Directory UMM :Data Elmu:jurnal:S:Small Ruminant Research:Vol36.Issue1.Apr2000:
Small Ruminant Research 36 (2000) 7±15
Antisperm response in rams experimentally
infected with Brucella ovis
F.A. Paolicchia,b,*, P.A. Casarob, E.J. Gimenoc,
L.G. Kortebanid, A.B. Mazzollid
a
Department of Animal Production, Faculty of Agricultural Sciences, National University of Mar del Plata,
PO Box 276, (7620) Balcarce, Argentina
b
Department of Animal Production, National Institute of Agricultural Technology, Argentina
c
Institute of Pathology, Faculty of Veterinary Sciences, University of La Plata, PO Box 296, (1900) La Plata, Argentina
d
Center for Immunology, Section Sterility and Reproduction, Paunero 2810 (1024) Buenos Aires, Argentina
Accepted 4 August 1999
Abstract
Brucella ovis infection causes chronic epididymitis in rams and may result in the formation of spermatic granulomas and a
major reduction in fertility. To study the relationship between bacterial genital infection and antisperm response, 14 adult rams
were experimentally inoculated via conjunctival (n 7; G1) or preputial route (n 7; G2) with a B. ovis strain. Serological
response to B. ovis was evaluated by means of complement ®xation (CF) and gel diffusion (GD). Autoimmunity was estimated
by determination of antisperm antibodies (ASab) and the leukocyte migration inhibition test (MIT) using three different
antigens: autologous sperm, ovine testicle, and B. ovis antigen. Immune responses were analyzed in relation to clinical signs,
histopathological features, and bacterial isolations from semen and genital organs. Seven non-infected rams served as controls.
Speci®c antibody titers were detected by CF during the second week post-inoculation (PI) in 100% of the inoculated rams. The
CF values reached a peak during the 8th and 6th week PI for groups G1 and G2, respectively. The percentage of seropositive
animals decreased progressively in both groups until weeks 45 and 30 PI. Speci®c antisperm reaction was veri®ed by detection
of immobilizing ASab and by MIT. ASab were detected in the serum of 85.7% and 71.4% of rams in G1 and G2, respectively,
from week 3 PI onwards. Genital alterations were clinically detected in 71.4% of the inoculated rams. Gross epididymal
lesions consisted of multiple caseous masses and cysts with purulent content. In those rams that presented clinical lesions,
numerous spermatic granulomas were observed. 80% of these granulomas were located in the epididymis tail. Epididymitis,
seminal vesiculitis, and chronic ampullitis were histologically detected in 71.4% of the animals of G1 and G2. B. ovis was
isolated from 57% of semen cultures and was recovered from genital tissues in 75% of rams in both G1 and G2. At week 50
PI, four rams from each inoculated group (G1 and G2) received antibiotic treatment with long-acting oxytetracycline. In those
animals treated with antibiotics, all semen and tissue cultures became negative to B. ovis at the time of the necropsy. However,
cellular immunity was positive for autologous, testicular, and bacterial antigen and all rams demonstrated to be positive for
ASab until week 80 PI. These data showed that rams with genital lesions caused by B. ovis developed a long-standing
*
Corresponding author. Tel.: 54-2266-422040; fax: 54-2266-421756.
E-mail address: fpaolicchi@inta.gov.ar (F.A. Paolicchi).
