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Chemical Composition of Mammary Gland During Experimental Mastitis
CHEMICAL
COMPOSITION OF MAMMARY GLAND DURING EXPERIME:NTAL MASTITIS
JOEL. BITMAN, HELENE C. CECIL, D. R. GILLIAM, AND T. R. WRENN Dairy Cattle Research Branch, U.S.D.A., Beltsville, Maryland SUi~IMAR¥
Water, fat, Na, K, C1, DNA, RNA, histamine, and glycogen were determined on sheep manunary gland samples to characterize the tissue changes occurring during the experimental induction of mastitis with Escherichia coli and S t a p h y l o c o c c u s aureus. Inoculations were made on one-half of the udder and chemical changes determined during initial stages of inflammation. One-half to five hours after inoculation treated udder halves showed great increases in size and weight. This inflammatory response resulted in dilution of adipose tissue from 20% of total weight in the control to 10% in mastitic glands. W a t e r content increased markedly, from 83 to 88%. The response appeared to be very similar in glands from either estrous or luteal phases of the reproductive cycle, while ovariecto~fized ewes showed a lesser response. Na and C1 increased greatly and K decreased, indicating an inflammatory edema. ]-Iistamine concentration of the inoculated glands decreased. The most dramatic change oecurred in glycogen content, which increased three- to fomffold in inoculated glands. This glycogen response appeared to be correlated with leukocytic invasion. Chemical changes induced by S. aureus were not as marked as those occurring after E. coli infection.
Mastitis is one of the principal problems of the dairy industry (15). Although important advances have been made in treatment of the disease since the advent o f modern drugs and antibiotics, preventive measures, a p a r t from good management, have not been developed. The state of normal mammary gland tissue and the chemical and physiological changes which take place during mastitis infection have not been characterized. Methods of prevention, treatment, and control of mastitis could be devised more readily if this basic knowledge of mammary gland physiology and biochemistry during mastitic infection were available. Previous work from this laboratory on the early stages of uterine infection (9, 10) indicated that the uterus responds differently to bacterial infection, depending upon the hormonal state of the animal. Since the ovarian hormones, estrogen and progesterone, are also primarily responsible for mammary gland growth, development, and lactation, as they are for uterine growth, it seemed possible that the nmmnmry gland defenses might also be controlled by these hormones. Similar responses during infection could influence the incidence, duration, and course of the inflammatory response necessary to control the irritating nficroorganisms causing mastitis. Received for publication June ~, 1963.
The purpose of the present investigation, therefore, was to study the physiological and biochemical processes involved in mastitis infection, particularly as related to the endocrine status of the animal. Since the initial stages of the infection were of particular interest, the study was carried out during the first 5 hr of the induced mastitie infection. The histopathological changes occurring in the tissues were also studied and are reported separately in a companion p a p e r (8). Mastitis infection was induced experimentally in nonlactating sheep that were either in estrus, in the luteal phase of the cycle, or ovarieetomized. Two pathogens, Escherichia coli and S t a p h y l o c o c c u s aureus~ were used to induce mastitis in both mature and young sheep. This paper reports the results of these initial studies on the chemical composition of the mamma~7 gland during experimental mastitis. ~ A T E 2 I A L S AI~-D METHODS
The young ewes used in these experiments were of mixed breeding and were about 11-13 months old (average body weight 110 lb). The older sheep were from 2 to 6 y r old. Fifty-two nonlactating sheep were used in this study of the experimental induction of mastitis. Estrus was detected by checking with a vasectomized ram and the ewe was used on the day of heat. Luteal sheep were used seven, eight, or nine 933
934
JOEL
BITMAN
days after heat. The ovariectomized sheep were spayed approximately 30 days before the experiment. I n each experiment, one mammary gland was inoculated with the bacteria by intramammary injection through the teat canal. The end of the nipple was washed with sterile saline and an average of 6.03 billion E. coli or 423 million S. aureus (Strain 209D) in 5-10 ml of 0.9% saline injected into the gland with a syringe. The other gland served as an internal untreated control. At various time intervals after inoculation, the sheep were killed by shooting with a captive bolt pistol and bled from the jugular veins. The inoculated and uninoculated udder halves were quickly removed, weighed, and tissue samples taken for analysis within 0.5 to 1 hr after slaughter. Specimens were selected to obtain as much duct and glandular tissue as possible from the area directly above the teat. Attempts were made to avoid samples which were apparently mostly fatty tissue. I n a series of control experiments, the effect of inoculation of an udder half with saline alone was ascertained. One gland of each of 15 sheep (estrous, ]uteal, and ovariectomized) was inoculated with saline and the tissue studied 4 hr later, a time at which compositional tissue changes in response to experimental infection are large. The saline inoculated mammary gland tissue was indistinguishable from the uninoculated tissue, no statistical differences being noted for any of the chemical constituents studied. These control experiments demonstrated that the saline medium was not responsible for any of the chemical changes found in this study. The chemical procedures employed for the determination of water, fat, No, K, C1, glycogen, histamine, DNA, and RNA have been described previously (2). Either triplicate or quadruplicate determinations were made for all constituents and the results expressed on a fat-free wet-tissue weight basis. Mammary glands from young and old sheep were compared, using Student's "t" test with correction for unequal group size. Statistical comparisons of groups after inoculation was impractical, because of the small group size in any time period. RESULTS
Total mammary gland weight, water, solids~ and fat. Changes in the inoculated mammary gland halves in total mammary gland weight, dry weight, and fat are presented in Figure 1. The weight of the mammary glands of older sheep (uninoculated) was about 50% greater than that of young sheep (P ~- .05 for estrous
ET
AL
MAMMARY GLAND WEIGHT, WATER SOLIDS, AND AFTER INOCULATION WITH E. COLI MATURE
FAT
yOUNG
15q
WEIGHT mo GRAMS so
o
~
[~
u
~
u
3
i
~
3
HOURS r-'lwArER I I so~os r--IFAT U-UN~NOCULATE0 E- ESTROU$ L-LUTEAL
FIO. 1. Gross mammary gland composition. The proportion of total weight that was water, solids, or fat is shown as the open, black, or hatched areas, respectively. and luteal) and the fat content was considerably less, being only about 14% as compared to 28% in the young ewes (P ----.001 for estrous and P----.005 for luteal). Mammary gland weight more than doubled 1 to 1.5 hr after inoculation with E. coli in both old and young estrous and luteal sheep. This inflammatory increase was maintained at the 3-hr time period. When the increase is considered with regard to the major components-fat, dry matter, and water--the total amount of fat per gland was observed to decrease, whereas dry matter increased about 50% and total water doubled. To examine the time course of the inflammatory response in more detail, animals were killed 0.5 hr after inoculation, but no changes from the untreated control glands were seen. The response to infection with S. aureus was different from that observed with E. eoli (Figure 2). The glands showed much smaller increases in weight at the early time periods, in marked contrast to the large increases produced by E. coll. I t was not until 5 hr that a 50-80% increase in total gland weight was evident. Considering the distribution of the increases at the 2-, 3-, and 5-hr periods, the estrous and luteal glands showed an increase in both total solids and total water. The ovariectomized sheep, however, exhibited only a, very slight increase in total water and solids until the 5-hr period. The ovarieetonfized sheep thus appeared to respond more slowly than the sheep under hormonal control. Sodium~ chloride~ potassium~ DNA~ ~ N A , histamine~ and glycogen. The quantitative chemical composition of the sheep mammary gland and the changes induced by inoculation with
COMPOSITION
MASTITIS MAMMARY GLAND
IN YOUNG SHEEP WEIGHT. WATER, SOLf0S, Aft0
O/~~ M A M M A R Y
FAT
125
ESTROUS
LUTEAL
r-
OVARIECTOMIZED
IoO
HOURU 1. . .2. . . .3 J~IWATER
O. . . . .
UON 2W13TH5 . . . . . . .
