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Immunoglobulin levels, protein concentrations and alkaline phosphatase activity in uterine flushings from mares with endometritis
THERIOCENOLOGY
IMMUNOGLOBULIN LEVELS, PROTEIN CONCENTRATIONS AND ALKALINE PHOSPHATASE ACTIVFlY IN UTERINE FLUSHINGS FROM MARES WITH ENDOMETRITIS P. Williamson, Alison Dunning, Julie O'Connor & W.J. Penhale School of Veterinary Studies Murdoch University Murdoch Western Australia 6150 Received for Publication: Accepted:
October 12, 1982 November 24, 1982
ABSTRACT Uterine samples and flushings were obtained from 87 mares to compare endometrial bacteriology and biopsy with immunoglobulin and protein concentration and alkaline phosphatase activity in uterine flushings. Mares were designated as infected if both bacteriology and biopsies were positive. The imoglobulin levels, protein concentration and alkaline phosphatase activity in uterine flushings from infected and non-infected mares were compared. Twenty (23%) of the mares were classified as infected. A significantly higher proportion of infected mares (cf. non-infected) had elevated IgA and protein concentrations. Levels Of IgG, IgGT or alkaline phosphatase were not significantly elevated in infected mares. These results suggest that IgA and protein levels are elevated in the uterus in the presence of active infection. INTRODUCTION Genital tract infection is considered to be an important cause of infertility in the mare lr2. However, diagnosing active uterine infection in the mare is difficult. The outcome of uterine infection depends on both the susce ;pility of the mare and the pathogenicity of the invading organismsf . This complex interaction has led to controvers over the significance of different organisms isolated from the uterusY 16. Interpreting the results of bacterial culture of endometrial swabs is difficult; therefore, other parameters which might indicate uterine infection have been investigated, including endometrial biopsy"' and the cellular content of uterine exudateg. The concurrent use of uterine bacteriology and either cytological smears or the presence of inflammatory cells in endometrial biopsies provides a reasonable basis for diagnosing acute endometritis in the mareg""il. Recent studies have indicated that increased immunoglobyiiv3concentrations may occur in the uterine exudate of infected mares This research was supported by a grant from the Australian Equine Research Foundation. Mrs. P. Clifford, Mr. R. McEenna and Mr. D. Brockway provided valuable assistance.
MARCH
1983 VOL. 19 NO. 3
441
THERIOGENOLOGY
The present study was undertaken to evaluate immunoglobulin levels, protein concentrations and alkaline phosphatase activity in uterine flushings as aids to diagnosing active uterine infection in the mare and to investigate the response of the uterus to infection. The established diagnostic techniques of endometrial bacteriology and biopsy were used to diagnose uterine infection, and the levels of iuununoglobulins,protein and alkaline phosphatase in the flushings of infected and non-infected mares were compared. MATERIALS & METHODS Animals. Eighty-seven non-pregnant standardbred and thoroughbred mares aged between two and seventeen years and of unknown breeding history were used in the present study, which was undertaken in the Perth, Western Australian region (Lattitude 32O south). Collection of Samples. Mares were injected with 20mg acepromazine (Aceprome, V.R. Laboratories [Aust)) and 30mg Probantheline bromide (Probanthine, SearleLaboratories) 20 min before sampling to facilitate the protracted rectal manipulation necessary during collection procedures. Following initial rectal examination, the mares' tails were wrapped in clean bandages and the perineal regions were washed twice with surgical scrub. This cleaning procedure was repeated between sampling operations. Rndometrial Swabs. A guarded uterine swab (V.R. Laboratories [Au&]) was introduced into the lumen of the uterus, where it was exposed and gently rotated for 15-20 seconds. The swab was then withdrawn and either sealed in its protective plastic packaging (42 mares) or placed immediately into Stuarts Transport Medium and refrigerated (45 mares). All swabs were plated onto blood agar (10% sheep's blood) and McConkeys agar plates within 4 hours of collection and incubated overnight at 37OC in air. The organisms which grew were identified by routine biochemical procedures. When more than 20 colonies of a particular bacteria grew per culture plate, the growth was considered to be significant2. Uterine Flushing. Uterine flushings were collected from each mare using a modified Foley catheter". Eighty ml of sterile normal saline (10 mares) or isotonic mannitol solution (77 mares) were infused slowly into the uterus and the uterus was gently massaged per rectum for 4 minutes. The fluid was then allowed to flow from the uterus into sterile containers. When sufficient fluid for testing (2Oml) had been collected, the procedure was halted to minimise the time and trauma of collection. Flushings were immediately cooled to 4'?, frozen and stored at -20°C until required for analysis. With the last 45 mares sampled, 2ml aliquotes of flushings were decanted before freezing, centrifuged and stored at 4OC to estimate protein concentration.
