l e m a s t i t i s program, i t i s unnecessary to study i n d e t a i l the rod forms isolated, from apparently healthy quarters or from p o s i t i v e quarters i n which pathogenic staphylococci and s t r e p t o c o c c i are present. A t o t a l of 424 cultures were c l a s s i f i e d f o r herd #1 and 523 f o r herd #2. Because of the f a c t t h a t , i n a great many cases, i t was found that surface and sub-surface colonies of the same organism had been picked from the o r i g i n a l p l a t e s , i t was decided to study the general f i n d i n g s on the c l a s s i f i  c a t i o n of the organisms from the- point of view of the pre dominant types of b a c t e r i a found i n each quarter, taking i n t o consideration the p r o b a b i l i t y of the organisms present being - 112 - p o t e n t i a l producers of m a s t i t i s . These general r e s u l t s are found i n Table 15. TABLE 15. PREDOMINANTTYPES OF.ORGANISMS IN QUARTER MILK SAMPLES FROM ALL OF THE ANIMALS IN HERDS #1 AND #2 PERCENT PERCENT PERCENT HERD TYPES OF OF TOTAL OF TOTAL OF TOTAL ORGANISMS NEGATIVE SUSPICIOUS POSITIVE QUARTERS QUARTERS QUARTERS No. of Quarters 4? 41 19 No Organisms 10.64 4«83 Saprophytic 56.10 Staph, Streps, Rods 57.44 10.53 Staph, aureus 29.79 JL 2 * -1-9 5.26 S t r . agalactiae 17.07 52.63 #1 S t r . dysgalactiae ___ 4.88 5.26 S t r , u b e r i s 2.44 5.26 U n c l a s s i f i e d S t r . 2.13 — Combinations of Streptococci 10,53 Combinations of Stre p t o c o c c i with Staph, aureus 10.53 No. of Quarters 74 34 20 No Organisms 6.76 —— Saprophytic 20.59 5.00 Staph, Streps, Rods 33.73 Staph, aureus 35.14 1.35 32.35 30.00 S t r . agalactiae 5.3S 25.00 #2 S t r . dysgalactiae 3.11 2.94 10.00 S t r . uberis 1.35 U n c l a s s i f i e d S t r . 4.05 2 » Combinations of Streptococci 11.77 5.00 Combinations of Streptococci with Staph, aureus 9.46 23.53 25.00 - 113 - In considering the f i n d i n g s f o r herd #1, i t i s seen that the predominant organisms i n the negative quarters were saprophytic types and Staph, aureus. In the p o s i t i v e quarters, however, the most commonly found organism was S t r . a g a l a c t i a e . I t would thus appear l i k e l y that the m a s t i t i s i n t h i s herd was due almost e n t i r e l y to S t r . a g a l a c t i a e . In herd #2, on the other hand, Staph, aureus, S t r . a g a l a c t i a e , and combinations of Staph, aureus and s t r e p t o c o c c i are seen to be present i n about equal numbers i n the p o s i t i v e q u a r t e r s . I t would seem probable, then, that a number of i n  f e c t i v e agents were present i n t h i s herd, a f a c t which might render d i f f i c u l t an attempt to c o n t r o l the disease i n t h i s herd by a management program. The high incidence of Staph, aureus i n the samples studied i s noteworthy. Chapman et a l (23), i n a study of staphylococci i s o l a t e d from human sources, concluded that any staphylococcus which was hemolytic on cow's blood agar, which coagulated human plasma and which fermented mannitol should be considered as a possible pathogen. P l a s t r i d g e et a l (84) came to the same conclusion regarding staphylococci i s o l a t e d from m a s t i t i c animals, and stated that i n the routine examination of m i l k samples, any staphylococcus which produced hemolysis on cow's blood agar or which was associated w i t h over 500,000 leucocytes per c.c. should be considered as probably patho genic. The s i g n i f i c a n c e which i s to be attached to the i s o  l a t i o n of a large proportion of Staph, aureus from otherwise - 114 - normal, quarters i s d i f f i c u l t to determine. No conclusions can be drawn from the study of only two herds, but i t i s probable that t h i s organism may be more commonly d i s t r i b u t e d i n normal udders than i s generally recognized. 5. Studies on the Control of. M a s t i t i s . Various workers have demonstrated that e n t i r e herds can be freed from S t r . a g a l a c t i a e I n f e c t i o n , and can be main tained in such a c o n d i t i o n f o r considerable periods of time, by a r i g i d management, segregation and treatment program. Among these workers are P l a s t r i d g e et a l (83) and Minett et a l (74)• Such a program involves the segregation of i n f e c t e d animals from healthy ones, the e l i m i n a t i o n from the herd o f . the animals considered u n l i k e l y to respond to treatment, the treatment of animals harbouring S t r . a g a l a c t i a e , a r i g i d con t r o l of the s a n i t a r y p r a c t i c e s employed during the mi l k i n g pro cedure to prevent the spread of the organism, and general measures to eliminate f a c t o r s which might predispose to I n f e c  t i o n of the udder. Sucker and Harrison (52) found i n t h e i r studies that while they could, not maintain a herd free from animals d i s  charging m a s t i t i c s t r e p t o c o c c i i n t h e i r m i l k , they could main t a i n a herd free from animals secreting abnormal m i l k . I t should be emphasized, however, that t h e i r studies were con f i n e d to three herds only. "Watts (108) states t h a t , i f the Incidence of S t r . agalactiae I n f e c t i o n i n a herd i s below 15$, the management - 115 - p r a c t i c e s are not responsible f o r the amount of i n f e c t i o n i n the herd, but t h a t , I f the incidence i s over t h i s f i g u r e , the system of management i s c o n t r i b u t i n g to the incidence of the disease i n the herd, and that i f the management i s r i g i d l y con t r o l l e d , a decrease i n the amount of i n f e c t i o n should r e s u l t . In the experimental herd study reported h e r e i n , i