0921-4488/00/$ ± see front matter # 2000 Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 1 0 8 - X
8
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
antisperm immune reaction. This autoimmune process could be signi®cant in the pathogenesis of reduced fertility observed in
B. ovis infected rams. # 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords: Rams; Male infertility; Genital infections; Brucella ovis; Antisperm autoimmunity
1. Introduction
There is increasing evidence that genital infections
in males are an important cause of infertility both in
animals and men (Fowler, 1981; Foster, 1987; Megory
et al., 1987; Eggert Kruse et al., 1996; Hinting et al.,
1996). Venereal infectious agents are characteristically protected or harboured along the genital tract
(Alexander and Anderson, 1987). Brucella ovis is a
facultative intracellular bacterium that causes epididymitis, seminal vesiculitis, and ampullitis in rams,
followed by orchitis and a progressive loss of the
animal's reproductive performance (Foster, 1987;
Ladds, 1993). Blasco (1990) described the serological
response against B. ovis in spontaneous and experimental infections. Cell-mediated immune responses
also have been evaluated in rams experimentally
infected with B. ovis (Afzal et al., 1986) and the
presence of several different types of immunoglobulin
and immunocompetent cells was demonstrated in
tissues and ¯uids of the male genital tract (Alexander
and Anderson, 1987; Foster et al., 1988). Shott and
Young (1971) described an autoimmune response in
rams induced by injection of a testicular±epididymal
homogenate. In humans, antisperm antibodies (ASab)
are considered one of the important factors in male
infertility (Yamamoto et al., 1996; Bates, 1997). In
male dogs with chronic B. canis infection, an antisperm response with development of serum and seminal plasma antibodies was detected (George and
Carmichael, 1984). However, the development of
antisperm autoimmune phenomena in rams infected
with B. ovis has not yet been described in detail. The
formation of sperm granulomas has been used to
indicate infertility in men with epididymal-orchitis
of infectious or traumatic origin, or in vasectomized
men (Hinting et al., 1996). The infectious agents
associated with these granulomas can act as adjuvants
in developing antisperm autoimmunity and consecutive sterility (Purvis and Christiansen, 1993).
This autoimmune response in men is characterized
by the presence of ASab, positive cellular immunity,
altered seminal quality, high counts of cells from
sperm progeny, and leukocytospermia in ejaculates
(Mazzolli et al., 1988; GonzaÂlez et al., 1992; Kamada
et al., 1998).
In this study, we used an experimental model of
genital infection with B. ovis in rams to evaluate the
speci®c antibacterial immune response and to compare these results with the appearance of post-infection (PI) antisperm immune reaction.
2. Materials and methods
2.1. Animals
21 adult Corriedale rams from a Brucellosis-free
¯ock were used. All animals were clinically normal,
and serologically and bacteriologically negative to B.
ovis, with normal seminal parameters determined two
weeks before the beginning of the experiment. Basal
level of ASab was determined in serum. Rams were
fed on natural pasture, alfalfa hay, maize-corn concentrate negative to mycotoxins or zearalenone, and
water ad-libitum.
2.2. Inoculum and experimental inoculation
The B. ovis strain 88E1 INTA, isolated from semen
of a ram with epididymitis, was used as inoculum. It
was cultivated on Columbia blood agar at 378C under
a 10% CO2 atmosphere for 6 days. The inoculum
concentration was adjusted to 3.5 1010 colony forming units (CFU) mlÿ1 using the technique described by
Miles and Misra (1938). The average CFU was calculated from the arithmetic means of duplicate plates
and the number of bacteria was expressed as log10.
Seven adult male rams were allocated at random to
any of three groups:
Group 1 (G1): conjunctival. The animals (n 7)
were each inoculated with 1 ml of inoculum
conjunctivally.
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Group 2 (G2): preputial. The rams (n 7) were
administered a 5 ml inoculum into the prepuce,
according to Webb et al. (1980).
Group 3 (G3): non-infected controls. Control rams
(n 7) were given the equivalent volume of sterile
PBS by both routes.
At week 50 four randomly selected animals from
each group (G1, G2 y G3) were treated with longacting oxytetracycline (Terramicina LA, P®zer Lab.,
Argentina) at a dose of 20 mg/kg every 72 h during 30
days to eliminate the B. ovis infection in the genital
tract. Each group was maintained in separated corrals
during the experimental period. All rams were killed
at week 80 PI for bacteriological and pathological
studies.
2.3. Clinical examination
All animals were clinically examined on inoculation day and sampled for blood and/or semen. Genital
organs were carefully observed and palpated.
2.4. Semen analysis
Semen samples were obtained through electroejaculation at weeks 4, 8, 12, 40, 60, 70, and 80 PI.
Smears were stained with May Grunwald±Giemsa
stain to evaluate sperm morphology, and also to detect
spermatic progeny cells and white blood cells (WBC).
The amount of WBC was determined in a 400
microscopic ®eld; it was quali®ed from 1 to 5 according to Kott et al. (1988). The smears were stained with
Gram and modi®ed Ziehl Neelsen (ZN) strain (Stamp
et al., 1950) to observe the presence of Brucella.