442
MARCH
1983 VOL. 19 NO. 3
THERIOGENOLOGY Endometrial Biopsy. Endometrial biopsy vfasperformed on each mare using the technique described by Greenhoff and Kenney15. Biopsy tissue was fixed in Bouin's fluid and sections were stained with haematoxylin and eosin. Biopsy sections were assessed for inflammatory cells. Biopsies were classified as being 'negative' if no inflammation or a light mononuclear leucocyte infiltration was present in the endometrium. Mares were classified as 'positive' if two of the following inflammatory changes were present in the endometrium: 1.
moderate to severe infiltration of the endometrium with mononuclear leucocytes,
2.
abundant neutrophils in the uterine blood vessels, the endometrial connective tissue, or the uterine lumen,
3.
moderate to severe erosion of the uterine lumenal epithelium with underlying foci of inflammatory cells.
Laboratory Analysis of Uterine Flushings. Imnmnoglobulin Concentrations. The concentrations of immunoglobulins IgM, A, G and GT in flushings were determined by the single radial diffusion method16 using both commercial (Miles Laboratories Inc) and laboratory prepared antisera. Where purified standard preparations of immunoglobulin were available, inununoglobulinconcentrations in the samples were determined in absolute quantities. In those cases where purified standards were unavailable, samples were compared to a colostrum standard and recorded as a percentage of this standard. Protein Concentration. Protein levels in the uterine washin s of 45 of the mares were determined by Spectrophotometric Assay17'l4 Alkaline Phosphatase Levels. Alkaline Phosphatase levels in the uterine flushings were determined on a Gemeni Minature Centrifugal Analyzer (Electra-Nucleonics, Inc, Fairfield, J.J. 07006). Statistical Comparison of Uterine Measurements. The relationship between the occurrence of positive culture and biopsy results was analysed using a Sign Test". To assess immunoglobulin concentration, protein concentration and alkaline phosphatase activity in uterine flushings as aids for diagnosing uterine infection, mares were arbitarily designated as being infected or non-infected using the bacteriology and biopsy results. Mares were classified as being infected if they had both of the following: 1.
a significant growth of bacteria (more than 20 colonies/plate from an endometrial s~ab)~,
2.
positive inflammatory changes in the endometrial biopsy as defined previously.
MARCH
1983 VOL. 19 NO. 3
443
THERIOGENOLOGY
Mares were placed in an 'uncertain infection' category if only one of the above criteria was positive and they were considered clear of uterine infection if neither of these findings occurred. The numbers of mares classified as infected and non-infected with measurable levels using Fischers of the immunoglobulins IgA, IgG, and IgGT were COnpared Complete analysis. The concentration of protein and mean alkaline phosphatase levels in flushings from the mares classified as infected and noy;infected were compared using students t-test for unequal group sizes . RESULTS The bacteriological investigation of swabs (Table 1) revealed the presence of a number of bacterial species. Thirty-two percent of mares sampled had positive bacteriology. Considerable variation in the numbers of bacterial colonies grown from different mares with positive bacteriology was observed. Table 1.
Bacteria grown from uterine swabs.
Organism
8 haemolytic Strep.
Number of Mares with Positive Bacteriology 11 (12.5%)
E. coli
4 (4.5%)
Corynebacterium spp.
1 (1%)
E. coli + pseudomonas spp.
1 (1%)
E. coli + 8 haemolytic strep.
2 (2%)
Light mixed growth
9 (10.5%)
TOTAL
28 (32%)
4 Examination of the biopsies showed that 36 mares (41%) had moderate or severe inflammation of the endometrium, 28 (32%) had mild inflammation and 23 (26%) had no inflammatory changes present. The relationship between positive scores for culture and endometrial biopsy (p
444
MARCH
1983 VOL. 19 NO. 3
THERIOGENOLOGY
Table 2.