t had been hoped to demonstrate that the fi n d i n g s of P l a s t r i d g e et al (83) and Minett et al (74), that S t r . agalactiae could be eliminated completely from a herd, could be duplicated i n t h i s area. The r e s u l t s from the f i r s t three routine t e s t i n g s of herds #1 and #2 and from the f i r s t two t e s t i n g s of herds #3 and #4 are shown i n Table 16. I t i s observed that i n a l l ex cept herd #3 there was no s i g n i f i c a n t reduction i n the amount of i n f e c t i o n as the study proceeded. In order to expla i n some of these f i n d i n g s , a b r i e f d i s c u s s i o n on each of the four herds i s given below. Herd #1 was found to be h i g h l y Infected with S t r . aga l a c t i a e , although only three c l i n i c a l cases of m a s t i t i s were encountered during the course of the study. I t would appear that t h i s herd should respond to a management program f o r the e l i m i n a t i o n of S t r . a g a l a c t i a e . The f a c t s that no treatment measures had been employed i n t h i s herd during the period of the work reported h e r e i n , and that none of the c l i n i c a l cases had been eliminated from the herd, may explain why the i n c i  dence of m a s t i t i s i n t h i s herd has not decreased. I t i s e v i  dent, however, that the management program has at l e a s t pre-- 116 - vented the disease from spreading to a greater extent through out the herd. TABLE 16. PERCENTAGE INFECTION IK THE EXPERIMENTAL HERDS AT THE TIME OF THE I4EI0US„R0UTINE_T PERCENT OF COWS IN HEED HERD #1 HERD #2 HERD #3 HERD No, of Cows 28 35 14 23 FIRST TEST Neg. Susp. Pos. 4.2.86 28.57 28,57 54-29 II.42 34.29 35.71 42.86 21.43 43.48 17.39 39.13 No. of Cows 27 33 10 27 SECOND TEST Neg. Susp. 33.34 33.33 15.16 42.42 40.00 50.00 48.15 14.81 Pos. 33.33 42.42 10.00 37.04 No. of Cows 23 33 THIRD TEST Neg. Susp. Pos. 34.78 34.78 30.44 27.28 36,36 36.36 As has been shown i n the previous section on the c l a s s i  f i c a t i o n of the organisms associated with m a s t i t i s , herd #2 was found to harbour a v a r i e t y of m a s t i t i c organisms - S t r . a g a l a c t i a e , S t r . dysga l a c t i a e , S t r . u b e r i s , and Staph, aureus. Several cases of c l i n i c a l m a s t i t i s were recorded during the - 117 - period of study. The presence i n t h i s herd of a number of o r  ganisms i n a d d i t i o n to S t r . agalactiae probably explains why the herd has not responded to the c o n t r o l program. Herd #3, which i s a small and c a r e f u l l y supervised herd, has shown a considerable lessening In the incidence of m a s t i t i s during the course of the study. None of the common m a s t i t i c organisms could be i s o l a t e d from the milk samples from t h i s herd at e i t h e r of the routine t e s t i n g s . A number of pseudomonas-type rods were observed to be present i n the milks from most animals i n the herd, but no r e l a t i o n s h i p could be es t a b l i s h e d between the amount of i n f e c t i o n i n the herd and the d i s t r i b u t i o n of these organisms. The predominant organisms i n herd #4 were found, to be s t a p h y l o c o c c i . The absence of S t r . agalactiae probably ac counts f o r the f a c t that l i t t l e success has been attained with the c o n t r o l program i n t h i s herd. I t should be re-emphasized that any c o n t r o l program i s of value only i f the predominant organism i n the herd i s S t r . a g a l a c t i a e . Such a c o n d i t i o n was found i n only one of the ex perimental herds. Complete success, therefore, cannot be ex pected i n the other three herds, although some lessening of the incidence of the disease may be a n t i c i p a t e d i n these herds, because of the f a c t that the adoption of the r i g i d management p r a c t i c e s , o u t l i n e d i n the schedule to the agreement and r e  f e r r e d to previously i n t h i s r e p o r t , should eliminate most of the f a c t o r s which might predispose the udder to i n f e c t i o n by - 118 - other m a s t i t i c organisms. 6. Studies on the Treatments Used f o r M a s t i t i s . . Ever since m a s t i t i s has become a serious problem to the d a i r y farmer, various i n v e s t i g a t o r s have worked towards the development of s u i t a b l e treatment measures f o r t h i s d i s  ease . Because of the v a r i e t y of organisms which have been found to be capable of causing m a s t i t i s , the problem of pre s c r i b i n g s u i t a b l e treatment measures becomes very complicated. I t i s , of course, necessary to determine whether the case at hand i s of the i n f e c t i o u s or non-infectious type of m a s t i t i s , since treatment with chemotherapeutic or a n t i b a c t e r i a l agents i s of l i t t l e value i n curing non-infectious m a s t i t i s . I t i s a l s o u s e f u l to know the causative organism i n a given case of m a s t i t i s , because s p e c i f i c agents are often best s u i t e d f o r e l i m i n a t i n g c e r t a i n b a c t e r i a , and may have l i t t l e e f f e c t on other organisms. Mixed b a c t e r i a l vaccines and autogenous vaccines were among the f i r s t treatment measures t r i e d . Their use, how ever, met with i r r e g u l a r success. In some herds the vaccines appeared to have a s s i s t e d i n the lessening of the incidence of the disease, but the r e s u l t s were not conclusive enough to prove d e f i n i t e l y that these methods were wholly responsible f o r the e l i m i n a t i o n of m a s t i t i s i n f e c t i o n . I t appears that the common m a s t i t i s s t r e p t o c o c c i and staphylococci do not induce