EFFECT OF SUSCEPTIBILITY TO ENDOMETRITIS

ON SPECIFIC ANTIBODY IN THE ENDOMETRIA OF MARES

E. D. Watson’ and C. R. Stokes2

‘Section of Reproductive Studies
School of Veterin Medicine, University of Pennsylvania
“E: ew Bolton Center
Kennett Square, PA 19348
‘Department of Veterina Medicine
University of Bristol School of Gyeterinary Science
Langford House, Ian ord
Bristol BS18 7DU, d K
Received for publication: November 20, 1989
Accepted: April 6, 1990

ABSTRACI
Endometrial biopsies were collected on two occasions from mares resistant
to (n = 3) and once from mares susceptible to persistent endometritis (n = 6). The
endometrial tissue was minced and cultured in vitro for 24 h. No hemolytic
complement activity was detected in the endometrial culture supernatant.
Endometrial culture su ematant from mares with persistent endometritis contained
titers of antibodies to & r eptococcus zooepidemicus similar to those from resistant
mares. However, the culture supematant of biopsies from mares with endometritis
was less effective (P < 0.05) at opsonizing % zooemdemicus in vitro.
Key words: mare, endometritis, specific antibody
INTRODUCTION
The ersistence of intrauterine infection in mares is a common cause of
subfertility P1). Susceptible mares become infected at parturition, or during
breeding or veterinary uterine examination and, instead of rapidly eliminating
invading microorganisms, remain infected and do not conceive (2).
The most common pathogen isolated from cases of endometritis is S,
Resistance to intrauterine infection requires an efficient
and although mobilization of neutrophils ap ears adequate in
phagocytosis by uterine neutrophrls may 1e defective (6,7).
system constitutes another Important uterine defense
mechanism. In most species, the systemic immune system is the major source of
immunoglobulins in genital tract secretions. In the mare, it appears that either local
s thesis of immunoglobulins or active transport from serum is more important
tK”
an passive diffusion (8). Concentrations of immunoglobulins in uterine secretions
from mares vary durm the estrous cycle (g-11), and hormone treatment of
ovariectomized mares af!ects endometrial antibody content (12). Mares susceptible
to persistent endometritis have higher concentrations of immunoglobulins present
____________________
.

This study was supported by the Horserace Betting Levy Board, London, UK.

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in uterine secretions than normal mares (9,13,14). This finding has cast doubt on
the functional capacity of immunoglobulins in the uterine secretrons of these mares,
but antibody titers to mfective organisms have not been measured to date.
In the resent study, we measured antibody response after intrauterine
inoculation of B. zooepidemia. Endometrial antibody content was assessed using
in vitro culture of tissue from resistant mares and from mares with persistent
endometritis. The capacity of o sonins in both serum and endometrial culture
supematant to opsonize S, zooeni
_B emicus was evaluated.
MATERIALS AND METHODS
Animals
Three ovariectomized mares were used which had no evidence of any
pathological changes on histological examination of endometrial biopsies. These
mares were being used in a related study on the effect of ovarian steroids on uterine
antibody content (12) and had been shown to be resistant to intrauterine infection
with S, Zs?oeDidemrcus Six other mares with intrauterine infections of S
ZooeDidemicus were studied. These mares were classified as having persistent
endometritis and had a history of subfertility. Examination of endometrial biopsies
revealed Category III endometria by the criteria of Kenney (15).
In Vitro Release of Immunoglobulins from Endometria
The procedure was modified from a method described
Biopsies were collected into serum-free incubation medium
pemcillin/streptomycin 5,000 units/ml; L-glutamine 200 mM/I; pH
me. The biopsies were dissected wrth a seal el into approximately 2-mm pieces.
The fragments were weighed and 300 mg oP tissue was suspended in 120 ml of
medium in a flat-bottomed incubation flaska and incubated at 37’C in an
atmosphere of 95% air : 5% CO2 After 24 h, the flask was frozen at -2OOC. When
the tissues were thawed, the resultant disruption of cells released the endogenous
immunoglobulins. The endometrial culture supematants were stored in aliquots at
-7ooc.
Preparation of s+ ZoocDidemicus Antigen
was inoculated into 1 1 of brain-heart infusion
broth am!
_W incubated for h at 37 C in an atmosphere of 95% air : 5% CO2 The
broth was centrifuged at 10,000 x g for 15 mm to pellet the bacteria. The bacteria
were then washed three times in sterile phosphate buffered saline PBS, pH 7.3).
The bacteria were resuspended in PBS and boiled in a water bath $or 1 h. After
washing the bacteria three times with 15 ml carbonate buffer (pH 9.6), the
supematants were pooled.
Quantitation of Specific Antibodies to S, zooeoidemicus
Specific IgG, IgM and IgA antibodies were measured by an indirect enzyme
linked immunosorbent assay (ELISA . All three classes were measured in
endometrial culture supematant and Ig& and IgM in serum.