Number of mares classified as being 'infected', 'doubtful' and 'uninfected', based on the results of bacteriology and biopsy. c
Classification of Mares
Infected*
Doubtful+
Uninfected'
Number of mares
20
21
46
(23%)
(24%)
TOTAL.
(53%)
87
* both criteria +ve + only one +ve o non +ve Uterine flushings were collected successfully from all mares in the Immunoglobulins of class IgA, IgG and IgGT were detected in may. many of the uterine flushings tested. IgA was the predominant immunoglobulin present. The minimum level of sensitivity of the respective irmnunoglobulinassays were IgA, 22mg%; IgG, 15 mg%; and IgGT, 10% of the colostrum standard. The number of uterine flushings from mares classified as infected or non-infectedwhich contained measurable quantities of the different immunoglobulins is shown in Table 3. A significantly higher proportion of flushings from mares designated as infected (as judged by bacteriology and histological criteria) compared with non-infected mares had measurable amounts of IgA (P < 0.01). The proportion of infected and non-infected mares with measurable amounts of IgG and IgGT did not differ significantly/ Table 3.
The number of mares in infected and non-infected groups with measurable concentrations of IgA, IgG and IgGT in uterine flushings.
The mean protein concentration in uterine flushings from the mares designated as infected was also significantly higher than in the flushings from non-infected mares (3.17 + 5.12 mg per ml cf. 0.35 f 0.24 mg per ml; P < 0.01). On the other hand, alkaline phosphatase activity did not differ significantly between non-infected and infected groups (142.1 f 155.6 vs 92.2 + 74.0, N.S.).
MARCH 1983 VOL. 19 NO, 3
445
THERIOGENOLOGY
DISCUSSION
Bacteria grown from endometrial swabs in the present trial are Similar in type and frequency of 9 c7urrence to uterine pathogens reported in other studies on the mare . The highly significant relationship (p
446
MARCH 1983 VOL. 19 NO. 3
THERIOGENOLOGY
appear to consistently indicate established infection. High IgA levels in the uterine flushings of infected mares does not, therefore, ensure that the infection will be overcome. Both the results of the present study and those of Asbury et a1.12 suggest that mares resistant to infection have low IgA levels in uterine washings. That a generalised failure of IgA secretion renders a mare susceptible to infection seems unlikely. The presence of immunoglobulins directed specifically against the invading organisms in infected mares is currently under investigation. The increased protein concentrations in the uterine flushings from infected mares may be due to increased levels of secretory proteins and cellular debris, damaged tissues and lysed micro-organisms. Three of the four doubtful mares with positive cultures had low immunoglobulin and protein concentrations in uterine washings, indicating that the invading organisms had not (as yet) become established. High IgA and protein concentrations in uterine washings may be useful adjuncts to bacteriology, cytology and biopsy in diagnosing active uterine infection and providing markers in future research aimed at defining the causes and effects of endometritis in mares. REFERENCES 1.
Bain, M.A. The role of infection in infertility in the Thoroughbred mare. Vet. Rec. -78 : 168-173 (1966).
2.
Shin, S.J., Lein, A., Aronson, A.L. and Nusbaum, S.R. The bacteriological culture of equine uterine contents, in vitro sensitivity of organisms isolated and interpretation. J. Reprod. Fert., Suppl. 27 : 307-315 (1979).
3.
Crouch, J-F., Atherton, J.G. and Platt, H. Venereal transmission of KlebsieZZa aerogenes in a Thoroughbred stud from a persistently infected stallion. Vet. Rec. -90 : 21-24 (1972).
4.
Hughes, J.P. and Loy, R.G. The relation of infection to infertility in the mare and stallion. Equine Vet. J. -7 : 155-159 (1975).
5.
Hughes, K.L. Streptococcus zooepidemicus and infertility in horses. Aust. vet. J. -51 : 281 (1975).
6.
Woolcock, J.B. Equine bacterial endometritis. of North America, -2 : 241-251 (1980).
7.
Ricketts, S-W. Endometrial biopsy as a guide to diagnosis of endometrial pathology in the mare. J. Reprod. Fert., Suppl. -23 : 241-247 (1975).
8.
Penney, R.M. Cyclic and pathological changes in the mare endometrium as detected by biopsy, with a note on early embryonic death. J.Am.Vet.Med.Assoc. 172 : 241-262 (1978).
MARCH
1983 VOL. 19 NO. 3
The Vet Clinics
447
THERIOGENOLOGY 9.