the production of a very high l e v e l of antibodies i n the blood. - 119 - Numerous chemotherapeutic and several a n t i b i o t i c agents have been employed from time to time i n the treatment of mas t i t i s . The most widely used of these are discussed b r i e f l y below. One of the f i r s t of these chemo the rap e u t i c agents to be used i n the treatment of chronic m a s t i t i s was c o l l o i d a l s i l v e r oxide which i s commonly known as N o v o x i l . Very favourable r e  ports have been made regarding the effectiveness of udder i n  fusions of t h i s product i n the removal of m a s t i t i c organisms from the udder. However, the f a c t that t h i s preparation i s very t o x i c to udder t i s s u e , and frequently produces a m i l d i n  duration i n the udder and t e a t s , precludes i t from extensive use. This compound i s d e f i n i t e l y contraindicated i n cases of acute m a s t i t i s . C e r t a i n of the a c r i d i n e dyes, namely a c r i f l a v i n (trypa- f l a v i n ) and p r o f l a v i n , have also been used extensively i n the treatment of m a s t i t i s . Schalm (93) reported favourable r e s u l t s from i n f u s i n g the udder with t r y p o f l a v i n i n hypertonic (20$) sugar s o l u t i o n i n e l i m i n a t i n g S t r . a g a l a c t i a e . Johnson (.53) was successful i n t r e a t i n g dry and almost dry cows harbouring S t r . a g a lactiae with a c r i f l a v i n e . Schalm (92) reports having used Entozon i n the t r e a t  ment of m a s t i t i s due to S t r . agalactiae by udder i n f u s i o n methods. This preparation has the f o l l o w i n g composition; 2, 3-dimethoxy-6-nitro-9-(y-diethyl-amino-(3-oxy~ propylamine) acridene dihydro-chloride 5.38$ - 120 2-ethoxy-6, 9-diamino-acridine l a c t a t e amyl saccarine sodium biborate 29.44$ 58.80$ 5.88$ The treatment was successful i n most cases with very l i t t l e re s u l t i n g i r r i t a t i o n to the udder. However, the m i l k from the treated cows.could not be used f o r one to three weeks a f t e r treatment, and the presence of milk or other exudate 'was found to p r e c i p i t a t e the acrichine d e r i v a t i v e s i n the preparation, n e c e s s i t a t i n g the frequent i n j e c t i o n of the diseased quarter w i t h the drug. Bryan et a l (15) reported the successful treatment of s u b - c l i n i c a l s t r e p t o c o c c i c m a s t i t i s with i n f u s i o n i n t o the aff e c t e d quarter of 75 c.c. q u a n t i t i e s of a 1:1000 aqueous so l u t i o n of phemerol (para-tertiary-octy1-phenoxy-ethoxy-et h y l - dimethyl-benzyl ammonium c h l o r i d e monohydrate). The use of t h i s product, however, tends to induce the production of f l a k y m ilk f o r several days, and the mucous membranes l i n i n g the teat and mil k c i s t e r n s become temporarily thickened. Because of these f a c t s , i t i s recommended that t h i s treatment be ap p l i e d near the end of the l a c t a t i o n p e r i o d . The s u l f a drugs, e s p e c i a l l y sulfanilamide and s u l f a - t h i a z o l e , have been extensively used i n the treatment of mas t i t i s , the former compound being more e f f e c t i v e against s t r e p  t o c o c c i and the l a t t e r against s t a p h y l o c o c c i . These drugs are best administered as udder i n f u s i o n s , and are very s u i t a b l e f o r i n j e c t i o n i n t o dry udders, the preparation being l e f t i n the udder u n t i l the cow freshens. I t i s often recommended i n - 121 - acute cases that sulfanilamide be administered o r a l l y u n t i l the acute symptoms i n the udder have subsided. Favourable r e s u l t s were obtained by K l e i n et a l (62) i n the treatment of S t r . agalactiae and staphylococcic i n f e c t i o n w i t h s u l f a n i l a m i d e . S w e t t et a l (105) found sulfanilamide and s u l f a d i a z i n e alone and i n combination to be e f f e c t i v e i n curing m a s t i t i s due to s t r e p t o c o c c i , staphylococci and c o l i  form organisms. These drugs, however, were only about 50$ e f  f e c t i v e i n e l i m i n a t i n g Ps. aeruginosa i n f e c t i o n . T y r o t h r i c i n and i t s two components, t y r o c i d i n and grami c i d i n , the products of the organism B a c i l l u s b r e v i s , have also been used with considerable success i n the treatment of mas t i t i s . These products are h i g h l y t o x i c to the udder t i s s u e , and care must be taken i n determining the dosage to be used, and i n deciding how long the preparations are to be l e f t i n the udder. L i t t l e (65) found t y r o t h r i c i n and gramicidin to be e f  f e c t i v e i n curing chronic streptococcic m a s t i t i s . Bean et a l (3) recorded favourable r e s u l t s i n t r e a t i n g chronic streptococcic m a s t i t i s i n l a c t a t i n g cows with the f o l  lowing products arranged i n order of decreasing e f f i c i e n c y : a c r i f l a v i n , Movoxll, Entozon, and t y r o t h r i c i n . In dry cows, t y r o t h r i c i n was found to be most e f f e c t i v e i n e l i m i n a t i n g s t r e p t o c o c c i , with Entozon, a c r i f l a v i n , and Wovoxil f o l l o w i n g i n order of decreasing e f f e c t i v e n e s s . Schalm (94) reported on the use of Entozon, t r y p a f l a v i n , n e u t r a l a c r i f l a v i n , t y r o t h r i c i n and Novoxil i n the treatment - 122 - of chronic s t r e p t o c o c c i c m a s t i t i s , and found Entozon, t y r o - t h r i c i n and Novoxil to be of most value f o r t h i s purpose. He found staphylococcic i n f e c t i o n r e l a t i v e l y r e s i s t a n t to chemo therapy, Schalm