Aliquots of semen also were cultivated immediately
after collection. Each sample was cultivated on
Columbia blood agar, Skirrow agar, and modi®ed
Thayer Martin agar (Terzolo et al., 1991) at 378C
under a 10% CO2 atmosphere for 10 days. B. ovis was
identi®ed by the morphology of colonies and by Gram
and modi®ed ZN stains. Suspicious colonies were
referred to the Pan American Zoonosis Centre
(CEPANZO) in Buenos Aires for further typing by
standard procedures (Corbel and McHendry, 1985).
Aliquots of semen were centrifuged at 500G for
10 min. The sperm pellets obtained were stored at
ÿ208C; they were later employed as autologous
9
spermatic antigen in the cell mediated immunity test
(CMI).
2.5. Anti-Brucella serology
Blood samples were obtained at different PI periods: during the ®rst four weeks samples were taken
every 3 days, weekly from week 5 to 8, every 2 weeks
until week 16, and once a month until week 40. Three
additional blood samples were obtained from the
animals at 60, 70 and 80 weeks. Sera were tested
by complement ®xation (CF) and gel diffusion (GD)
tests for B. ovis. The antigen used was that described
by Myers et al. (1972).
2.6. Autoimmunity determinations
Antisperm antibodies (ASab): One drop of hemolysis-free inactivated serum (568C for 30 min) from
each animal was tested against one drop of semen
obtained from a normal donor ram [3 106 sperm/ml]
to identify antisperm immobilizing or agglutinating
antibodies as described by Mazzolli et al. (1983).
Those sera that agglutinated or immobilized 50% or
more of the present sperms were considered as positive at a dilution higher than 1 : 8, considering this as
basal ASab titer for all rams included in this study at
the beginning of the experiment.
Cell mediated immunity (CMI): This was evaluated
through the leukocyte migration inhibition test (MIT)
using heparinized peripheral blood samples (Mazzolli,
1971; Mancini et al., 1972). Three different antigens
were used: (a) autologous sperm (obtained from the
semen of each ram at the beginning of the experiment), (b) ovine testicular homogenate, and (c) bacterial antigen of the B. ovis 88E1 INTA strain.
Inhibition of migration was expressed as a percentage;
it was the mean value of several determinations per
animal (eight tubes). Inhibition levels of 25% or more
were considered signi®cant in our experimental conditions and indicated the presence of positive cellular
immunity.
2.7. Post-mortem examination
Samples for bacteriological determinations were
taken from testes, epididymides, seminal vesicles,
ampullae, bulbourethral, and prostatic glands. Each
10
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
tissue sample was ground separately and cultivated as
described above. Specimens from the same tissues
were ®xed in Bouin's, solution embedded in paraf®n,
cut at 5 mm, and stained using haematoxylin and eosin.
3.2. Anti-Brucella serology
All inoculated rams were positive to B. ovis for both
tests, i.e., GD and CF (1/10 or higher). All G1 rams
presented positive titers from week 3 PI onwards. The
maximum percentage of rams positive to CF was
reached from week 8 PI onwards, then descended
and became negative in some rams, or retained low
titers in others until week 80 PI.
57% of G2 animals had positive CF titers from week
3 PI onwards and all (100%) the G2 rams from week 6
PI onwards. The maximum percentage of positive CF
was reached at week 12 PI onwards. The percentage of
seropositive animals in G1 and G2 groups decreased
progressively until week 40 and 30 PI, respectively.
Disappearance of antibody titers coincided with
remission of epididymal lesions in 4 out of 10 rams
from both groups. After antibiotic treatments all
rams in G1 (n 4) and G2 (n 4) were CF negative,
(Fig. 1a and b).
2.8. Statistical evaluation
The geometric means of CF titers were calculated
for each group of rams. The simple linear correlation
was calculated at different moments of the experimental period between the percentage of rams with CF
and with ASab positive titers for each sampling time.
3. Results
3.1. Bacteriological and serological ®ndings
The results corresponding to G1 and G2 are shown
in Tables 1 and 2. Control rams (G3) were clinically
normal, serologically negative for GD and CF antiBrucella, ASab and CMI. They had neither macroscopic nor histological lesions in their genital organs.
All the bacteriological cultures of semen and tissues
were negative.
3.3. ASab and cell mediated immunity
Positive immobilizing ASab were detected in
85.7% and 71.4% of ram serum from groups G1
Table 1
Serologic anti-Brucella titers (CF), presence of ASab, and isolates of B.ovis of genital tissues in rams inoculated via conjunctival (G1) or
preputial route (G2)a
Inoculation
route
Ram
No.