ISOLIDS
F~IFAT
~C(~C2US3
approached that of the estrous animals. When the water increase and total mammary gland weight increase were taken into account, and the total amount of the constituent per gland examined, similar patterns were observed (Figures 4 and 7). E. coil-young sheep. The composition of the uninoculated mammary gland in the young sheep was found to be different in several respects from that of the older ewes. It~O content was higher and the amount of fat was considerably greater, being about 35% as corn-
......
MASTITIS IN MATURE SHEEP CONSTITUENTS AFTER INOCULATION WIIN
GLAND
U - UNINOCULATED
~G. 2. Gross mammary gland composition. The
Na
proportion of total weight that was water, solids, or fat is shown as the open, black, or hatched areas, respectively. E. coli and S. aureus are presented in Table 1 and Figures 3-7. All changes are described in comparison with the uninoculated control group. E. coil-mature sheep. The compositional changes observed during the early stages of infection were similar quantitatively and qualitatively in both the estrous and luteal sheep, with the exception of changes in glycogen. The mammary gland became edematous as measured by the large increase in per cent water which occurred 1 hr after inoculation. Shifts in the extracellular electrolytes, Na and C1, were correlated with this increase in water. K and fat content decreased, due to the corresponding tissue dilution. Glycogen exhibited large progressive increases in the estrous sheep, but the increase in the luteal sheep was somewhat slower. At 4 hr post-inoculation, however~ the luteal sheep glycogen concentration increase
935
GLAND
CI
AUREUS
YOUNG SHEEP OvARIECTOmizEOWTEAL EStROUS
.......
13
13
u13 L- LUTEAL MASTITIS
GLAND
mM PER I: GLAND
,.. 123)
UNINOCULATED20o
"
E
_,°%-
ul3
YOUNG
u13
SHEEP
CI
HISTAMINE
K
~g PER G L A N D
HOURS L- LUTEAL IN
U-UN!NOCULATED
YOUNG
SHEEP
GLAND CONSTitUENTS AFTER INOCULATIONWITH S. AUREUS
MATURESHEEp YOUNGSHEEp ESTROUS LUTEALESTROUS LUTEAL
',,/
~-.~ 134
l
-
Na
:133
-
=
CI
I''ji L K"
\L J
)I)3
HISTAMINE mg PER G L A N D L O
"E
35
U1235
U1235
LUI235
u1235
HOURS Estrous
1~I~. 3.. Per cent change in chemical composition
of mastitic glands.
IN
L
E eSTROOS
ESCHERICHIA COLI
1235HOUR$ I ~ 4
.......:oof j j
U13
i-m
GLAN
1235
UI3
U-UNINOCULATED
CONSTITUENTS AFTER INOCULATION WiTH E COLI
Na
mM
V
U13
HOURS
E- ESTROUS
12
::o[
liil
mM PER GLAN
PER
......
HISTAMINIE
I
Jmg PER GLAND..J
MASTITIS STAPHYLOCOCCUS
K
E. COLI
u-IutEAL
O-OvAR~EcTOMIZE D
2~Ia. 4, 5, 6. Electrolyte and histamine concentration during experimental mastitis.
TABLE 1 C h e m i c a l c o m p o s i t i o n of m a m m a r y g l a n d d u r i n g e x p e r i m e n t a l m a s t i t i s
% Fat (as % N
FFWW
t~g/100 m g F F W W
/-Iours after inoculation
% FF H..O
fresh wt)
Na
E s c h e r i c h i a c o l i - - o l d sheep Estrous
Uninoculatcd
80.3
]2.7
102.0
Luteal
1 3 4 Uninoculated
86.2 86.5 80.8 82.0
--34 --42 --15 15.0
1 3 4
88.2 85.5 85.2
--48 --82 --44
Uninoculated
88.0
38.2
88.5 88.0 90.4 84.2
4 2 --56 --57 31.2
86.9 89.2 89.6
--31 --14 --54
73.9 35.0 329 346 P e r cent c h a n g e f r o m c on tr o l v a l u e 4 5 -- 4 --]3 + 3 --30 4 7 412 --43 ........... + I + 13 --42 99.0 76.0 41.5 518 425 P e r cent c h a n g e f r o m c on tr o l v alu e 411 +14 --27 4 8 4 1 417 416 --52 ............ 411 + 8 --52 ............
85.6
22.6
97.7
1 2 3 5 Uninocu]ated
87.5 88.4 88.0 86.6 8~.8
+27 --36 --36 --46 28.2
1 2 3 5 Uninoculated
85.1 87.4
+6
+5
+6
-6
-48
+12
413
-29
88.6 88.0 86.4
--3'7 --66 21.5
411 423 98.5
1 2 3 5
86.4 88.4 87.5 87.4
--34 --10 + 6 --36
Endocrine group
E. c o l i - - y o u n g sheep Estrous
½ 1½ Luteal
3 Uninoculated
½ 1½ 3 S t a p h y l o c o c c u s a u r e u s - - y o u n g sheep Estrous Uninoculated
Lntea] 1 4 2 2 10
m~/kg
Ovariectomized
V a l u e s a r e means. FFWW = Fat-free wet weight.
C1
:K
DNA
RNA
78.9 33.8 624 43q P e r cent c h a n g e f r o m control v alu e 416 413 --32 -- 6 -- 1 410 + 8 --27 ............ --12 --15 94.0 75.2 37.9 643 553 P e r cent c h a n g e f r o m c on tr o l v alu e 421 417 --47 -- 2 --29 412 +11 --53 ............ 412 427 --44 ............