l a t e r (95) concluded t h a t , i n order to reduce to a minimum the amount of milk l o s t during the treatment of l a c t a t i n g animals, only c l i n i c a l cases should be treated during l a c t a t i o n w ith t y r o t h r i c i n or sulfanilamide i n o i l , and that these cases should be treated only enough to r e l i e v e the c l i n i  c a l symptoms. The organisms should be completely eliminated from the udder by the treatment of the dry cow with such pro ducts as s i l v e r oxide. The 194-2 report of the Chief of the United States Bureau of Animal Industry (18) records f i n d i n g s s i m i l a r to those of Bean et a l and Schalm. I t was found that a c r i f l a v l n , c o l l o i d a l s i l v e r oxide, Entozon, and t y r o t h r i c i n , i n order of decreasing e f f i c i e n c y , to be e f f e c t i v e i n curing l a c t a t i n g cows, and t y r o t h r i c i n , Entozon, a c r i f l a v i n and s i l v e r oxide, i n order of decreasing e f f e c t i v e n e s s , to be of value i n t r e a t  ing dry cows. P e n i c i l l i n , the a n t i b i o t i c agent formed by P e n i c i l l i u m notaturn, i s being very widely used at the present time i n the treatment of m a s t i t i s . This substance i s active against gram p o s i t i v e organisms but apparently has very l i t t l e e f f e c t on gram negative b a c t e r i a . I t i s very s u i t a b l e f o r ad m i n i s t r a t i o n by udder i n f u s i o n , because i t i s r e l a t i v e l y non-toxic and d i s  solves r e a d i l y i n water and s a l i n e . - 123 - Foley et a l (38) and Seeley et a l (99) have c a r r i e d out i n v i t r o studies on the e f f e c t of p e n i c i l l i n on the organisms commonly associated with m a s t i t i s , and have found that the f o l l o w i n g organisms were susceptible to small amounts of the drug: S t r . a g a l a c t i a e , S t r . u b e r i s , S t r . d y s g a l a c t i a e , S t r . zooepidemicus, Staph, aureus and C. pyogenes. E. c o l i was not destroyed by the preparation. Schalm (91) found staphylococcic m a s t i t i s more r e s i s  tant to p e n i c i l l i n therapy than the streptococcic form of the disease. He also found that a greater proportion of dry udders responded, to p e n i c i l l i n treatment than di d l a c t a t i n g udders. He recommends an i n i t i a l treatment of 5 doses to be adminis tered at 5 successive m i l k i n g s . I f the organism i s s t i l l present 10-14 days a f t e r treatment, he suggests repeating the treatment w i t h 4 i n j e c t i o n s 12-24 hours apart using double the o r i g i n a l dosage. The 1945 report of the Chief of the United States Bureau of Animal Industry (19) records v a r i a b l e success with the a d m i n i s t r a t i o n of one treatment only of 50,000 u n i t s of p e n i c i l l i n , and strongly suggests that p e n i c i l l i n should not be considered a panacea f o r m a s t i t i s , but that prevention i s s t i l l the best treatment f o r t h i s disease. Piiddet (31) suggests that the use of a bacteriophage may be e f f e c t i v e i n the treatment of m a s t i t i s due to staphy l o c o c c i . He i s o l a t e d several phages against m a s t i t i c staphy l o c o c c i , and found that the phage preparations were non-toxic — 124- ~ to the udder t i s s u e . He d i d not r e p o r t , however, how succes s f u l the phage preparations were i n e l i m i n a t i n g the organisms from the udder. Washburn (107) concluded from h i s studies that i f cows are fasted f o r a f i v e day period once every l a c t a t i o n p e r i o d , there i s a d e f i n i t e tendency towards the e l i m i n a t i o n of mas t i t i s . As a r e s u l t of t h i s procedure, he found the pH and percent c h l o r i n e of the milk of m a s t i t i c animals to r e t u r n to normal, and f o r f l a k e s and c l o t s i n the m i l k to disappear. He d i d not r e p o r t , however, how w e l l the m a s t i t i c organisms were eliminated by t h i s treatment. Pounden (85) recommends the cessation of m i l k i n g i n acute attacks of m a s t i t i s due to S t r . agalactiae u n t i l the in flammatory symptoms have subsided. He found that the cessa t i o n of m i l k i n g , f o r up to three days, had no i l l e f f e c t s on the animal, and that the causative organisms were e f f e c t i v e l y eliminated by t h i s procedure. Such a treatment, however, was considered to be of no value i n cases of m a s t i t i s due to staphylococci and C. pyogenes. Despite a l l the progress which has been made In the treatment of m a s t i t i s , the best treatment f o r t h i s disease i s prevention. A c e r t a i n treatment may eliminate the m a s t i t i c organisms from an udder, but i t does not protect that udder from becoming r e i n f e c t e d i n the future with the same or a d i f f e r e n t organism. I t has u s u a l l y been found that cows which - 125 - have had attacks of m a s t i t i s and have been s u c c e s s f u l l y cured are more susceptible to i n f e c t i o n than healthy animals (58). Hence, care i n the prevention of the spread of m a s t i t i s i n  f e c t i o n from already diseased to healthy cows i n the herd, and v i g i l a n c e i n the e l i m i n a t i o n of f a c t o r s which might pr e  dispose the udder to i n f e c t i o n , are the best treatments.which can be recommended f o r t h i s disease. I t should also be emphasised that the greater the ex tent of i n d u r a t i o n and f i b r o s i s i n the udder, the l e s s l i k e l y i s the treatment applied to be s u c c e s s f u l . I t i s thus ap parent that the best r e s u l t s w i l l be obtained