Conjunctival
Preputial
a
Serology
Brucella ovis isolation
CFb
ASab
(>1/8)
Epididymal
tail
Epidiymal
head
Seminal
vesicle
Ampullae
Testicle
Bulbouretral
gland
1
2
3
4c
5c
6c
7c
1/320
1/80
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
8
9
10
11c
12c
13c
14c
1/320
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Results at the time of necropsy, 80 weeks post-inoculation.
Complement ®xation reciprocal titers (>1/10).
c
Rams treated with oxytetracycline (20 mg/kg) during weeks 50±60 post-inoculation.
b
ÿ
ÿ
ÿ
11
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Table 2
Serologic anti-Brucella titers (CF), presence of ASab, and isolates of B. ovis of semen (BoS) or genital tissues (BoGT) in rams inoculated via
conjunctival (G1) or preputial route (G2)
Inoculation route
Conjunctival
Preputial
a
b
Ram No.
During experimental period
At necropsy
CFa
ASab (>1/8)
BoS
CF
ASab (>1/8)
BoGT
1
2
3
4b
5b
6b
7b
1/320
1/320
1/320
1/80
1/80
1/40
1/320
ÿ
1
ÿ
ÿ
1/320
1/80
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
8
9
10
11b
12b
13b
14b
1/320
1/320
1/10
1/80
1/40
1/160
1/320
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
1/320
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Complement ®xation, maximal reciprocal titers.
Rams treated with oxytetracycline (20 mg/kg) during weeks 50±60 post-inoculation.
and G2 respectively, starting from week 3 PI onwards.
In G1 rams the positive ASab decreased to 42.8% from
week 6 to 15 PI, whereas in animals from G2 ASab
diminished in week 30 PI and kept their positive titers
until week 80 PI. Simultaneously, the percentage of
rams with positive anti-Brucella antibodies decreased
until all were negative. In all rams from both groups
G1 and G2 a negative correlation between number of
rams with positive CF titers and positive ASab titers
was determined at the beginning (10±20 weeks postinoculation) and at the end of the experimental time
(60±80 weeks post-inoculation). In 7/8 of the inoculated animals treated with antibiotics, the cellular
immunity was positive (25% or more CMI) for spermatic autologous, testicular, and bacterial B. ovis
antigens until week 80 PI. These rams showed ASab
positive, but negative anti-Brucella titers and negative
isolation of B. ovis.
3.4. Semen examination
In¯ammatory WBC were detected in the semen of
71.4% of the rams in both G1 and G2 and coincided
with clinical detection of testicular±epididymal
lesions. The score reached 4 and 5 (5±10 and >10
cells values for microscopic ®eld respectively) in all
cases and remained unchanged during the assay. The
semen presented oligospermy and spermatic progeny
cells increased in 71.4% and 66%, in G1 and G2 rams
respectively, at week 9 PI. B. ovis was initially recovered from semen of two G2 rams (29%) at week 4 PI,
but from week 8 PI onwards it was recovered from
57% of the animals in each group. In both groups, B.
ovis was recovered from the genital tissues of 43% and
29% in G1 and G2 rams, respectively. Occasionally,
the ZN technique applied to seminal smears showed B.
ovis in phagocytic cells or around them. Neither WBC
nor bacteria were detected in the semen of antibiotic
treated rams.
3.5. Clinical and pathological ®ndings
Genital alterations were detected in 71.4% of the
inoculated rams. Two rams belonging to G1 showed
bilateral epididymal lesions and another two animals
presented unilateral lesions from the 6th week PI
onwards. The other rams manifested epididymitis
from week 8 onwards. Two animals from group G2
with clinical lesions from week 3 onwards were
detected while the others showed lesions from week
6 onwards. The lesions comprised testicular adherences, deformation and increase in epididymal tone.
12
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Fig. 1. Percentage of seropositive rams for anti-Brucella ovis (CF 1/10) and antispermatic antibodies (ASab 1/8) in eight rams after: (a)
conjunctival (G1) and (b) preputial (G2) experimental inoculation with B. ovis during 80 weeks. Antibiotic treatment was administered on
week 50 in four rams in both G1 and G2.