107.4
79.5 44.3 599 458 P e r cent c h a n g e f r o m c on tr o l v a l u e -- 2 0 --14 410 411 --34 --26 --23 414 414 --37 --12 --28 + 8 + 6 --20 + 3 --30 95.4 78.0 41.9 605 470 P e r c e nt c h a n g e f r o m c on tr o l v alu e
~1
~5
410 --13 -- 3 --23 421 --38 --22 --33 79.9 44.1 481 453 P e r cent c h a n g e f r o m c ontr o l v a l u e 4 3 + 4 --10 4 9 423 + 6 + 6 --29 -- 7 --16 + 9 + 8 --20 -- 1 --11 413 + 7 --35 4 4 --34
mg/kg FFWW Itistanlille
mg/100 g FFWW Glycogen
39.4
44.0
--34 --63 --18 31.2
+ 54 4246 4369 45,.5
--44 --49 --6,8
4 16 4 89 4334
10.6
38.4
+116 --19 --2'0 ].5.8
+ 8 -- 6 + 25 34.8
431 --4,5 --53
-- 8 -- 6 479
16.1
28.5
--18 --43 --10 --30 16.1
+ 16 + 45 4322 4245 40.5
-3 - 9
+13 + 56
--49 --57 14.3
+ 68 4213 ~5.6
--56 --23 + 2 --12
-- 32 + 29 + 57 +108
OY
CO/V£POSITION
GLYCOGEN MA~Re SHEEP
RESPONSES
yOUNG
IN
IN(~UCRO
SHtEP
MASTITIS
VOUUG
SHEEP
200
150
• ESTROUS
13LUTEAL
/
U
I
3
U
~
HOURS
1~
3
AFTER
U
1
2
3
INOCULATION
Fro. 7. Glycogen concentration of mammary gland during experimental mastitis. pared to 14% of the total mammary gland weight. ~Iistamine content was also at a different level than that in the older sheep, being about 50% lower. Several differences were also observed between the response of young and mature sheep to the mastitis infection. The young estrous gland exhibited smaller alterations in the concentration of the constituents than did the young ]uteal gland; whereas, the older estrous and luteal glands had been similar in response. H20 concentration did not change appreciably until 3 hr after inoculation and the Na and C1 shifts were correspondingly small. I n contrast, the luteal inoculated gland exhibited immediate and much larger changes in almost all components. A H~O response was seen at 0.5 hr and Na and C1 concentration changes were immediate. Glycogen concentration also increased more ( + 8 0 % ) than in the estrous gland ( + 2 4 % ) . When the results were expressed on a per gland basis, however, it was seen that the absolute increases were usually greater in the estrous gland, due to the larger weight increases which occurred during the inflammation. Thus, the estrous gland had responded to the infection to as great or to a greater degree than the luteal. S. aureus-young sheep. The estrous sheep showed greater and more immediate alterations in concentration of constituents than the luteal, which in turn showed greater change than the ovariectomized sheep. The usual increases in H~O, Na, and C1, and decreases in K, fat, and histamine concentrations were noted as the tissue became edematous. Glycogen concentration followed the usual pattern of increasing after inoculation. The relative concentration relationships among the three groups, estrous luteal ~ ovariectomized, were still evident when absolute glycogen increases were examined. As was observed in the sheep infected with E. coli, after a lag, the luteal and ovariectomized sheep caught up with the estrous with
M A M M A R Y
937
GLAND
regard to the amount o£ glycogen hi the tissue, so that there was little difference at 5 hr. The glycogen response probably reflects the progressive influx of leukocytes in the defense mechanism of the tissue to the bacterial invasion and infection. The differences in amounts of constituents at these early times may indicate a difference in the rate of response of the defensive mechanisms. They show a consistent pattern, in that the estrous sheep always showed a quicker increase, possibly due to variations in vaseulax permeability. I n addition, the ovariectomized sheep were the least responsive when compared to the ewes under ovarian hormonal control. DISCUSSIO~
Chemical composition of mammary gland during experimental mastitis. Shortly after the experimental induction of mastitis by bacterial inoculation, the treated mammary gland showed extensive changes in chemical composition. The general pattern which emerges is characteristic of the infective process in the early stages of experimental mastitis. The increase in total gland weight elicited by E. coli infection was three or four times greater than that produced by S. aureus. This inflammatory response resulted in dilution of the adipose tissue to about half of the control level. Examination of the chemical composition of the mammary gland (water, fat, Na, C1, K, histamine, and glycogen) during mastitis infection revealed marked changes in several of the constituents. The water content increased markedly, and this response was somewhat greater in glands under estrous control than in animals in the luteal phase of the estrous cycle; ovarieetomized ewes showed the least change. Na and C1 showed a large increase and K a decrease, indicating that the fluid increase probably occurs primarily in the extracellular compartment and is undoubtedly an inflammatory edema. A relationship between K and DNA concentration was expected, since both of these constituents axe associated with cellular elements and relatively constant values were obse~wed. Thus, the K / D N A ratio in the old sheep was 0.54 for estrous and 0.59 for luteal sheep. The young sheep inoculated with E. coll had values of 1.06 for estrous and 0.80 for the ]uteal (mean 0.93). The experiments on the young sheep infected with S. aureus were carried out about a month after the E. coli experiments and the maturation of the gland is seen by a change from the mean ratio of 0.93 to 0.74 for the estrous and 0.69 for Iuteal, values which ap-
938
JOEL
BITMAN
proach those of the older sheep. The ovariectomized sheep had their ovaries removed about one month earlier and the K / D N A ratio was arrested at the higher level, being 0.92 and corresponding to those of the first group of younger sheep. Itistamine was followed in the present study because this substance has been postulated as being released at times of i n j u r y and damage to cells (16). Because of its biological action and participation in inflanmmtion, it seemed possible that histamine shifts might parallel the course of the inflammation. Histamine concentration decreased routinely in all the inoculated mammary glands at all time periods, with the exception of the 0.5-hr samples. These samples showed an increase before exhibiting the usual decline in concentration caused by dilution of the tissue histamine as the gland increased in size. Whether these 0.5-hr increases were significant and a consistent p a r t of the inflammatory response could not be determined with the small numbers involved in these experiments. The most dramatic changes were observed in glycogen concentration and content between control and infected glands. Glycogen concentration increased three- to fourfold in inoculated glands and total glycogen content increased up to eight times over the control level. Correlation of chemical changes with histopathological alterations. Comparison of the chemical changes with the histological and pathological alterations in tissue (8) indicated a high degree of general agreement. Thus, leukocytic infiltration of the tissue appeared to be fairly well correlated with the glycogen response in the nmtnre sheep. The estrous ewes showed a slightly more r a p i d leukocytic response than the luteal phase ewes to the E. eoli infection, which agreed with the earlier increase noted in total glycogen in estrous sheep. No differences were noted in estrous vs. luteal leukocytic infiltration in the Staphylococcus infection, although both were higher than the ovariectomized animals. The quantitative glycogen results, however, showed a gradation in response, estrous being more r a p i d than luteal which, in turn, was faster than ovariectomized. In several instances in the young ewes, there were discrepancies between the leukoeytic density in the tissue and the glycogen concentration (S). The inflammatory response, a change in capillary permeability, edema (extracellular fluid influx-It~O, Na, and CI) and infiltration of leukocytes from the circulation into the tissue, is a p r i m a r y feature of the early stages of