by treatment i n the e a r l y stages of the disease. Further, the f a c t that the i n f e c t i o n of the udder with s t r e p t o c o c c i i s thought to begin at the base of the teat and to work upwards i n the udder, whereas staphylococcic and c o l l i o r m i n f e c t i o n s take place i n the parenchymatous t i s s u e i t s e l f , may help to e x p l a i n why mas t i t i s due to s t r e p t o c o c c i i s u s u a l l y more responsive to t r e a t  ment measures than that caused by staphylococci and c o l i f o r m organisms. I t i s generally agreed that the most e f f e c t i v e method of administering the various therapeutic agents used f o r the treatment of m a s t i t i s i s by i n f u s i o n d i r e c t l y i n t o the udder. Oral or intravenous methods appear to be of l i t t l e value, with the possible exception of the o r a l administration of s u l f a  nilamide i n acute cases of the disease. The dosage and num ber of treatments to be prescribed vary, depending on the s i z e - 126 - of the udder, the i n f e c t i n g organism, the s e v e r i t y of the a t  tack, the extent of the udder t i s s u e i n v o l v e d , and the length of time the disease has been present i n the udder. In the general survey reported h e r e i n , a number of therapeutic agents were used by the p r a c t i s i n g v e t e r i n a r i a n s i n the treatment of m a s t i t i s . The most widely used treatment measure was the i n f u s i o n of the udder with a mixture i n min e r a l o i l of sulfanilamide and s u l f a t h i a z o l e . Unfortunately, no data could be c o l l e c t e d on the effectiveness of the various treatments a p p l i e d . In the experimental herd study, an experiment using s i x cows i n herd #2 was performed, i n order to study the e f  fectiveness of p e n i c i l l i n i n the treatment of streptococcic m a s t i t i s . The animals selected f o r study were a l l s e r i o u s l y i n f e c t e d with streptococcic m a s t i t i s . 5 animals were given an i n i t i a l treatment of 25,000 u n i t s of p e n i c i l l i n per quarter, and the s i x t h was given 37,500 u n i t s per quarter. 4. of the f i r  st 5 animals were given a second treatment of 25,000 u n i t s per quarter 24 hours a f t e r the f i r s t . 2 of these 4 cows were given a t h i r d treatment of 25,000 u n i t s per quarter 24 hours a f t e r the second. M i l k samples were obtained from a l l 6 animals one week a f t e r the l a s t treatment. The r e s u l t s of the examination of the milk samples showed that there was no apparent reduc t i o n i n the numbers of str e p t o c o c c i i n the i n f e c t e d quarters as a r e s u l t of the treatment. This may have been because too l i t t l e p e n i c i l l i n was administered, or because the treatments - 127 - were not given frequently enough, or because the corns treated were too severely i n f e c t e d with m a s t i t i s to respond to t r e a t  ment. These r e s u l t s i n d i c a t e that there i s scope f o r con side r a b l e work on the treatment of m a s t i t i s i n the herds under study by the Research C o u n c i l . GENERAL DISCUSSION... The r e s u l t s of the studies reported herein i n d i c a t e that the extent of m a s t i t i s i n f e c t i o n i n the d a i r y herds of B r i t i s h Columbia i s of the same order as that reported f o r other parts of the world. This disease, then, i s so wide spread and produces such serious consequences as to create a d e f i n i t e problem f o r the d a i r y i n d u s t r y . Of the many t e s t s which have been employed f o r the de t e c t i o n of m a s t i t i s I n f e c t i o n , none appears to give a complete p i c t u r e of the case at hand. The use of a large number of te s t s y i e l d s considerable information regarding the status of a p a r t i c u l a r I n f e c t i o n . Yet, i t i s probable that a s i n g l e or a few t e s t s applied very f r e q u e n t l y , e.g. at weekly or h a l f - weekly i n t e r v a l s , would give more information regarding the changes taking place i n the udder, than would a large number of t e s t s applied at l e s s frequent i n t e r v a l s . The f i n d i n g of abrupt changes or trends towards more p o s i t i v e reactions with one or two t e s t s would be of greater value i n the study of a case of chronic m a s t i t i s than would be the f i n d i n g of a sus p i c i o u s l y p o s i t i v e r e a c t i o n with a number of t e s t s on a sin g l e - 128 - occasion. The value of such a procedure i s demonstrated i n the work of Johns and Hastings (55), who found that there was a tendency i n normal cows f o r the biochemical reactions of the m i l k to be s l i g h t l y higher at the afternoon m i l k i n g , and f o r the t o t a l b a c t e r i a l count to be higher at the morning m i l k i n g . They thus recommended the examination of the m i l k of an animal from several consecutive milkings as being neces sary f o r the proper diagnosis of m a s t i t i s i n f e c t i o n . Some t e s t s , however, appear to be of greater value than others f o r the dete c t i o n of m a s t i t i c m i l k . The Hotis t e s t i s u s e f u l only where there i s a high incidence of m a s t i t i s due to S t r . a g a l a c t i a e . D e f i n i t e p o s i t i v e f i n d i n g s are probably 100$ accurate i n the diagnosis of t h i s type of m a s t i t i s , but negative f i n d i n g s do not n e c e s s a r i l y s i g n i f y freedom from disease. In view of the f a c t that i t should be possible to observe the presence of s t r e p t o c o c c i on the microscopic exami na t i o n of incubated milk samples, the Hotis