Wingfield-Digby, N.J. The techniques and clinical application of endometrial cytology in mares. Equine Vet. J. -10 : 167-170.
10. Blanchard, T.L., Cummings, M-R., Garcia, M-C., Hurtgen, J.P. and Kenney, R.M. Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares. Theriogenology,-16 : 541-552 (1981). 11. Ricketts, R.W. Bacteriological examination of the mare's cervix: Techniques and interpretation of results. Vet.Rec. 108 : 46-51 (1981). 12. 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,-14 : 299-308 (1980). 13. Liu, I.K.M., Mitchell, G., Perryman, L.E. and Stewart, E.W. Immunological defense machanisms of the uterus in the mare. Proc. Sot. Theriogenology : 265-267 (1981). 14. Zavy, M-T., Baser, F.W., Sharp, D.C. and Wilcox, C.J. Uterine luminal proteins in the cycling mare. Biol.Reprod. -20 : 689-698 (1977). 15. Greenhoff, G.R. and Kenney, R.M. status of the non-pregnant mare. 449-458 (1975).
Evaluation of reproductive J.Am.Vet.Med.Assoc.167 :
16. Mancini, G., Carbonara, A.D. and Heremans, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry,2 : 235-254 (1965). 17. Bradford, M.M. A rapid and sensitive method for quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. -72 : 248-254 (1976). 18. Spector, T. Refinement of the Comassie Blue method of protein quantitation. Anal. Biochem. -86 : 142-147 (1978). 19. Snedecor, G.W. and Cochran, W.G. Statistical methods, 6th ed. Iowa State Uni. Press, 1967, pp.104-127. 20. Witherspoon, D., Goldston, R. and Aslit, M. Uterine culture and biopsy in the mare. J.Am.Vet.Med.Assoc.161 : 1365-1366 (1972). 21. Peterson, F.B., McFeely, R.A. and David, J.S.E. Studies on the pathogenesis of endometritis in the mare. Proc. 15th Am. Assoc. Eq. Pratt., pp.279-287 (1969). 22. Bergman, R.V. and Kenney, R.M. Representativenessof a uterine biopsy of the mare. Proc.Zlst Am. Ass.Eq.Pract. pp.355-361 (1975).
448
MARCH 1983 VOL. 19 NO. 3
IMMUNOGLOBULIN LEVELS, PROTEIN CONCENTRATIONS AND ALKALINE PHOSPHATASE ACTIVFlY IN UTERINE FLUSHINGS FROM MARES WITH ENDOMETRITIS P. Williamson, Alison Dunning, Julie O'Connor & W.J. Penhale School of Veterinary Studies Murdoch University Murdoch Western Australia 6150 Received for Publication: Accepted:
October 12, 1982 November 24, 1982
ABSTRACT Uterine samples and flushings were obtained from 87 mares to compare endometrial bacteriology and biopsy with immunoglobulin and protein concentration and alkaline phosphatase activity in uterine flushings. Mares were designated as infected if both bacteriology and biopsies were positive. The imoglobulin levels, protein concentration and alkaline phosphatase activity in uterine flushings from infected and non-infected mares were compared. Twenty (23%) of the mares were classified as infected. A significantly higher proportion of infected mares (cf. non-infected) had elevated IgA and protein concentrations. Levels Of IgG, IgGT or alkaline phosphatase were not significantly elevated in infected mares. These results suggest that IgA and protein levels are elevated in the uterus in the presence of active infection. INTRODUCTION Genital tract infection is considered to be an important cause of infertility in the mare lr2. However, diagnosing active uterine infection in the mare is difficult. The outcome of uterine infection depends on both the susce ;pility of the mare and the pathogenicity of the invading organismsf . This complex interaction has led to controvers over the significance of different organisms isolated from the uterusY 16. Interpreting the results of bacterial culture of endometrial swabs is difficult; therefore, other parameters which might indicate uterine infection have been investigated, including endometrial biopsy"' and the cellular content of uterine exudateg. The concurrent use of uterine bacteriology and either cytological smears or the presence of inflammatory cells in endometrial biopsies provides a reasonable basis for diagnosing acute endometritis in the mareg""il. Recent studies have indicated that increased immunoglobyiiv3concentrations may occur in the uterine exudate of infected mares This research was supported by a grant from the Australian Equine Research Foundation. Mrs. P. Clifford, Mr. R. McEenna and Mr. D. Brockway provided valuable assistance.