ET
AL
mastitis infection. The leukocytic influx represents a defense mechanism of the tissue to combat the invading microorganisms. Since leukocytes are known to contain large amounts of glycogen (1), the glycogen increase is probably a direct reflection of the presence of leukocytes. The possibility exists, however, that the polysaccharide increase in the tissue might represent the presence of serum or tissue glycoproreins or depolymerization of ground substance (7), either mobilized or synthesized at the site of tissue i n j u r y (17). The slight increase in total K per gland noted at some of these early time periods may also be correlated with leukocytic infiltration and might represent an increase due to the cellular elements of the leukocytes rather than tissue cellular elements. The DNA and R N A determinations were not instituted at the start of the experiments and it was not possible to determine to what extent cell growth, hypertrophy, and hyperplasia were involved in the inflammatory tissue changes in all of these experiments. After inoculation the D N A / R N A ratio went up, which could signify an i n f u x of DNA from leukocytes with no increase in cytoplasmic RNA. An alternative interpretation could be that dissolution of cytoplasmic constituents occurred while DNA remained constant. The first possibility is favored by the data, however, since total DNA increased while total RNA per gland remained relatively constant. Correlation of inflammatory response with age and stage of estrous cycle. The somewhat greater ability of the estrous mammary gland to respond to the infection is qualitatively but not quantitatively similar to the greater response which the estrous uterus as compared to the luteal uterus exhibits to bacterial infection (9, 10). I n the uterus the estrous defense mechanism is much more responsive than the luteal, while the differences noted here in the mammary gland were small. Differences in chemical response between young and old sheep were noted, but it is difficult to relate these to mastitis susceptibility. I t is well known that the rate of mastitis increases with age (15). The greater response of the old animals demonstrated here, if correlated with defensive elements, should also make the older gland more able to combat the infection than the young udder. Relation of mastitis inflammation to inflammation in other tissues. The chemical changes which occur during inflammation in other tissues have not been studied extensSvely. The extent to which the inflammatory changes oh-
COMPOSITION OF MAMMARY GLAND
served in experimental mastitis correspond to those occurring in inflammation of other types is, therefore, difficult to determine. I n previous studies on deciduoma in the rat uterus (18), the chemical changes accompanying the inflammation due to the stimulus were determined. The inflammato~T changes in mastitis are very similar to the alterations seen in this type of uterine inflmmnation. I n the uterine studies, the electrolyte changes were also found to be quite similar to those occurring in the early stages of growth, and it was not possible to separate the i n j u r y effects from those due to the concomitant growth of the uterine tissue. I n mastitis, the infection--with its consequent degeneration of tissue, and healing and repair--with regeneration and growth of new tissue, are processes which go on concurrently within the gland. I t is likely that a similar situation prevails in the mammary gland as it does in the uterus, and the existence of these concomitant phenomena with similar chemical patterns will probably make it difficult to differentiate between the early phases of these processes. Forscher and Cecil (6) have studied the tissue alterations of some biochemical constituents occurring in the rat palate after a burn. Although the electrolyte changes were not reported, water increased immediately. However, glycogen did not rise significantly until one to three days after injury. I n this study, the glycogen response was assmned to be due to leukocytic infiltration. Further che.m.ical approaches to the study of mastitis. While there have been many reports of the histological changes which take place during the development of mastiffs, quantitative chemical studies of the tissue have not been made. Several investigators have induced mastitis with bacteria (4, 12, 14), but the initial inflammatory response was not examined. I n the present study, the times of sampling were chosen to yield information on the early stages of mastitis infection and the general chemical and histological pattern described is thus thought to be characteristic of the early stages of mastitis. The subsequent course of the disease, as measured by the chemical parameters determined in this study, could not, of course, be determined. The relationship of variations in the levels of constituents to the defense of the gland, their effects and interactions upon the severity and duration of the infection, would make controlled studies at longer time periods highly interesting. The characterization of differences in the
939
nature of the response with E. coli and Staphylococcus infection could be due either to (a) a basic difference in pathogenicity--the nature and virulence of the two microorganisms, (b) a difference in response caused by the difference in numbers of bacteria infused (6 billion vs. 0.4 billion, respectively), or (e) both of these factors. The nature of the chemical response to the mastitis produced with killed bacteria, bacterial extracts, or with any foreign material serving as an irritant was not studied in the present investigation and can only be determined by future research. Mastitis is commonly a disease of lactation and the animals used did not have extensive development of glandular secreting tissue. I n this preliminary investigation, however, in which methodology, techniques, and certain characteristics of the mastiffs infective process were worked out, the uncomplicated nature of the nonlactating gland was a welcome benefit. The presence of milk and glandular secretions in the tissue presents a problem which can be more readily approached as a result of the present experiments. The changes which take place as secreting cells decline in milk production during mastitis, and the alterations which occur as the glandular tissue involutes and is replaced by connective tissue, are other processes of mastitis infection which merit attention. The chemics~l alterations occurring in mastitic milk have been studied and a rise in chloride and decline in lactose concentration have been associated with the appearance of leukocytes in the milk (5). More recently, the whey proteins of mastitic milk have been studied in detail (3, 11, 13). The extent to which the chemical changes occurring in the tissue and described in this report are responsible for or are part of similar alterations in milk is an interesting question which we hope to answer in further investigations. Such information might provide an approach to very early diagnosis as well as affording more objective measures of following the course of the infection and treatment of the disease. Thus, the relationship of the extracellular rise in tissue chloride to the increase in milk chloride, the decline in milk lactose to the rise in tissue glycogen (or polysaecharide) are specific objectives of future research. ACK~O~LEDGMENT
Grateful acknowledgment is extended to the Sheep and Fur Animals l~esearch Branch, A]~S, Agricultural Research Center, United States Department of Agriculture, for generously supplying the sheep used in this study.