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Multiple caseous masses and cysts with purulent
contents were observed in the sectioned epididymis.
Epididymitis, seminal vesiculitis, and chronic ampullitis were observed histologically in 71.4% of the
animals of G1 and G2 in coincidence with the presence of macroscopic lesions. In those rams that
presented clinical lesions, numerous spermatic granulomas, 80% of them in the tail of the epididymis,
were observed as a mass of neutrophils, macrophages
with phagocyted spermatozoa, and giant cells in direct
contact with mononuclear cells. Degeneration, atrophy, and mineralization were frequently found in
testicles. The histological ®ndings were similar to
those presented by other authors in cases of epididymitis due to B. ovis (Biberstein et al., 1964; Shott and
Young, 1971; Searson, 1987; Foster, 1987; Blasco,
1990; Ladds, 1993).
4. Discussion
The results of the present study indicate that genital
brucellosis in rams is associated with a long-standing
antispermatic response that could be con®rmed by the
presence of ASab and CMI against autologous sperm
and testicular tissue. The inoculations via mucosal
routes was very effective is establishing the disease.
This was con®rmed by a high percentage of rams
with speci®c positive titers to B. ovis, the isolation
of the agent from semen and genital tissues, as well
as by the development of typical lesions. Similar
®ndings were reported by other authors in both
naturally and experimentally B. ovis infected rams
(Shott and Young, 1971; Afzal et al., 1986; Foster
et al., 1988).
Allergic orchiepididymitis has been induced in
bulls and guinea pigs (Parsonson et al., 1971;
Mazzolli, 1971) after inoculation of testicular
homogenates, with lesions similar to those of our
study. The exposure of the immune system to
spermatic antigens, as a result of trauma or infection
in the genital tract, stimulates a high titer of ASab
production that could persist for many years
(Alexander and Anderson, 1987; Purvis and
Christiansen, 1993; Hinting et al., 1996).
The presence of severe macroscopic and histological lesions in the genital tissues of rams infected with
B. ovis indicates a possible break in the immune
13
tolerance and a response to spermatic autosensitization. Infection does not necessarily initiate an autoimmune phenomenon, but it is known that infections
in the genital tract are often associated with positive
cellular immunity, and ASab in animals as well as in
men (Afzal et al., 1986; Foster, 1987; Ladds, 1993;
Hinting et al., 1996). Dogs chronically infected with
B. canis with a bacteremia lasting for more than four
months had cutaneous delayed-type hypersensitivity
reactions when tested with soluble canine testicular
extracts. Those dogs with testicular atrophy had the
most severe skin test responses. Seemingly, isoimmune responses to sperm antigens are involved in
infertility caused by B. canis infection of male dogs
(George and Carmichael, 1984).
Although antibodies do not necessarily mean infertility, many cases showed a close correlation between
reproductive tract infection and autosensitization with
positive cellular immunity and ASab in serum, seminal plasma or both (Mazzolli and Barrera, 1989).
Moreover, ASab in infertile men were inversely correlated with gestation percentage (Rumke et al.,
1974), conception rate (Fuchs and Alexander,
1983), poor initial spermatic motility, and decreased
cervical mucus penetration (Mathur et al., 1986). The
ASab were demonstrated in men with prostatitis,
positive seminal cultures, and testicular obstruction
(Hendry, 1989). Obstruction of the male genital tract,
whatever the cause, is often associated with antisperm
immune reaction and infertility (Hinting et al., 1996;
Yamamoto et al., 1996).
GonzaÂlez et al., 1992 found that 50% of men with
ASab in the seminal plasma showed astenozoospermia, teratozoospermia, leukocytospermia, and hypofunction of seminal vesicles with high prevalence of
IgA in the seminal plasma. Also, chronic genital
infection could have an adjuvant role in spermatic
autoimmunity (Purvis and Christiansen, 1993). Shott
and Young (1971) attempted to demonstrate the
contribution made to the pathological process by
addition of testicular±epididymal homogenates in
rams inoculated with B. ovis, but they did not ®nd
a correlation between the pathological and the
immunological processes. However, other authors
communicate that B. canis infection in dogs induced
an immune response of variable intensity against
spermatozoa due to absorption of sperm antigens
(Serikawa et al., 1984).