t e s t i s found to be non-essential as a diagnostic t e s t f o r m a s t i t i s . The value of the leucocyte count depends on the ob t a i n i n g of a uniform smear. When t h i s determination, properly c a r r i e d out, i s combined w i t h the examination of the smear f o r the presence of m a s t i t i c organisms, considerable information i s obtained regarding the extent of inflammation i n the udder, and the organisms producing such disturbances. Of the te s t s which might be applied to f i e l d use, as i n a c o n t r o l program, the determination of the pH of the milk - 129 - appears to be u n r e l i a b l e . This t e s t undoubtedly reveals the most s e r i o u s l y i n f e c t e d coxys i n a herd, but i t f a i l s to de t e c t a large number of samples which should be p o s i t i v e as revealed by other t e s t s . The determination of the c h l o r i n e content of the m i l k , on the other hand, appears to be too sen s i t i v e to s l i g h t changes i n the composition of the milk to be of value as a f i e l d t e s t . On the basis of the r e s u l t s obtained i n the present study, the modified 'Whiteside t e s t i s recommended f o r use i n the f i e l d . Although some d i f f i c u l t y has been encountered i n the i n t e r p r e t a t i o n of some of the f i n d i n g s of t h i s t e s t as a r e s u l t of the d i f f e r e n t ages of the samples t e s t e d , the use of t h i s t e s t a t f r e q u e n t I n t e r v a l s on s t r i c t l y f r e s h samples of m ilk i s to be recommended as an a i d i n detecting abnormali t i e s i n the udder. This t e s t could be r e a d i l y c a r r i e d out i n the barn with very l i t t l e equipment by a non-technical person. A p h y s i c a l examination of the udder i s a u s e f u l adjuct to studies on the milk from a suspicious animal. Such an examination gives an i n d i c a t i o n of the extent of the udder which has been a f f e c t e d by the disease, and so would a i d i n determining whether or not i t would be advisable to apply treatment to such an udder. The present state of our knowledge of m a s t i t i s indicates that m a s t i t i s caused by S t r . agalactiae i s the only form of the disease which spreads extensively through an e n t i r e herd. S t r . agalactiae i s the only m a s t i t i c organism, with the pos s i b l e exceptions of S t r . dysgalactiae and S t r . u b e r i s , which - 130 - l i v e s almost e x c l u s i v e l y i n the udder and i t s s e c r e t i o n . The other organisms capable of producing m a s t i t i s i n f e c t i o n are constantly present i n the environment of the animal and spe c i a l conditions appear to operate when these organisms pro duce disease. Because of t h e i r ubiquitous nature, i t would be impossible to t r y to eliminate these organisms from a herd. I t has been demonstrated by a number of workers that S t r . a g a l actiae can be eliminated from a herd, and that the herd can be maintained, i n t h i s c o n d i t i o n f o r considerable periods of time. In order to accomplish t h i s o b j e c t i v e , how ever,, i t i s necessary to maintain a r i g i d management program i n the herd w i t h s p e c i a l a t t e n t i o n to m i l k i n g procedures, and to combine t h i s program with the segregation of i n f e c t e d a n i  mal s and the prompt treatment of animals found to harbour S t r . a g a l a c t i a e . The average farmer does not appreciate the contagious nature of common chronic m a s t i t i s , and he considers the pro gram necessary f o r i t s e r a d i c a t i o n to be beyond p r a c t i c a l ap p l i c a t i o n . However, there i s no other known Tray to c o n t r o l the disease, since the successful treatment of an animal f o l  lowing an attack of m a s t i t i s does not prevent r e i n f e c t i o n of the udder at a l a t e r date. In the case of m a s t i t i s due to organisms other than S t r . a g a l a c t i a e , a r i g i d c o n t r o l program as described above, appears to be of l i t t l e value i n c o n t r o l l i n g the incidence of the disease i n a herd, except i n s o f a r as the sanitary practices - 131 - employed, a i d i n the maintenance of more h e a l t h f u l conditions i n the herd. In herds #3 and #4 i n the experimental herd study r e  ported h e r e i n , the i n f e c t i v e agents were found not to be S t r . a g a l a c t i a e , and i t would appear that the p r o b a b i l i t y of com p l e t e l y e r a d i c a t i n g the disease from these herds by a c o n t r o l program only i s very s l i g h t . The f i n d i n g of a v a r i e t y of s t r e p t o c o c c i i n the milk samples from herd #2 may help to ex p l a i n why t h i s herd has not responded as favourably as might have been expected to the management program. Herd #1 i s ap parently s u i t a b l e f o r a management program study, since the dominant organism i n the herd i s S t r . a g a l a c t i a e . An important problem i n the study of m a s t i t i s i s the question of what c o n s t i t u t e s i n f e c t i o n . I f the term i n f e c t i o n i s taken to mean the invasion of the t i s s u e s of the host by ' pathogenic agents, and the production of p a t h o l o g i c a l changes i n such t i s s u e s , does the mere f i n d i n g of p o t e n t i a l m a s t i t i c organisms i n the milk of a cow, i n the absence of other e v i  dence of abnormality, i n d i c a t e that the cow i s infected? Is the organism merely e x i s t i n g i n the udder In a commensal state under such conditions? What f a c t o r s are responsible f o r i t s m u l t i p l i c a t i o n or i t s disappearance from the udder? Should