940
JOEL BITI~IAN ET AL
REFERENCES (1) BAZIN, S., R~BIN~VX, R., A~]) D~I~AVN~Z, A. The Glycogen Content of Polynuclear Leukocytes. Compt. rend. soc. biol., Paris, 14.5: 1855. 1951. (2) BITI~iAN, J., CECIL, I~. C., GILLIAI~, D. R., AND WR~.N~, T. R. Chemical Composition of Sheep Mammary Gland. J. Dairy Sci., 46: 941. 1963. (3) GAmao~, E. J. Whey Proteins of DryingOff Secretions, Mastitic Milk, and Colostrum Separated by Ion-Exchange Cellulose. J. Dairy Sei., 44: 219~. 196.1. (4) D~I~BYSHIRE, 5. B. The Experimental Production of Staphylococcal Mastiffs in the Goat. J. Comp. Pathol. Therap., 68: 232. 1958. (5) Es~E, D., AND SMITH, V. R. Secretion of Milk. p. 186. Iowa State College Press, Ames, Iowa. 1952. (6) FO~SCHER, B. K-, AN]) CECib, t{. C. Biochemical Studies of Acute Inflammation. I. Chemical Changes in the Normal Animal. Arch. Bioehem. Biophys., 70: 367. 1957. (7) GEESH, I., AN]) C~
(10) HAWK, It. W., TUI~NEE, G. D., AND SYKES, J. F. Variation in the Inflammatory Response and Bactericidal Activity of the Sheep Uterus During the Estrous Cycle. Am. J. Vet. Research, 22:689. 1961. (11) LECCE, J. G., AND ~EDATE,S, J. E. Changes in the Paper Electrophoretic Whey-Protein P a t t e r n of Cows with Acute Mastitis. J. Dairy Sci., 4~2: 698. 1959. (12) MURPHY, J. M., AN]) STUARt, O. M. The Effect of Introducing Small Numbers of Streptococcus agalactiae (Cornell 48 Strain) Directly into the Bovine Teat Cavity. Cornell Vet., 43: 290. 1953. (13) NIbSSON, T. Blood and Milk Proteins and Protein-Bound Carbohydrates in Bovine Chronic Mastitis. Acta Path. Biol. Scandinav. Suppl., 125. 1958. (14) PATTIS0N, I. H., AND HOL~[AN, H. H. Studies on Experimental Streptococcal Mastltis. IV. The Disease Caused by Inoculation of Streptococcus Agalactiae Strain $13 into the Teat Canal of Goats. J. Comp. Pathol. Therap., 61: 26. 1951. (15) PLASTalI)~E, W. N. Bovine Mastitis: A Review. J. Dairy Sci., 41: 1141. 1958. (16) ROCHE E SILVA, M. Histamine---Its Role in Anaphylaxis and Allergy. Charles C Thomas, Springfield, Illhlois. 1955. (17) Wi~rzh~a, R. 5. I n Methods of Biochemical Analysis. Vol. 2. (ed., D. Glick) p. 279. Academic Press, N. Y. 1955. (18) WrmNN, T. R., C~OlL, H. C., CONNOLLY, M. R., Am) BIT~AN, J. Ionic Studies of Decidual Tissue Growth in the Rat Uterus. Acta Endoerinol., 4.0: 472. 1962.
COMPOSITION OF MAMMARY GLAND DURING EXPERIME:NTAL MASTITIS
JOEL. BITMAN, HELENE C. CECIL, D. R. GILLIAM, AND T. R. WRENN Dairy Cattle Research Branch, U.S.D.A., Beltsville, Maryland SUi~IMAR¥
Water, fat, Na, K, C1, DNA, RNA, histamine, and glycogen were determined on sheep manunary gland samples to characterize the tissue changes occurring during the experimental induction of mastitis with Escherichia coli and S t a p h y l o c o c c u s aureus. Inoculations were made on one-half of the udder and chemical changes determined during initial stages of inflammation. One-half to five hours after inoculation treated udder halves showed great increases in size and weight. This inflammatory response resulted in dilution of adipose tissue from 20% of total weight in the control to 10% in mastitic glands. W a t e r content increased markedly, from 83 to 88%. The response appeared to be very similar in glands from either estrous or luteal phases of the reproductive cycle, while ovariecto~fized ewes showed a lesser response. Na and C1 increased greatly and K decreased, indicating an inflammatory edema. ]-Iistamine concentration of the inoculated glands decreased. The most dramatic change oecurred in glycogen content, which increased three- to fomffold in inoculated glands. This glycogen response appeared to be correlated with leukocytic invasion. Chemical changes induced by S. aureus were not as marked as those occurring after E. coli infection.
Mastitis is one of the principal problems of the dairy industry (15). Although important advances have been made in treatment of the disease since the advent o f modern drugs and antibiotics, preventive measures, a p a r t from good management, have not been developed. The state of normal mammary gland tissue and the chemical and physiological changes which take place during mastitis infection have not been characterized. Methods of prevention, treatment, and control of mastitis could be devised more readily if this basic knowledge of mammary gland physiology and biochemistry during mastitic infection were available. Previous work from this laboratory on the early stages of uterine infection (9, 10) indicated that the uterus responds differently to bacterial infection, depending upon the hormonal state of the animal. Since the ovarian hormones, estrogen and progesterone, are also primarily responsible for mammary gland growth, development, and lactation, as they are for uterine growth, it seemed possible that the nmmnmry gland defenses might also be controlled by these hormones. Similar responses during infection could influence the incidence, duration, and course of the inflammatory response necessary to control the irritating nficroorganisms causing mastitis. Received for publication June ~, 1963.
The purpose of the present investigation, therefore, was to study the physiological and biochemical processes involved in mastitis infection, particularly as related to the endocrine status of the animal. Since the initial stages of the infection were of particular interest, the study was carried out during the first 5 hr of the induced mastitie infection. The histopathological changes occurring in the tissues were also studied and are reported separately in a companion p a p e r (8). Mastitis infection was induced experimentally in nonlactating sheep that were either in estrus, in the luteal phase of the cycle, or ovarieetomized. Two pathogens, Escherichia coli and S t a p h y l o c o c c u s aureus~ were used to induce mastitis in both mature and young sheep. This paper reports the results of these initial studies on the chemical composition of the mamma~7 gland during experimental mastitis. ~ A T E 2 I A L S AI~-D METHODS
The young ewes used in these experiments were of mixed breeding and were about 11-13 months old (average body weight 110 lb). The older sheep were from 2 to 6 y r old. Fifty-two nonlactating sheep were used in this study of the experimental induction of mastitis. Estrus was detected by checking with a vasectomized ram and the ewe was used on the day of heat. Luteal sheep were used seven, eight, or nine 933
934
JOEL
BITMAN
