Lysozyme, alkaline phosphatase and neutrophils in uterine secretions of mares with differing resistance to endometritis

THERIOGENOLOGY

LYSOZYME, ALKALINE PHOSPHATASE AND NEUTROPHILS IN UTERINE SECRETIONS OF MARES WITH DIFFERING RESISTANCE TO ENDOMETRITIS T. Katila,112 T.F. Lock,1 W.E. Hoffmannl and A.R. Smith1 1Department of Veterinary Clinical Medicine University of Illinois Urbana, IL 61801 USA Received for publication: Ju2.q31, 1986 Accepted: December 21, 1989 ABSTRACT A study was conducted to 1) determine differences in the inflammatory response following bacterial challenge between normal mares and mares with chronic endometritis and 2) to determine if enzyme activity in uterine fluid can be used to evaluate degree of inflammation in the equine uterus. Six normal mares (Group 1) and four mares with chronic end.ometritis(Group 2) received an intrauterine infusion of B-hemolytic streptococci on the second day of estrus. Neutrophil concentration as well. as lysozyme and alkaline phosphatase activity were determined in uterine secretions obtained by placing tampons in the uterus of mares. All mares had a similar inflammatory response following bacterial challenge of the uterus, as indicated by a neutrophil response of the same magnitude. Neutrophil numbers, lysozyme and alkaline phosphatase corcentrations were all increased 12 h postinoculation and declined rapidly to normal preinoculation values by 48 h after inoculation. In spite of the similarity of the clinical signs, neutrophil concentratic,ns and enzyme activity, mares in group 1 demonstrated a markedly higher ability to eliminate the infection than mares in group 2. It is corcluded that factors other than neutrophil numbers, lysozyme and alkaline phosphatase activity account for the inability of the mare to eliminate uterine infections. Key words:

lysozyme, alkaline phosphatase, neutrophils INTRODUCTION

Although it has been well documented that mares differ in their susceptibility to uterine infection (l), it is still unclear why resistance of the uterus decreases in some mares. Only recently has interest been focused on the defense mechanisms of the uterus. Hughes and Loy (2) showed very convincingly that young maiden mares are highly resistant to bacterial inoculation of the uterus. Successive intrauterine infusions of Streptococcus zooeoidemicus over a period of 1 yr did not decrease the fertility of three young mares. In another experiment, two mares (one ovariectomized maiden and one chronically infected mare) received intrauterine infusions of Streptococcus zooenidemicus and were sacrificed and necropsied 5 h later. The

Acknowledgments 2Present address: EKK, 04840 Hautjarvi, Finland. This study was supported in part by funds provided by the Illinois Department of Agriculture Horse Racing Programs.

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maiden mare had exudate severe edema of the in the uterine lumen, The chronically infected endometrium, and relaxation of the cervix. mare exhibited very little cervical or uterine reaction to the bacterial challenge (2). This may indicate that the failure to eliminate bacteria could be due to a deficient or delayed inflammatory endometrial biopsies response. Peterson et al. and (3) studied cytological smears after intrauterine inoculation of bacteria. Four normal mares and two barren mares developed a similar kind of inflammatory response, but the barren mares required more than one estrous whereas the normal mares cleared the cycle to eliminate the bacteria, The authors assumed that bacteria within 4 to 15 d postexposure. endometritis results from a breakdown in the noncellular defense mechanisms or the failure of intracellular killing of bacteria by phagocytic cells (3). Blood leukocytes had higher phagocytic and streptocidal activity in mares than in progesterone-treated estrogen-treated ovariectomized mares at 2 d after inoculation with Streutococcus intrauterine zooepidemicus (4). Streptococcal phagocytosis was measured by chemiluminescence and streptocidal activity of peripheral blood leukocytes was Clearance of streptococcal infectmeasured, by a bactericidal assay. Cultures were negative for ion was determined by endometrial culture. estrogen-treated mares but positive for progesterone-treated mares 7 d after bacterial challenge. Additional studies have shown that phagocytosis and bacterial clearance from the uterus are enhanced by estrogens and depressed by progestens (5-6). Neutrophils from the uterus of susceptible mares have significantly less phagocytic and killing capacity than neutrophils from the uterus of normal mares (7). It has been suggested that a critical difference in chemiluminescence between resistant and susceptible animals does not exist (8). Immunoglobulins IgG, IgA and IgG(T) have been demonstrated in uterine secretions of mares (9-12). Higher concentrations of immunoglobulins are present in mares with impaired resistance to endometritis The importance than in normal mares of immunoglobulins in (11). uterine secretions remains to be determined. The purpose of our study was to determine the difference in inflammatory response of the uterus between normal mares and mares with chronic endometritis following intrauterine streptococcal challenge. The inflammatory response was quantitated by determining the number of neutrophils in uterine secretions and by measuring neutrophil lysosomal enzymes (alkaline phosphatase and lysozyme) present in the uterine secretions. Alkaline phosphatase and lysozyme are important products of neutrophils (13, 14) and high concentrations of these enzymes occur in inflammatory exudates. These observations suggest that measurement of alkaline phosphatase and lysozyme in uterine secretions may be a simple and sensitive means of assessing degree of inflammatory response of the uterus, and for this purpose they were evaluated along with the numbers of neutrophils present in the secretions.