14
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Male Beagles infected with B. canis for 3 months or
more developed serum antibodies that agglutinated
normal canine spermatozoa, and antibodies were
observed in seminal plasma of chronically infected
dogs (George and Carmichael, 1984). In our work, the
antispermatic response in rams had a similar behaviour in both groups (G1 and G2) and coincided with
the presentation of epididymal lesions. Moreover, with
the antibiotic treatment a gradual decrease of titers
against B. ovis as well as the elimination of the
bacterium in semen and genital tissues allowed the
identi®cation of the rams with positive ASab and
cellular immune responses. The ASab were detected
early after infection, remaining higher than the basal
titer during a long period of time, as could be observed
for 80 weeks PI. In men presenting obstruction and/or
infection of genital organs, ASab have been considered the cause of prolonged sterility and the period of
infertility was estimated to be of 5 years in average
(Hinting et al., 1996).
The relevant leukocytospermia together with the
alterations in seminal quality observed in our study
coincides with the ®ndings of other authors in similar
experimental studies (Kott et al., 1988; Kortebani
et al., 1992). It was demonstrated that leukocytes
produce cytokines, tumor necrosis factor-a and free
radicals, and also reduce motility and the fertilizing
potential of spermatozoa (Tomlinson et al., 1992). In
fact, polymorphonuclear leukocytes are a major
source of harmful oxygen-derived free radicals, especially after interaction with bacterial products and
cytokines (Wang et al., 1997). Additionally, B. ovis
infection in rams modi®ed the carbohydrate composition of organs participating in maturation, transport,
and storage of spermatozoa. Glycoconjugates are
instrumental in receptor±ligand interactions. These
alterations have been postulated to be involved in
the impaired fertility observed in B. ovis infected rams
(Paolicchi et al., 1995).
Today, ASab detection is one of the most important
steps in the evaluation of infertility in men (Dondero et
al., 1997; Lenzi et al., 1997), and ASab could become
an important tool in the diagnosis of animal infertility.
Continued investigation of infertility in rams will lead
to novel treatment regimens as well as to the development of new prophylactic measures. Finally, this
ovine disease could be a useful model in comparative
pathology and biomedical research.
Acknowledgements
The authors would like to thank Dr. C. GarcõÂaCarrillo and Dr. N. Lucero for biotyping of the Brucella strains. We also thank Dr. C. Campero, Dr. H.
Terzolo, and the staff of the histology and bacteriology
laboratories for their collaboration with our work. We
wish to thank Mr. R. Gregoret and Ms. E. Piazza
(INTA Castelar, Argentina) for their assistance in GD
and CF serologic diagnostics. We thank Dr. Silvina
Viviana Macnie for skilled assistance in language
revision. This work was partially supported by a grant
from the National Research Council (CONICET),
Argentina to FAP. EJG is a Research Career Member
of CONICET.
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Nagoya J. Med. Sci. 59, 25±29.
Antisperm response in rams experimentally
infected with Brucella ovis
F.A. Paolicchia,b,*, P.A. Casarob, E.J. Gimenoc,
L.G. Kortebanid, A.B. Mazzollid
a
Department of Animal Production, Faculty of Agricultural Sciences, National University of Mar del Plata,
PO Box 276, (7620) Balcarce, Argentina
b
Department of Animal Production, National Institute of Agricultural Technology, Argentina
c
Institute of Pathology, Faculty of Veterinary Sciences, University of La Plata, PO Box 296, (1900) La Plata, Argentina
d
Center for Immunology, Section Sterility and Reproduction, Paunero 2810 (1024) Buenos Aires, Argentina
Accepted 4 August 1999
Abstract
Brucella ovis infection causes chronic epididymitis in rams and may result in the formation of spermatic granulomas and a
major reduction in fertility. To study the relationship between bacterial genital infection and antisperm response, 14 adult rams
were experimentally inoculated via conjunctival (n 7; G1) or preputial route (n 7; G2) with a B. ovis strain. Serological
response to B. ovis was evaluated by means of complement ®xation (CF) and gel diffusion (GD). Autoimmunity was estimated
by determination of antisperm antibodies (ASab) and the leukocyte migration inhibition test (MIT) using three different
antigens: autologous sperm, ovine testicle, and B. ovis antigen. Immune responses were analyzed in relation to clinical signs,
histopathological features, and bacterial isolations from semen and genital organs. Seven non-infected rams served as controls.