a Titertek, Plow Laboratories, Irvine, UK.

40 JULY 1990 VOL. 34 NO. 1

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The assay was performed usin methods described previously (12). Twelve
five-fold dilutions of serum, and six Brve-fold dilutions of culture supematant were
placed individually in wells which had been coated with S, zooeuidem’w ’
A ositive standard (serum) was included in each assa . The antiseia uf$rg$
ei tfler aflimity- urilied sheep antisera (prepared in the e niversity of Bristol Schoq
of Veterinary 5 cience) to equine IgG or equine IgA, or rabbit anti-equine IgM.
These antrsera were shown to be monospecific for the heavy cham of their
respective isotypes. Alkaline phos hatase conjugates of pig antiserum to sheee IgG
or sheep antiserum to rabbit Ig 8 were added to each well. The substrate was
added after a final wash. olor was allowed and the plates were read on
a Titertek multiscan Mcs using the dual wavelen mode at 405 and 494 nm.
Background readings were obtained by the of incubation buffer in
indivrdual wells for the primary antiserum, the sample, and the antigen coating,
respectively.
Results were expressed as optical density for all portions of the experiment
which permitted assays to be performed on the same day. Otherwise, the titers were
calculated relative to the positive standard and presented as sample titer/standard
titer x 100%.
Bactericidal Assay
This was performed as described previously (17) using heat-treated (56OC for
30 min) endometrial culture supematant or dO% heat-treated horse serum a? the
opsonin (0.3 ml) for & zooepidemicus (3 x 10 /ml; 0.1 ml). Neutrophils (1 x 10 /ml;
0.2 ml from a geldin were added and the tubes were incubated on a roller at 37’C
for 2 h . Tri hcate drops (0.02 ml) of serial dilutions of the sus ension were then
laced on bPood agar plates and incubated overnight at 37’ 8 . The surviving
Eacteria were uantrtated by counting the number of colony-forming units. Results
were expresse 8 as a percenta
supematant was replaced by r!
by HEPES buffered Hanks balanced salt solution
included in which endometrial culture supematant was added, but the neutro hils
were replaced by HBSS and in which culture supematant was replaced by me dpium.
Before the endometrial culture su ematants were used in the assay, they were
dialyzed overnight at 4’C against PFlS pH 7.3 to remove antibiotics. The exclusion
limit on the dialysis tubing was a molecular weight of 15,000? Results used were
the mean of two replicate assays performed on separate days.
Hemolytic Complement Assay
Porcine red blood cells were sensitized with a subagglutinating titer of rabbit
anti-pig red blood cell serum and endometrial culture supematant (0.3 ml) was
added (18). The mixture was incubated at 37’C for 30 min. The degree of
hemolysis was calculated by using a spectrophotometer at 541 nm and comparing
the reading with a tube containing ammonia solution (0.04%) in which there was
100% hemolysis. Positive controls of serially diluted fresh horse serum were
included. Negative controls were included in which the antiserum or endometrial
culture supematant was replaced with buffer.

b Nordic Laboratories, Maidenhead? UK

cd Alkaline phosphatase substrate, Sigma, Poole, UK.
Plow Laboratories, Irvine, UK.
e Medicell Int’l Ltd., London, UK.