MARCH
1983 VOL. 19 NO. 3
441
THERIOGENOLOGY
The present study was undertaken to evaluate immunoglobulin levels, protein concentrations and alkaline phosphatase activity in uterine flushings as aids to diagnosing active uterine infection in the mare and to investigate the response of the uterus to infection. The established diagnostic techniques of endometrial bacteriology and biopsy were used to diagnose uterine infection, and the levels of iuununoglobulins,protein and alkaline phosphatase in the flushings of infected and non-infected mares were compared. MATERIALS & METHODS Animals. Eighty-seven non-pregnant standardbred and thoroughbred mares aged between two and seventeen years and of unknown breeding history were used in the present study, which was undertaken in the Perth, Western Australian region (Lattitude 32O south). Collection of Samples. Mares were injected with 20mg acepromazine (Aceprome, V.R. Laboratories [Aust)) and 30mg Probantheline bromide (Probanthine, SearleLaboratories) 20 min before sampling to facilitate the protracted rectal manipulation necessary during collection procedures. Following initial rectal examination, the mares' tails were wrapped in clean bandages and the perineal regions were washed twice with surgical scrub. This cleaning procedure was repeated between sampling operations. Rndometrial Swabs. A guarded uterine swab (V.R. Laboratories [Au&]) was introduced into the lumen of the uterus, where it was exposed and gently rotated for 15-20 seconds. The swab was then withdrawn and either sealed in its protective plastic packaging (42 mares) or placed immediately into Stuarts Transport Medium and refrigerated (45 mares). All swabs were plated onto blood agar (10% sheep's blood) and McConkeys agar plates within 4 hours of collection and incubated overnight at 37OC in air. The organisms which grew were identified by routine biochemical procedures. When more than 20 colonies of a particular bacteria grew per culture plate, the growth was considered to be significant2. Uterine Flushing. Uterine flushings were collected from each mare using a modified Foley catheter". Eighty ml of sterile normal saline (10 mares) or isotonic mannitol solution (77 mares) were infused slowly into the uterus and the uterus was gently massaged per rectum for 4 minutes. The fluid was then allowed to flow from the uterus into sterile containers. When sufficient fluid for testing (2Oml) had been collected, the procedure was halted to minimise the time and trauma of collection. Flushings were immediately cooled to 4'?, frozen and stored at -20°C until required for analysis. With the last 45 mares sampled, 2ml aliquotes of flushings were decanted before freezing, centrifuged and stored at 4OC to estimate protein concentration.
442
MARCH
1983 VOL. 19 NO. 3
THERIOGENOLOGY Endometrial Biopsy. Endometrial biopsy vfasperformed on each mare using the technique described by Greenhoff and Kenney15. Biopsy tissue was fixed in Bouin's fluid and sections were stained with haematoxylin and eosin. Biopsy sections were assessed for inflammatory cells. Biopsies were classified as being 'negative' if no inflammation or a light mononuclear leucocyte infiltration was present in the endometrium. Mares were classified as 'positive' if two of the following inflammatory changes were present in the endometrium: 1.
moderate to severe infiltration of the endometrium with mononuclear leucocytes,
2.
abundant neutrophils in the uterine blood vessels, the endometrial connective tissue, or the uterine lumen,
3.
moderate to severe erosion of the uterine lumenal epithelium with underlying foci of inflammatory cells.
Laboratory Analysis of Uterine Flushings. Imnmnoglobulin Concentrations. The concentrations of immunoglobulins IgM, A, G and GT in flushings were determined by the single radial diffusion method16 using both commercial (Miles Laboratories Inc) and laboratory prepared antisera. Where purified standard preparations of immunoglobulin were available, inununoglobulinconcentrations in the samples were determined in absolute quantities. In those cases where purified standards were unavailable, samples were compared to a colostrum standard and recorded as a percentage of this standard. Protein Concentration. Protein levels in the uterine washin s of 45 of the mares were determined by Spectrophotometric Assay17'l4 Alkaline Phosphatase Levels. Alkaline Phosphatase levels in the uterine flushings were determined on a Gemeni Minature Centrifugal Analyzer (Electra-Nucleonics, Inc, Fairfield, J.J. 07006). Statistical Comparison of Uterine Measurements. The relationship between the occurrence of positive culture and biopsy results was analysed using a Sign Test". To assess immunoglobulin concentration, protein concentration and alkaline phosphatase activity in uterine flushings as aids for diagnosing uterine infection, mares were arbitarily designated as being infected or non-infected using the bacteriology and biopsy results. Mares were classified as being infected if they had both of the following: 1.
a significant growth of bacteria (more than 20 colonies/plate from an endometrial s~ab)~,
2.
positive inflammatory changes in the endometrial biopsy as defined previously.