days after heat. The ovariectomized sheep were spayed approximately 30 days before the experiment. I n each experiment, one mammary gland was inoculated with the bacteria by intramammary injection through the teat canal. The end of the nipple was washed with sterile saline and an average of 6.03 billion E. coli or 423 million S. aureus (Strain 209D) in 5-10 ml of 0.9% saline injected into the gland with a syringe. The other gland served as an internal untreated control. At various time intervals after inoculation, the sheep were killed by shooting with a captive bolt pistol and bled from the jugular veins. The inoculated and uninoculated udder halves were quickly removed, weighed, and tissue samples taken for analysis within 0.5 to 1 hr after slaughter. Specimens were selected to obtain as much duct and glandular tissue as possible from the area directly above the teat. Attempts were made to avoid samples which were apparently mostly fatty tissue. I n a series of control experiments, the effect of inoculation of an udder half with saline alone was ascertained. One gland of each of 15 sheep (estrous, ]uteal, and ovariectomized) was inoculated with saline and the tissue studied 4 hr later, a time at which compositional tissue changes in response to experimental infection are large. The saline inoculated mammary gland tissue was indistinguishable from the uninoculated tissue, no statistical differences being noted for any of the chemical constituents studied. These control experiments demonstrated that the saline medium was not responsible for any of the chemical changes found in this study. The chemical procedures employed for the determination of water, fat, No, K, C1, glycogen, histamine, DNA, and RNA have been described previously (2). Either triplicate or quadruplicate determinations were made for all constituents and the results expressed on a fat-free wet-tissue weight basis. Mammary glands from young and old sheep were compared, using Student's "t" test with correction for unequal group size. Statistical comparisons of groups after inoculation was impractical, because of the small group size in any time period. RESULTS
Total mammary gland weight, water, solids~ and fat. Changes in the inoculated mammary gland halves in total mammary gland weight, dry weight, and fat are presented in Figure 1. The weight of the mammary glands of older sheep (uninoculated) was about 50% greater than that of young sheep (P ~- .05 for estrous
ET
AL
MAMMARY GLAND WEIGHT, WATER SOLIDS, AND AFTER INOCULATION WITH E. COLI MATURE
FAT
yOUNG
15q
WEIGHT mo GRAMS so
o
~
[~
u
~
u
3
i
~
3
HOURS r-'lwArER I I so~os r--IFAT U-UN~NOCULATE0 E- ESTROU$ L-LUTEAL
FIO. 1. Gross mammary gland composition. The proportion of total weight that was water, solids, or fat is shown as the open, black, or hatched areas, respectively. and luteal) and the fat content was considerably less, being only about 14% as compared to 28% in the young ewes (P ----.001 for estrous and P----.005 for luteal). Mammary gland weight more than doubled 1 to 1.5 hr after inoculation with E. coli in both old and young estrous and luteal sheep. This inflammatory increase was maintained at the 3-hr time period. When the increase is considered with regard to the major components-fat, dry matter, and water--the total amount of fat per gland was observed to decrease, whereas dry matter increased about 50% and total water doubled. To examine the time course of the inflammatory response in more detail, animals were killed 0.5 hr after inoculation, but no changes from the untreated control glands were seen. The response to infection with S. aureus was different from that observed with E. eoli (Figure 2). The glands showed much smaller increases in weight at the early time periods, in marked contrast to the large increases produced by E. coll. I t was not until 5 hr that a 50-80% increase in total gland weight was evident. Considering the distribution of the increases at the 2-, 3-, and 5-hr periods, the estrous and luteal glands showed an increase in both total solids and total water. The ovariectomized sheep, however, exhibited only a, very slight increase in total water and solids until the 5-hr period. The ovarieetonfized sheep thus appeared to respond more slowly than the sheep under hormonal control. Sodium~ chloride~ potassium~ DNA~ ~ N A , histamine~ and glycogen. The quantitative chemical composition of the sheep mammary gland and the changes induced by inoculation with
COMPOSITION
MASTITIS MAMMARY GLAND
IN YOUNG SHEEP WEIGHT. WATER, SOLf0S, Aft0
O/~~ M A M M A R Y
FAT
125
ESTROUS
LUTEAL
r-
OVARIECTOMIZED
IoO
HOURU 1. . .2. . . .3 J~IWATER
O. . . . .
UON 2W13TH5 . . . . . . .
ISOLIDS
F~IFAT
~C(~C2US3
approached that of the estrous animals. When the water increase and total mammary gland weight increase were taken into account, and the total amount of the constituent per gland examined, similar patterns were observed (Figures 4 and 7). E. coil-young sheep. The composition of the uninoculated mammary gland in the young sheep was found to be different in several respects from that of the older ewes. It~O content was higher and the amount of fat was considerably greater, being about 35% as corn-
......
MASTITIS IN MATURE SHEEP CONSTITUENTS AFTER INOCULATION WIIN
GLAND
U - UNINOCULATED
~G. 2. Gross mammary gland composition. The
Na
proportion of total weight that was water, solids, or fat is shown as the open, black, or hatched areas, respectively. E. coli and S. aureus are presented in Table 1 and Figures 3-7. All changes are described in comparison with the uninoculated control group. E. coil-mature sheep. The compositional changes observed during the early stages of infection were similar quantitatively and qualitatively in both the estrous and luteal sheep, with the exception of changes in glycogen. The mammary gland became edematous as measured by the large increase in per cent water which occurred 1 hr after inoculation. Shifts in the extracellular electrolytes, Na and C1, were correlated with this increase in water. K and fat content decreased, due to the corresponding tissue dilution. Glycogen exhibited large progressive increases in the estrous sheep, but the increase in the luteal sheep was somewhat slower. At 4 hr post-inoculation, however~ the luteal sheep glycogen concentration increase
935
GLAND
CI
AUREUS
YOUNG SHEEP OvARIECTOmizEOWTEAL EStROUS
.......
13
13
u13 L- LUTEAL MASTITIS
GLAND
mM PER I: GLAND
,.. 123)
UNINOCULATED20o
"
E
_,°%-
ul3
YOUNG
u13
SHEEP
CI
HISTAMINE
K
~g PER G L A N D
HOURS L- LUTEAL IN
U-UN!NOCULATED
YOUNG
SHEEP
GLAND CONSTitUENTS AFTER INOCULATIONWITH S. AUREUS
MATURESHEEp YOUNGSHEEp ESTROUS LUTEALESTROUS LUTEAL
',,/
~-.~ 134
l
-
Na
:133
-
=
CI
I''ji L K"
\L J
)I)3
HISTAMINE mg PER G L A N D L O
"E
35
U1235
U1235
LUI235
u1235
HOURS Estrous
1~I~. 3.. Per cent change in chemical composition
of mastitic glands.
IN
L
E eSTROOS
ESCHERICHIA COLI
1235HOUR$ I ~ 4
.......:oof j j
U13
i-m
GLAN
1235
UI3
U-UNINOCULATED
CONSTITUENTS AFTER INOCULATION WiTH E COLI
Na
mM
V
U13
HOURS
E- ESTROUS
12
::o[
liil
mM PER GLAN
PER
......
HISTAMINIE
I
Jmg PER GLAND..J
MASTITIS STAPHYLOCOCCUS
K
E. COLI
u-IutEAL
O-OvAR~EcTOMIZE D
2~Ia. 4, 5, 6. Electrolyte and histamine concentration during experimental mastitis.
TABLE 1 C h e m i c a l c o m p o s i t i o n of m a m m a r y g l a n d d u r i n g e x p e r i m e n t a l m a s t i t i s
% Fat (as % N
FFWW
t~g/100 m g F F W W
/-Iours after inoculation
% FF H..O
fresh wt)
Na
E s c h e r i c h i a c o l i - - o l d sheep Estrous
Uninoculatcd
80.3
]2.7
102.0
Luteal
1 3 4 Uninoculated
86.2 86.5 80.8 82.0
--34 --42 --15 15.0
1 3 4
88.2 85.5 85.2
--48 --82 --44
Uninoculated
88.0
38.2
88.5 88.0 90.4 84.2
4 2 --56 --57 31.2
86.9 89.2 89.6
--31 --14 --54
73.9 35.0 329 346 P e r cent c h a n g e f r o m c on tr o l v a l u e 4 5 -- 4 --]3 + 3 --30 4 7 412 --43 ........... + I + 13 --42 99.0 76.0 41.5 518 425 P e r cent c h a n g e f r o m c on tr o l v alu e 411 +14 --27 4 8 4 1 417 416 --52 ............ 411 + 8 --52 ............