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Experimental Design
The three ovariectomized mares were treated daily for 14 d sequentially with
intramuscular injections (4 ml) of estradiol benzoate (1 mg) or oily vehicle (arachis
oil), with a period of 14 d between treatments. A previous study had shown that
trea.tment wrth estradi etrial antibody content or o sonizing
abrlrty of culture supem 7 of bqth.treatment errods, tKe mares
were infected with live S, x 10 ) m 50 ml P1 S b mtrauterme
infusion via a Foley 1 cuff). Endometrial 4:iopsies were
collected on Da 14. Endometrial swabs collected on Days 7, 10 and 14 were
streaked onto bPood agar plates which were then pcubated overnight at 37oC.
Blood samples were collected into evacuated tubes on Days 7 and 14, and the
serum was stored at -2OOC. Uterine flushings were centrifuged at 2000 g and stored
in aliquots at -2OOC.

ese mares and from another two persistently infected

mares were stored at -2OOC.
Statistical Analyses
Results were analyzed by a Student’s l-test.
RESULTS
When bacteria were opsonized with culture su ernatant, omission of
neutro hils resulted in 100% bacterial survival. Bacteria P survival was 96% when
uncon 1 itioned culture medium was added to bacteria in the presence of neutrophils.
Opsonization of bacteria usin culture supematant from endometrial biopsies of
susce tible mares resulted in kower bactencidal activity of neutrophils (54 + 4.5%,
P < g .05) than supematant from the biopsies of resistant mares (67 t 3.9%).
Opsonization of bacteria with serum from susceptible mares resulted in bactericidal
activity (61 + 8.1%) which was higher but not significantly different from that using
serum from resistant mares (49 + 4.1%). No hemolytic complement activity could
be detected in any of the endometrial culture supematants.
Titers of specific antibody to S, wrnicus in biopsy cultures from
susceptible mares were compared with titers measured in culture supematants from
endometrial biopsies of resistant mares. Titers to & ZooeDidemicuS m IgG, IgM and
IgA were similar in both groups of mares (Table 1).
Titers to & zooeDidemicus in I G were not lower in serum of susceptible
(OD : l.O_+ 0.05) than in resistant 6 .9 + 0.03) mares. Similarly, titers to S
zooeprdemru in IgM were not lower in susceptible (OD = 0.5 + 0.03) than in
resistant (0.6 + 0.03) mares.

f Becton-Dickinson, &ford, England.

42 JULY 1990 VOL. 34 NO. 1

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DISCUSSION
Our study showed that although titers of antibody to S, ~ were
similar in endometrial culture supematant from susceptible and resistant mares,
culture supematant from susceptible mares was less effective at opsonizing bacteria
than supematant from resistant mares.
Previous studies have shown that treatment of ovariectomized mares with
estradiol did not affect antibody titers or o sonizing capacity of endometrial culture
supematant (12). In our stud , mares witE persistent endometritis tended to have
hi her concentrations of antil!odies in their endometria. High concentrations of
Ig6 and IgA have been measured in the uterine secretions of mares with persistent
endometrrtis (9,13,14), but antibodies to S, zooeDidemict&$have not been previously
quantitated in uterine secretions.
Table 1. Titer (optical densi at 405 and 494 nm) of antibody to &
zooepidemicus (z + ?&I) m * immunoglobulins present in culture
supematant of endometrial tissue from resistant and susceptible
mares.

Titer in Immunoglobulin
# of # of
mares samples IgG IgA IgM

Resistant 3 ;;M& + 8.;;; 0.235 + 0.021 0.734 + 0.117

Susceptible 4 : . 2 * 0.266 + 0.018 0.791+ 0.062

Despite the apparently adequate concentrations of opsonizing antibody, the

bactericidal activity of neutro hils was significantly lower when they were suspended
in culture supematant of en cfometria from mares with persistent endometritis than
when suspended in culture supematant of endometrial tissue from resistant mares.
Not all subclasses of IgG act as opsonins. One subclass of IgG in horses, Ig , does
not ffl corn lement, does not mediate opsonization, and may inhibit camp “f ement
fmtion an B opsomzation by IgGab (19,20). Therefore, although titers of IgG
antibodies in endometrial tissue may have been equivalent between groups,
roportions of antibodies m IgG subclasses ma have differed. By contrast, serum
P,om susceptible mares tended to be more e&ective at opsonizm bacteria than
serum from resistant mares, but this did not reach sigmficance i ecause of the
variance in the six susceptible mares tested.
Despite the hemolytic activity previously measured in uterine flushings (18), no
hemolytic complement activity could be detected in endometrial culture
supematant.
It is important that these results be extended to include a greater number of
resistant and susceptible mares and to investigate possible deficiencies in uterine
humoral and cellular interactions in opsonization and phagocytosis of bacteria.

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