MARCH
1983 VOL. 19 NO. 3
443
THERIOGENOLOGY
Mares were placed in an 'uncertain infection' category if only one of the above criteria was positive and they were considered clear of uterine infection if neither of these findings occurred. The numbers of mares classified as infected and non-infected with measurable levels using Fischers of the immunoglobulins IgA, IgG, and IgGT were COnpared Complete analysis. The concentration of protein and mean alkaline phosphatase levels in flushings from the mares classified as infected and noy;infected were compared using students t-test for unequal group sizes . RESULTS The bacteriological investigation of swabs (Table 1) revealed the presence of a number of bacterial species. Thirty-two percent of mares sampled had positive bacteriology. Considerable variation in the numbers of bacterial colonies grown from different mares with positive bacteriology was observed. Table 1.
Bacteria grown from uterine swabs.
Organism
8 haemolytic Strep.
Number of Mares with Positive Bacteriology 11 (12.5%)
E. coli
4 (4.5%)
Corynebacterium spp.
1 (1%)
E. coli + pseudomonas spp.
1 (1%)
E. coli + 8 haemolytic strep.
2 (2%)
Light mixed growth
9 (10.5%)
TOTAL
28 (32%)
4 Examination of the biopsies showed that 36 mares (41%) had moderate or severe inflammation of the endometrium, 28 (32%) had mild inflammation and 23 (26%) had no inflammatory changes present. The relationship between positive scores for culture and endometrial biopsy (p
444
MARCH
1983 VOL. 19 NO. 3
THERIOGENOLOGY
Table 2.
Number of mares classified as being 'infected', 'doubtful' and 'uninfected', based on the results of bacteriology and biopsy. c
Classification of Mares
Infected*
Doubtful+
Uninfected'
Number of mares
20
21
46
(23%)
(24%)
TOTAL.
(53%)
87
* both criteria +ve + only one +ve o non +ve Uterine flushings were collected successfully from all mares in the Immunoglobulins of class IgA, IgG and IgGT were detected in may. many of the uterine flushings tested. IgA was the predominant immunoglobulin present. The minimum level of sensitivity of the respective irmnunoglobulinassays were IgA, 22mg%; IgG, 15 mg%; and IgGT, 10% of the colostrum standard. The number of uterine flushings from mares classified as infected or non-infectedwhich contained measurable quantities of the different immunoglobulins is shown in Table 3. A significantly higher proportion of flushings from mares designated as infected (as judged by bacteriology and histological criteria) compared with non-infected mares had measurable amounts of IgA (P < 0.01). The proportion of infected and non-infected mares with measurable amounts of IgG and IgGT did not differ significantly/ Table 3.
The number of mares in infected and non-infected groups with measurable concentrations of IgA, IgG and IgGT in uterine flushings.
The mean protein concentration in uterine flushings from the mares designated as infected was also significantly higher than in the flushings from non-infected mares (3.17 + 5.12 mg per ml cf. 0.35 f 0.24 mg per ml; P < 0.01). On the other hand, alkaline phosphatase activity did not differ significantly between non-infected and infected groups (142.1 f 155.6 vs 92.2 + 74.0, N.S.).
MARCH 1983 VOL. 19 NO, 3
445
THERIOGENOLOGY
DISCUSSION
Bacteria grown from endometrial swabs in the present trial are Similar in type and frequency of 9 c7urrence to uterine pathogens reported in other studies on the mare . The highly significant relationship (p
446
MARCH 1983 VOL. 19 NO. 3
THERIOGENOLOGY
appear to consistently indicate established infection. High IgA levels in the uterine flushings of infected mares does not, therefore, ensure that the infection will be overcome. Both the results of the present study and those of Asbury et a1.12 suggest that mares resistant to infection have low IgA levels in uterine washings. That a generalised failure of IgA secretion renders a mare susceptible to infection seems unlikely. The presence of immunoglobulins directed specifically against the invading organisms in infected mares is currently under investigation. The increased protein concentrations in the uterine flushings from infected mares may be due to increased levels of secretory proteins and cellular debris, damaged tissues and lysed micro-organisms. Three of the four doubtful mares with positive cultures had low immunoglobulin and protein concentrations in uterine washings, indicating that the invading organisms had not (as yet) become established. High IgA and protein concentrations in uterine washings may be useful adjuncts to bacteriology, cytology and biopsy in diagnosing active uterine infection and providing markers in future research aimed at defining the causes and effects of endometritis in mares. REFERENCES 1.