85.6
22.6
97.7
1 2 3 5 Uninocu]ated
87.5 88.4 88.0 86.6 8~.8
+27 --36 --36 --46 28.2
1 2 3 5 Uninoculated
85.1 87.4
+6
+5
+6
-6
-48
+12
413
-29
88.6 88.0 86.4
--3'7 --66 21.5
411 423 98.5
1 2 3 5
86.4 88.4 87.5 87.4
--34 --10 + 6 --36
Endocrine group
E. c o l i - - y o u n g sheep Estrous
½ 1½ Luteal
3 Uninoculated
½ 1½ 3 S t a p h y l o c o c c u s a u r e u s - - y o u n g sheep Estrous Uninoculated
Lntea] 1 4 2 2 10
m~/kg
Ovariectomized
V a l u e s a r e means. FFWW = Fat-free wet weight.
C1
:K
DNA
RNA
78.9 33.8 624 43q P e r cent c h a n g e f r o m control v alu e 416 413 --32 -- 6 -- 1 410 + 8 --27 ............ --12 --15 94.0 75.2 37.9 643 553 P e r cent c h a n g e f r o m c on tr o l v alu e 421 417 --47 -- 2 --29 412 +11 --53 ............ 412 427 --44 ............
107.4
79.5 44.3 599 458 P e r cent c h a n g e f r o m c on tr o l v a l u e -- 2 0 --14 410 411 --34 --26 --23 414 414 --37 --12 --28 + 8 + 6 --20 + 3 --30 95.4 78.0 41.9 605 470 P e r c e nt c h a n g e f r o m c on tr o l v alu e
~1
~5
410 --13 -- 3 --23 421 --38 --22 --33 79.9 44.1 481 453 P e r cent c h a n g e f r o m c ontr o l v a l u e 4 3 + 4 --10 4 9 423 + 6 + 6 --29 -- 7 --16 + 9 + 8 --20 -- 1 --11 413 + 7 --35 4 4 --34
mg/kg FFWW Itistanlille
mg/100 g FFWW Glycogen
39.4
44.0
--34 --63 --18 31.2
+ 54 4246 4369 45,.5
--44 --49 --6,8
4 16 4 89 4334
10.6
38.4
+116 --19 --2'0 ].5.8
+ 8 -- 6 + 25 34.8
431 --4,5 --53
-- 8 -- 6 479
16.1
28.5
--18 --43 --10 --30 16.1
+ 16 + 45 4322 4245 40.5
-3 - 9
+13 + 56
--49 --57 14.3
+ 68 4213 ~5.6
--56 --23 + 2 --12
-- 32 + 29 + 57 +108
OY
CO/V£POSITION
GLYCOGEN MA~Re SHEEP
RESPONSES
yOUNG
IN
IN(~UCRO
SHtEP
MASTITIS
VOUUG
SHEEP
200
150
• ESTROUS
13LUTEAL
/
U
I
3
U
~
HOURS
1~
3
AFTER
U
1
2
3
INOCULATION
Fro. 7. Glycogen concentration of mammary gland during experimental mastitis. pared to 14% of the total mammary gland weight. ~Iistamine content was also at a different level than that in the older sheep, being about 50% lower. Several differences were also observed between the response of young and mature sheep to the mastitis infection. The young estrous gland exhibited smaller alterations in the concentration of the constituents than did the young ]uteal gland; whereas, the older estrous and luteal glands had been similar in response. H20 concentration did not change appreciably until 3 hr after inoculation and the Na and C1 shifts were correspondingly small. I n contrast, the luteal inoculated gland exhibited immediate and much larger changes in almost all components. A H~O response was seen at 0.5 hr and Na and C1 concentration changes were immediate. Glycogen concentration also increased more ( + 8 0 % ) than in the estrous gland ( + 2 4 % ) . When the results were expressed on a per gland basis, however, it was seen that the absolute increases were usually greater in the estrous gland, due to the larger weight increases which occurred during the inflammation. Thus, the estrous gland had responded to the infection to as great or to a greater degree than the luteal. S. aureus-young sheep. The estrous sheep showed greater and more immediate alterations in concentration of constituents than the luteal, which in turn showed greater change than the ovariectomized sheep. The usual increases in H~O, Na, and C1, and decreases in K, fat, and histamine concentrations were noted as the tissue became edematous. Glycogen concentration followed the usual pattern of increasing after inoculation. The relative concentration relationships among the three groups, estrous luteal ~ ovariectomized, were still evident when absolute glycogen increases were examined. As was observed in the sheep infected with E. coli, after a lag, the luteal and ovariectomized sheep caught up with the estrous with
M A M M A R Y
937
GLAND
regard to the amount o£ glycogen hi the tissue, so that there was little difference at 5 hr. The glycogen response probably reflects the progressive influx of leukocytes in the defense mechanism of the tissue to the bacterial invasion and infection. The differences in amounts of constituents at these early times may indicate a difference in the rate of response of the defensive mechanisms. They show a consistent pattern, in that the estrous sheep always showed a quicker increase, possibly due to variations in vaseulax permeability. I n addition, the ovariectomized sheep were the least responsive when compared to the ewes under ovarian hormonal control. DISCUSSIO~
Chemical composition of mammary gland during experimental mastitis. Shortly after the experimental induction of mastitis by bacterial inoculation, the treated mammary gland showed extensive changes in chemical composition. The general pattern which emerges is characteristic of the infective process in the early stages of experimental mastitis. The increase in total gland weight elicited by E. coli infection was three or four times greater than that produced by S. aureus. This inflammatory response resulted in dilution of the adipose tissue to about half of the control level. Examination of the chemical composition of the mammary gland (water, fat, Na, C1, K, histamine, and glycogen) during mastitis infection revealed marked changes in several of the constituents. The water content increased markedly, and this response was somewhat greater in glands under estrous control than in animals in the luteal phase of the estrous cycle; ovarieetomized ewes showed the least change. Na and C1 showed a large increase and K a decrease, indicating that the fluid increase probably occurs primarily in the extracellular compartment and is undoubtedly an inflammatory edema. A relationship between K and DNA concentration was expected, since both of these constituents axe associated with cellular elements and relatively constant values were obse~wed. Thus, the K / D N A ratio in the old sheep was 0.54 for estrous and 0.59 for luteal sheep. The young sheep inoculated with E. coll had values of 1.06 for estrous and 0.80 for the ]uteal (mean 0.93). The experiments on the young sheep infected with S. aureus were carried out about a month after the E. coli experiments and the maturation of the gland is seen by a change from the mean ratio of 0.93 to 0.74 for the estrous and 0.69 for Iuteal, values which ap-
938
JOEL
BITMAN
proach those of the older sheep. The ovariectomized sheep had their ovaries removed about one month earlier and the K / D N A ratio was arrested at the higher level, being 0.92 and corresponding to those of the first group of younger sheep. Itistamine was followed in the present study because this substance has been postulated as being released at times of i n j u r y and damage to cells (16). Because of its biological action and participation in inflanmmtion, it seemed possible that histamine shifts might parallel the course of the inflammation. Histamine concentration decreased routinely in all the inoculated mammary glands at all time periods, with the exception of the 0.5-hr samples. These samples showed an increase before exhibiting the usual decline in concentration caused by dilution of the tissue histamine as the gland increased in size. Whether these 0.5-hr increases were significant and a consistent p a r t of the inflammatory response could not be determined with the small numbers involved in these experiments. The most dramatic changes were observed in glycogen concentration and content between control and infected glands. Glycogen concentration increased three- to fourfold in inoculated glands and total glycogen content increased up to eight times over the control level. Correlation of chemical changes with histopathological alterations. Comparison of the chemical changes with the histological and pathological alterations in tissue (8) indicated a high degree of general agreement. Thus, leukocytic infiltration of the tissue appeared to be fairly well correlated with the glycogen response in the nmtnre sheep. The estrous ewes showed a slightly more r a p i d leukocytic response than the luteal phase ewes to the E. eoli infection, which agreed with the earlier increase noted in total glycogen in estrous sheep. No differences were noted in estrous vs. luteal leukocytic infiltration in the Staphylococcus infection, although both were higher than the ovariectomized animals. The quantitative glycogen results, however, showed a gradation in response, estrous being more r a p i d than luteal which, in turn, was faster than ovariectomized. In several instances in the young ewes, there were discrepancies between the leukoeytic density in the tissue and the glycogen concentration (S). The inflammatory response, a change in capillary permeability, edema (extracellular fluid influx-It~O, Na, and CI) and infiltration of leukocytes from the circulation into the tissue, is a p r i m a r y feature of the early stages of