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MATERIALS

AND METHODS

Ten mixed breed mares ranging in age from 2 to 22 yr were used in our study. The mares were kept on pasture until the experiment began. During the study, they were housed in separate stalls and fed a diet of oats and alfalfa hay. The study was conducted during the physiological b-ceeding season. The mares were divided into two groups based on endometrial biopsy categories as defined by Kenney (15). Six normal mares were assigned Biopsy Category 1 (Group 1). Four mares with chronic endometritis were placed in Biopsy Category 2 (Group 2). The mares were teased and palpated daily. Uterine secretions were obtained on Day 2 of estrus, Day 4 of estrus and on Day 12 of diestrus to obtain normal values on different days of the cycle. Mares were then allowed to go through one estrous cycle without any manipulations. On Day 2 of estrus of the following cycle, all mares received a uterine infusion of /3-hemolytic streptococci. The average inoculation dose was 3.5 x lo9 CFU (range 2 to 6 x 10 g CFU) in a total volume of 15 ml of Todd-Hewitt broth.a Uterine fluid samples and endometrial cultures were obtained immediately prior to inoculation and at 12, 24 and 48 hours after inoculation and on Day 12 of diestrus following inoculation. cultures were obtained using a guarded culture instrument, plated on blood agar and incubated for 24 h. Uterine fluid was collected using a slender, regular size Tampax tampon.c A 24-inch piece of umbilical tape was attached to the tampon and the tampon was passed through the vulva and vagina with a gloved hand. The tampon was then passed through the cervix and into the uterus. The tampon was allowed to absorb uterine fluid for 30 to 90 min and then withdrawn through a sterile vaginal speculum. The fluid was squeezed from the tampon with sterile forceps.

Endometiial

The total number of cells was determined with a cell counterd immediately after collection. The remaining fluid, including cellular components, was frozen for enzyme assays. Extracts for lysozyme determinations were obtained by repeating freezing and thawing (six times). The fluid was centrifuged and the supernatant was used for the assay. Lysozyme was assayed by a modification of the turbidimetric method (16). Alkaline phosphatase was measured using magnesium thymophthalein monophosphate as a substrate.e To analyze model was used

the statistical (17).

differences

between

groups,

a split-plot

"Todd Hewitt Medium. BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD 21030. "Guarded Culture Instrument. Kalayjian Industries, Inc., Appian Way, Long Beach, CA 90803. ':Tampax Inc., Palmer, MA 01069. '~Coulter Counter. Coulter Electronics, Inc., Hialeah, FL. "Boehringer-Mannheim Diagnostic, Indianapolis, IN 46250.

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THERIOGENOLOGY RESULTS Average fluid volume recovered from the tampon was approximately 2 ml before inoculation, while more than 4 ml was recovered 12 to 48 h after inoculation. On two occasions, no fluid was obtained, which gave an overall failure rate of 3%. All mares responses to the bacterial inoculation with acute inflammation of the uterus, which was evident on clinical examinations. All endometrial cultures yielded a moderate or heavy growth of bhemolytic streptococci 6 h after inoculation (Table 1). Eight mares still had positive cultures at 48 h after inoculation. One mare (16.6%) from Group 1 had a moderate growth of @-streptococci on Day 12 of diestrus following inoculation, while three (75%) mares in Group 2 had either moderate or heavy growth. Table 1.

Mare NO.

6 7 10 12 13 45 4 8 11 14

1 2 3 4

= = = =

Growth of /3-hemolyticstreptococci

Biopsy category 1 1 1 1 1 1 2 2 2 2

Days of the cycle and hours Before inoculation After inoculation E2 E4 D12 Oh 6h 12h 24h 48h D12 1111 1111 12 12 112 12 2 13 1111 1111 113

12 11 1 11 1

1

no growth. light growth (lOl colonies).

4 3 4 4 4 4 4 4 4 4

2 14 4 2 4 3 2 2 4 4

111 4 3 4 4 2 13 4 4

3 4 11 4 4 2 4 4

1 1 3 1 1 3 4 4

E2 = Day 2 of estrus. E4 = Day 4 of estrus. D12 = Day 12 of diestrus.

Neutrophil numbers and lysozyme and alkaline phosphatase activity in uterine fluids followed the same pattern of change after bacterial inoculation (Figure 1). The values were very high at 12 h and decreased rapidly, reaching pretreatment values at 48 h. The preinoculation values did not differ statistically from each other, but there were very highly significant differences among periods of time for all three parameters after bacterial inoculation. Group 2 had more neutrophils than did Group 1 at all times except at 24 h, but there was also more variation in Group 2, so the difference was not statistically significant (Figure 2). Group 2 had higher lysozyme activity at 12 and 48 h and on Day 12 of diestrus following inoculation, but the differences between groups was not statistically significant (Figure 3). Alkaline phosphatase activity was higher for Group 1 than for Group 2 at 12 h postinoculation, but the differences between groups were not statistically significant (Figure 4).