Bain, M.A. The role of infection in infertility in the Thoroughbred mare. Vet. Rec. -78 : 168-173 (1966).
2.
Shin, S.J., Lein, A., Aronson, A.L. and Nusbaum, S.R. The bacteriological culture of equine uterine contents, in vitro sensitivity of organisms isolated and interpretation. J. Reprod. Fert., Suppl. 27 : 307-315 (1979).
3.
Crouch, J-F., Atherton, J.G. and Platt, H. Venereal transmission of KlebsieZZa aerogenes in a Thoroughbred stud from a persistently infected stallion. Vet. Rec. -90 : 21-24 (1972).
4.
Hughes, J.P. and Loy, R.G. The relation of infection to infertility in the mare and stallion. Equine Vet. J. -7 : 155-159 (1975).
5.
Hughes, K.L. Streptococcus zooepidemicus and infertility in horses. Aust. vet. J. -51 : 281 (1975).
6.
Woolcock, J.B. Equine bacterial endometritis. of North America, -2 : 241-251 (1980).
7.
Ricketts, S-W. Endometrial biopsy as a guide to diagnosis of endometrial pathology in the mare. J. Reprod. Fert., Suppl. -23 : 241-247 (1975).
8.
Penney, R.M. Cyclic and pathological changes in the mare endometrium as detected by biopsy, with a note on early embryonic death. J.Am.Vet.Med.Assoc. 172 : 241-262 (1978).
MARCH
1983 VOL. 19 NO. 3
The Vet Clinics
447
THERIOGENOLOGY 9.
Wingfield-Digby, N.J. The techniques and clinical application of endometrial cytology in mares. Equine Vet. J. -10 : 167-170.
10. Blanchard, T.L., Cummings, M-R., Garcia, M-C., Hurtgen, J.P. and Kenney, R.M. Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares. Theriogenology,-16 : 541-552 (1981). 11. Ricketts, R.W. Bacteriological examination of the mare's cervix: Techniques and interpretation of results. Vet.Rec. 108 : 46-51 (1981). 12. 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,-14 : 299-308 (1980). 13. Liu, I.K.M., Mitchell, G., Perryman, L.E. and Stewart, E.W. Immunological defense machanisms of the uterus in the mare. Proc. Sot. Theriogenology : 265-267 (1981). 14. Zavy, M-T., Baser, F.W., Sharp, D.C. and Wilcox, C.J. Uterine luminal proteins in the cycling mare. Biol.Reprod. -20 : 689-698 (1977). 15. Greenhoff, G.R. and Kenney, R.M. status of the non-pregnant mare. 449-458 (1975).
Evaluation of reproductive J.Am.Vet.Med.Assoc.167 :
16. Mancini, G., Carbonara, A.D. and Heremans, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry,2 : 235-254 (1965). 17. Bradford, M.M. A rapid and sensitive method for quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. -72 : 248-254 (1976). 18. Spector, T. Refinement of the Comassie Blue method of protein quantitation. Anal. Biochem. -86 : 142-147 (1978). 19. Snedecor, G.W. and Cochran, W.G. Statistical methods, 6th ed. Iowa State Uni. Press, 1967, pp.104-127. 20. Witherspoon, D., Goldston, R. and Aslit, M. Uterine culture and biopsy in the mare. J.Am.Vet.Med.Assoc.161 : 1365-1366 (1972). 21. Peterson, F.B., McFeely, R.A. and David, J.S.E. Studies on the pathogenesis of endometritis in the mare. Proc. 15th Am. Assoc. Eq. Pratt., pp.279-287 (1969). 22. Bergman, R.V. and Kenney, R.M. Representativenessof a uterine biopsy of the mare. Proc.Zlst Am. Ass.Eq.Pract. pp.355-361 (1975).
448
MARCH 1983 VOL. 19 NO. 3