i t be necessary to t r e a t apparently normal cows found to be shedding S t r . agalactiae i n t h e i r milk? The f a c t that m a s t i t i c organisms can be i s o l a t e d with great r e g u l a r i t y from the milk of apparently healthy udders, - 132 - suggests that a study of the r e l a t i o n s h i p s between the organ ism and the host i s urgently required i n order to understand f u l l y the m a s t i t i s problem. I s i t that chronic m a s t i t i s , be cause of i t s slow development, at f i r s t a f f e c t s the quantity of m i l k secreted and not so much the composition of the m i l k , except In cases of a flare-up? Why do apparently harmless udder b a c t e r i a on occasion produce severe reactions i n the udder? Do these organisms, under some co n d i t i o n s , acquire i n  creased v i r u l e n c e ? Do c e r t a i n apparently normal udder staphy l o c o c c i predispose the udder to I n f e c t i o n by m a s t i t i c s t r e p  tococci? Can the l i m i t e d b a c t e r i a l f l o r a of the normal udder be explained by the s e l e c t i v e a c t i o n of the b a c t e r i c i d a l substances of the udder? Are these substances reduced i n quantity or a l  tered i n composition when disease i s produced i n the udder? Why, during the course of an attack of m a s t i t i s , i s i t some times impossible to i s o l a t e any organisms, and yet there i s d e f i n i t e evidence of inflammation as determined by chemical t e s t s and leucocyte count of the milk? Why, at other times, i n the same case, are the chemical reactions of the milk nor mal, but a very high count of organisms are shed, i n the milk? The b a c t e r i o l o g y of the f l o r a of the milk from m a s t i t i c animals has been pursued to the extreme from a p r a c t i c a l point of vie?/. I t has been found that' m a s t i t i s due to S t r . agalactiae can be c o n t r o l l e d by a r i g i d management program, and that the organisms commonly causing m a s t i t i s are susceptible to various - 133 - therapeutic agents which may be used to t r e a t cases of mas t i t i s . I t i s also known that m a s t i t i s due to organisms other than S t r . agalactiae cannot be completely held i n check by a c o n t r o l program. However, very l i t t l e i s known about what ac t u a l l y takes place i n the udder when the organism i s estab l i s h i n g i t s e l f p r i o r to the production of v i s i b l e evidence of disease. I t i s the w r i t e r ' s opinion that the explanation f o r many of the problems associated with the development of m a s t i  t i s i n f e c t i o n i n the udder would be answered i f a concerted, ef f o r t were made to determine some of the i n t e r - r e l a t i n g f a c t o r s between pathogen and host. This information could best be ob tained by the i n t e n s i v e study of a few i n f e c t e d animals under c o n t r o l l e d c o n d i t i o n s , observations on the chemical and bac t e r i a l contents of the milk being made at d a i l y or twice d a i l y i n t e r v a l s over a period of time. I t appears evident to the author t h a t , through such a study, a s i g n i f i c a n t c o n t r i b u t i o n would be made to our knowledge of m a s t i t i s and of the means to combat t h i s disease. Based on the f i n d i n g s of t h i s study and on those repor ted by other workers, i t i s c l e a r that a l a r g e - s c a l e c o n t r o l program f o r the lessening of m a s t i t i s i n f e c t i o n i n the d a i r y herds of B r i t i s h Columbia should be i n i t i a t e d without delay. One of the f i r s t problems i n such a program would be the edu c a t i o n of the farmer, i n order to make him r e a l i z e that the r i g i d management p r a c t i c e s recommended are absolutely essen t i a l to keep the disease i n check i n h i s herd. Besides f o l -- 134 ~ lowing s a n i t a r y m i l k i n g procedures and methods f o r the e l i m i  n a t i o n as f a r as p o s s i b l e of f a c t o r s which might predispose the udder to i n f e c t i o n , the farmer should, be encouraged to maintain the s i z e of h i s m i l k i n g herd Y/ith h e i f e r s r a i s e d on the farm or by the purchase of maiden or i n - c a l f h e i f e r s . The purchase of mature cows should be discouraged. I t i s also evident -that some a c t i o n should be taken to suppress the pur chase of m a s t i t i c animals by d e a l e r s , and the subsequent sale of these animals to farmers at auction s a l e s . To carry on such a program, a number of t r a i n e d f i e l d men vrould be required to supervise 'the work on the farm, to perform p h y s i c a l examinations of the udders of the cows, and to carry out f i e l d t e s t s on the milk from a l l animals i n the herds. I t i s recommended that the I n i t i a l study of a herd should c o n s i s t of a p h y s i c a l examination of the udders of a l l cows i n the herd, together w i t h a microscopic examination of incubated quarter samples of milk f o r numbers of leucocytes and types of organisms present. I f i t appears necessary i n c e r t a i n cases, blood agar pl a t e s could be made to d i s t i n g u i s h between non-heraolytic and hemolytic staphylococci and to I s o  l a t e s t r e p t o c o c c i . I f i t seems advisable to d i f f e r e n t i a t e be tween S t r . agalactiae and other m a s t i t i c s t r e p t o c o c c i , the colonies on the plates can be picked, and Brown's p r e c i p i t i n t e s t (10) applied without much d i f f i c u l t y . A f t e r t h i s i n i t i a l t e s t , i t proposed that the herds be examined r e g u l a r l y by the modified Whiteside t e