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THERIOGENOLOGY

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THERIOGENOLOGY

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THERIOGENOLOGY 3000

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tion in uterine fluid after the bacterial inoculation. 730

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DISCUSSION Objective evaluation of the degree of inflammation of the uterus vaginoscopy and endometrial culture is using palpation per rectum, Neutrophil counts and lysozyme and alkaline phosphatase d.ifficult. fluids gave results which could be evaluated a.ctivity in uterine Since lysozyme and alkaline phosphatase paralleled the objectively. concentrations of these enzymes increase and decrease in neutrophils, in uterine fluid appear to be a useful diagnostic indicator in assessment of the intensity of the acute inflammatory response of the uterus to bacterial challenge. The inflammatory response to intrauterine inoculation with bacteria and intense but not very long-lasting. In both groups, i.s rapid alkaline phosphatase and neutrophil concentrations in the lysozyme, uterine fluids showed significant increases by 12 h postinoculation, There was no but by 48 h they had returned to pretreatment values. statistically significant difference in neutrophil numbers in the However, on Day 12 of uterine fluid between Group 1 and Group 2 mares. P-streptococci was recovered from diestrus following the inoculation, the uterus of one mare (16.6%) in Group 1 compared with three mares (75%) in Group 2. This study supports the view that factors other than neutrophil numbers account for the inability of susceptible mares to eliminate Reductions bacteria from the uterus. in phagocytic and killing capacities (7) as well as deficiency in opsonins (8) in the uterus contribute to the inability of susceptible mares to eliminate bacteria. REFERENCES 1. Dimock, W.W. and Snyder, E. Bacteria of the genital tract and relation to the semen of stallions their and efficiency. JAVMA &:288-297 (1924).

of mares breeding

Investigations J.P. and Loy, of the effect of R.G. 2. Hughes, of Streotococcus zooenidemicus in the intrauterine inoculations Proc. 15th Ann. Conv. Am. Assoc. Equine Practnrs., pp. 289mare. 292 (1969). F.B., McFeely, R.A. and David, J.S.E. 3. Peterson, pathogenesis of endometritis in the mare. Proc. Am. Assoc. Equine Practnrs., pp. 279-287 (1969).

Studies on the 15th Ann. Conv.

S.M., Klesius, P.H., Ganjam, V.K. and Brown, B.G. Effect 4. Washburn, of estrogen and progesterone on the phagocytic response of ovariectomized mares infected in utero with P-hemolytic streptococci. Am. J. Vet. Res. a:1367-1370 (1982). J.N. and 5. Blue, M.G., Brady, A.A., Davidson, on the composition of antibacterial activity J. Reprod. Fertil., 32(suppl.):143-149 mares.

Studies Kenney, R.M. of uterine fluid from (1982).

Antibacterial P.J., Do, D. and Kenney, R.M. 6. Strzemienski, of mare uterine fluid. Biol. Reprod. 31:303-311 (1984).

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7. Cheung, A.T.W., Liu, I.K.M., Walsh, E.M. and Miller, M.E. Phagocytic and killing capacities of uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis. Am. J. Vet. Res. 461938-1940 (1985). 8. Asbury, A.C., Schultz, Klesius, K.T., P.H., Foster, G.W. and Washburn, S.M. Factors affecting phagocytosis of bacteria by neutrophils in the mare's uterus. Fertil. J. Reprod. 32(Suppl.):151-159 (1982). 9. Bowman, coccal 1972.

T. Early Response of the Equine Uterus to Induced StreptoEndometritis. Master's thesis, University of Maryland,

10. Kenney, R.M. and Khaleel, S.A. uterus: a progress report on 23(Suppl.):357-358 (1975).

Bacteriostatic activity of the mare immunoglobulins. J. Reprod. Fert.

11

Asbury, A.C., Halliwell, R.E.W., Foster, G.W. and Longino, S.J. Immunoglobulins in uterine secretions of mares with differing resistance to endometritis. Theriogenology &:299-308 (1980).

12

Mitchell, G., Liu, I.K.M., Perryman, L.E., Stabenfeldt, G.H. and Hughes, J.P. Preferential production and secretion of immunoglobulins by the equine endometrium--A mucosal immune system. J. Reprod. Fertil. 32(Suppl.):161-168 (1982).

13

Jain, N.C. Alkaline phosphatase activity in animal species. Acta Haemat. %:51-59 (1968).

leukocytes

14. Jensen, R.S., Tew, J.G. and Donaldson, D.M. Extracellular and muramidase in body fluids and inflammatory exudates. Sot. Exp. Biol. Med. =:545-549 (1967). 15. Kenney, R.M. Cyclic and pathologic changes as detected by biopsy, with a note on early u:241-261 (1978).

of

some

P-lysin Proc.

of the mare endometrium JAVMA embryonic death.

16. Feldman, B.F., Madewell, B.R. and Miller, (muramidase) activity in dogs with neoplastic Res. g:1319-1321 (1981).

R.B. Serum lysozyme disease. Am. J. Vet.

17. Gill, J.L. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 2. The Iowa State University Press. Ames, 1978.

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