ET
AL
mastitis infection. The leukocytic influx represents a defense mechanism of the tissue to combat the invading microorganisms. Since leukocytes are known to contain large amounts of glycogen (1), the glycogen increase is probably a direct reflection of the presence of leukocytes. The possibility exists, however, that the polysaccharide increase in the tissue might represent the presence of serum or tissue glycoproreins or depolymerization of ground substance (7), either mobilized or synthesized at the site of tissue i n j u r y (17). The slight increase in total K per gland noted at some of these early time periods may also be correlated with leukocytic infiltration and might represent an increase due to the cellular elements of the leukocytes rather than tissue cellular elements. The DNA and R N A determinations were not instituted at the start of the experiments and it was not possible to determine to what extent cell growth, hypertrophy, and hyperplasia were involved in the inflammatory tissue changes in all of these experiments. After inoculation the D N A / R N A ratio went up, which could signify an i n f u x of DNA from leukocytes with no increase in cytoplasmic RNA. An alternative interpretation could be that dissolution of cytoplasmic constituents occurred while DNA remained constant. The first possibility is favored by the data, however, since total DNA increased while total RNA per gland remained relatively constant. Correlation of inflammatory response with age and stage of estrous cycle. The somewhat greater ability of the estrous mammary gland to respond to the infection is qualitatively but not quantitatively similar to the greater response which the estrous uterus as compared to the luteal uterus exhibits to bacterial infection (9, 10). I n the uterus the estrous defense mechanism is much more responsive than the luteal, while the differences noted here in the mammary gland were small. Differences in chemical response between young and old sheep were noted, but it is difficult to relate these to mastitis susceptibility. I t is well known that the rate of mastitis increases with age (15). The greater response of the old animals demonstrated here, if correlated with defensive elements, should also make the older gland more able to combat the infection than the young udder. Relation of mastitis inflammation to inflammation in other tissues. The chemical changes which occur during inflammation in other tissues have not been studied extensSvely. The extent to which the inflammatory changes oh-
COMPOSITION OF MAMMARY GLAND
served in experimental mastitis correspond to those occurring in inflammation of other types is, therefore, difficult to determine. I n previous studies on deciduoma in the rat uterus (18), the chemical changes accompanying the inflammation due to the stimulus were determined. The inflammato~T changes in mastitis are very similar to the alterations seen in this type of uterine inflmmnation. I n the uterine studies, the electrolyte changes were also found to be quite similar to those occurring in the early stages of growth, and it was not possible to separate the i n j u r y effects from those due to the concomitant growth of the uterine tissue. I n mastitis, the infection--with its consequent degeneration of tissue, and healing and repair--with regeneration and growth of new tissue, are processes which go on concurrently within the gland. I t is likely that a similar situation prevails in the mammary gland as it does in the uterus, and the existence of these concomitant phenomena with similar chemical patterns will probably make it difficult to differentiate between the early phases of these processes. Forscher and Cecil (6) have studied the tissue alterations of some biochemical constituents occurring in the rat palate after a burn. Although the electrolyte changes were not reported, water increased immediately. However, glycogen did not rise significantly until one to three days after injury. I n this study, the glycogen response was assmned to be due to leukocytic infiltration. Further che.m.ical approaches to the study of mastitis. While there have been many reports of the histological changes which take place during the development of mastiffs, quantitative chemical studies of the tissue have not been made. Several investigators have induced mastitis with bacteria (4, 12, 14), but the initial inflammatory response was not examined. I n the present study, the times of sampling were chosen to yield information on the early stages of mastitis infection and the general chemical and histological pattern described is thus thought to be characteristic of the early stages of mastitis. The subsequent course of the disease, as measured by the chemical parameters determined in this study, could not, of course, be determined. The relationship of variations in the levels of constituents to the defense of the gland, their effects and interactions upon the severity and duration of the infection, would make controlled studies at longer time periods highly interesting. The characterization of differences in the
939
nature of the response with E. coli and Staphylococcus infection could be due either to (a) a basic difference in pathogenicity--the nature and virulence of the two microorganisms, (b) a difference in response caused by the difference in numbers of bacteria infused (6 billion vs. 0.4 billion, respectively), or (e) both of these factors. The nature of the chemical response to the mastitis produced with killed bacteria, bacterial extracts, or with any foreign material serving as an irritant was not studied in the present investigation and can only be determined by future research. Mastitis is commonly a disease of lactation and the animals used did not have extensive development of glandular secreting tissue. I n this preliminary investigation, however, in which methodology, techniques, and certain characteristics of the mastiffs infective process were worked out, the uncomplicated nature of the nonlactating gland was a welcome benefit. The presence of milk and glandular secretions in the tissue presents a problem which can be more readily approached as a result of the present experiments. The changes which take place as secreting cells decline in milk production during mastitis, and the alterations which occur as the glandular tissue involutes and is replaced by connective tissue, are other processes of mastitis infection which merit attention. The chemics~l alterations occurring in mastitic milk have been studied and a rise in chloride and decline in lactose concentration have been associated with the appearance of leukocytes in the milk (5). More recently, the whey proteins of mastitic milk have been studied in detail (3, 11, 13). The extent to which the chemical changes occurring in the tissue and described in this report are responsible for or are part of similar alterations in milk is an interesting question which we hope to answer in further investigations. Such information might provide an approach to very early diagnosis as well as affording more objective measures of following the course of the infection and treatment of the disease. Thus, the relationship of the extracellular rise in tissue chloride to the increase in milk chloride, the decline in milk lactose to the rise in tissue glycogen (or polysaecharide) are specific objectives of future research. ACK~O~LEDGMENT
Grateful acknowledgment is extended to the Sheep and Fur Animals l~esearch Branch, A]~S, Agricultural Research Center, United States Department of Agriculture, for generously supplying the sheep used in this study.
940
JOEL BITI~IAN ET AL
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