s t , which could be performed at about - 135 - monthly i n t e r v a l s by the f i e l d man at the farm. I t i s the w r i t e r ' s opinion that f u r t h e r preliminary ex perimental work on the general problem of m a s t i t i s as applied to B r i t i s h Columbia i s not warranted, that the time i s suitable f o r the i n i t i a t i o n of a widespread c o n t r o l program i n t h i s pro v i n c e , and that the methods o u t l i n e d above f o r i t s execution are adequate and e a s i l y performed, SUMMARY MP CONCLUSIONS. The s i g n i f i c a n t f i n d i n g s from two years' work by the B.C. I n d u s t r i a l and S c i e n t i f i c Research Council on the pro blem of m a s t i t i s i n the d a i r y herds of B r i t i s h Columbia have been reviewed. I t has been found that the incidence of m a s t i t i s i n B r i t i s h Columbia i s comparable to that reported f o r other parts of the world. I t has also been determined that the breed of the cow has no e f f e c t on the incidence of udder I n f e c t i o n , that i n  jury i s a predisposing f a c t o r to m a s t i t i s , and that the age of the cow i s r e l a t e d to I n f e c t i o n , i n that the older the cow, the more l i k e l i h o o d of I t s being i n f e c t e d . Of the various t e s t s employed f o r the detection of mas t i t i s , the pH determination appeared, l e a s t s e n s i t i v e and the chlorine determination most s e n s i t i v e to changes i n the udder. The Hotis t e s t was found unsuitable f o r general use where the incidence of organisms other than S t r . agalactiae i s high. - 136 - The leucocyte count and blood agar plat e technique were con sidered e f f i c i e n t but time consuming. The modified Whiteside t e s t shows promise as a f i e l d t e s t f o r the rapid d e t e c t i o n of diseased conditions i n the udder. The r e s u l t s obtained with respect to the c o n t r o l of m a s t i t i s were not extensive enough to denote progress. The f a c t t h a t , i n three of the four herds studied In t h i s program, the dominant organisms were found not to be S t r . a g a l a c t i a e , may help to account f o r the apparently negative r e s u l t s ob t a i n e d . As a r e s u l t of these studies i t i s concluded that a study should be made of the i n t e r - r e l a t i o n s h i p s between the Invading organism and the host In the period during which the organism i s e s t a b l i s h i n g i t s e l f i n the udder. I t i s also concluded that a l a r g e - s c a l e c o n t r o l program should be i n i t i a t e d at once i n t h i s province i n order to check the spread of the disease and to provide f o r I t s eventual e r a  d i c a t i o n . An o u t l i n e of a suggested program Is given i n the General D i s c u s s i o n . - 137 - BIBLIOGRAPHY Be t t e r m i l k i n g procedure and m a s t i t i s c o n t r o l . Canad. Dairy and Ice Cream J . , 24* 4-3-4-4 & 86, 1945. AVERY, 0.1. & CULLEN, G.E. The use of the f i n a l hydro gen i o n concentration i n d i f f e r e n t i a t i o n of Strep tococcus haemolyticus of human and bovine types. J . Exp. Med., 29, 215-234, 1919. BEAN, C.W., MILLER, W.T. & HEISHMAN, J.O. 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Proc. Soc. A g r i c . Bact. (Gt. B r . ) , 1942, p. 35- Abstr. J . Dairy S c i . , 26, p. A128, 1943. 109. WELCH, R.C. & DOAN, F.J. The heat s t a b i l i t y of evapo rated milk made from hard-curd milk, s o f t - c u r d milk and milk from m a s t i t i s i n f e c t e d udders. J . Dairy S c i . , 18, 237-294, 1935. - 146 - 110. WESCHE, G. T i e r a r z t l . Rdsch., J39, 4-78- , 1933. V i a b i e n n i a l reviews of the progress of d a i r y s c i e n c e , S e c t i o n E. The diseases of d a i r y c a t t l e . I. Mas t i t i s . J . Dai r y Res., 7, 291-299, 1936. 111. WHITE, G.C., COUTURE, G.W., ANDERSON, E.O., JOHNSON, R.E., PLASTRIDGE, W.N. & WEIRETHER, F . J . Chronic bovine m a s t i t i s and m i l k y i e l d . J . Dairy S c i . , 20, 171-180, 1937. 112. WIEDMANN, F. Z. Untersucli. Lebensmitt., 6%, 113- , 1932. V i a b i e n n i a l reviews of the progress of d a i r y science, S e c t i o n E. The diseases of d a i r y c a t t l e . I. M a s t i t i s . J . Dairy Res., 7, 291-299, 19J6. 113. YALE, M.W. & MARQUARDT, J.C. Factors a f f e c t i n g the s u r v i v a l of Streptococcus pyogenes i n cheese. J , M i l k Tech., 32.6-333, 1940. The author i s deeply indebted to the B r i t i s h Columbia I n d u s t r i a l and S c i e n t i f i c Research Council f o r permission to use the m a t e r i a l contained h e r e i n as a t h e s i s f o r the .Degree of Master of Science i n A g r i c u l t u r e at the U n i v e r s i t y of B r i t i s h Columbia. The work was undertaken as a Research Pro j e c t of the C o u n c i l under i t s Sub-Committee on m a s t i t i s with Dr. J . G. J e r v i s as chairman. The author a l s o wishes to express h i s s i n c e r e apprecia t i o n to Dr. B. A. Eagles, Head of the Department of D a i r y i n g at the U n i v e r s i t y of B r i t i s h Columbia, f o r h i s continued g u i  dance and encouragement during the course of t h i s study, and f o r h i s a s s i s t a n c e i n the p r e p a r a t i o n of t h i s paper. The a s s i s t a n c e of the f o l l o w i n g persons i n the l a b o r a  t o r y and s t a t i s t i c a l work p e r t a i n i n g to the studies reported h e r e i n i s g r a t e f u l l y acknowledged: Dr. S. h. Wood, Mr. Vernon H. Grigg, Miss S y l v i a R. H a l l , Miss Lene Lo u r i e , Mrs. M. Kath- ryn H a l l , Mr. Ian J . McDonald and Mr. F r e d e r i c k D. Smith. 

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