Speci®c antibody titers were detected by CF during the second week post-inoculation (PI) in 100% of the inoculated rams. The
CF values reached a peak during the 8th and 6th week PI for groups G1 and G2, respectively. The percentage of seropositive
animals decreased progressively in both groups until weeks 45 and 30 PI. Speci®c antisperm reaction was veri®ed by detection
of immobilizing ASab and by MIT. ASab were detected in the serum of 85.7% and 71.4% of rams in G1 and G2, respectively,
from week 3 PI onwards. Genital alterations were clinically detected in 71.4% of the inoculated rams. Gross epididymal
lesions consisted of multiple caseous masses and cysts with purulent content. In those rams that presented clinical lesions,
numerous spermatic granulomas were observed. 80% of these granulomas were located in the epididymis tail. Epididymitis,
seminal vesiculitis, and chronic ampullitis were histologically detected in 71.4% of the animals of G1 and G2. B. ovis was
isolated from 57% of semen cultures and was recovered from genital tissues in 75% of rams in both G1 and G2. At week 50
PI, four rams from each inoculated group (G1 and G2) received antibiotic treatment with long-acting oxytetracycline. In those
animals treated with antibiotics, all semen and tissue cultures became negative to B. ovis at the time of the necropsy. However,
cellular immunity was positive for autologous, testicular, and bacterial antigen and all rams demonstrated to be positive for
ASab until week 80 PI. These data showed that rams with genital lesions caused by B. ovis developed a long-standing
*
Corresponding author. Tel.: 54-2266-422040; fax: 54-2266-421756.
E-mail address: fpaolicchi@inta.gov.ar (F.A. Paolicchi).
0921-4488/00/$ ± see front matter # 2000 Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 1 0 8 - X
8
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
antisperm immune reaction. This autoimmune process could be signi®cant in the pathogenesis of reduced fertility observed in
B. ovis infected rams. # 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords: Rams; Male infertility; Genital infections; Brucella ovis; Antisperm autoimmunity
1. Introduction
There is increasing evidence that genital infections
in males are an important cause of infertility both in
animals and men (Fowler, 1981; Foster, 1987; Megory
et al., 1987; Eggert Kruse et al., 1996; Hinting et al.,
1996). Venereal infectious agents are characteristically protected or harboured along the genital tract
(Alexander and Anderson, 1987). Brucella ovis is a
facultative intracellular bacterium that causes epididymitis, seminal vesiculitis, and ampullitis in rams,
followed by orchitis and a progressive loss of the
animal's reproductive performance (Foster, 1987;
Ladds, 1993). Blasco (1990) described the serological
response against B. ovis in spontaneous and experimental infections. Cell-mediated immune responses
also have been evaluated in rams experimentally
infected with B. ovis (Afzal et al., 1986) and the
presence of several different types of immunoglobulin
and immunocompetent cells was demonstrated in
tissues and ¯uids of the male genital tract (Alexander
and Anderson, 1987; Foster et al., 1988). Shott and
Young (1971) described an autoimmune response in
rams induced by injection of a testicular±epididymal
homogenate. In humans, antisperm antibodies (ASab)
are considered one of the important factors in male
infertility (Yamamoto et al., 1996; Bates, 1997). In
male dogs with chronic B. canis infection, an antisperm response with development of serum and seminal plasma antibodies was detected (George and
Carmichael, 1984). However, the development of
antisperm autoimmune phenomena in rams infected
with B. ovis has not yet been described in detail. The
formation of sperm granulomas has been used to
indicate infertility in men with epididymal-orchitis
of infectious or traumatic origin, or in vasectomized
men (Hinting et al., 1996). The infectious agents
associated with these granulomas can act as adjuvants
in developing antisperm autoimmunity and consecutive sterility (Purvis and Christiansen, 1993).
This autoimmune response in men is characterized
by the presence of ASab, positive cellular immunity,
altered seminal quality, high counts of cells from
sperm progeny, and leukocytospermia in ejaculates
(Mazzolli et al., 1988; GonzaÂlez et al., 1992; Kamada
et al., 1998).
In this study, we used an experimental model of
genital infection with B. ovis in rams to evaluate the
speci®c antibacterial immune response and to compare these results with the appearance of post-infection (PI) antisperm immune reaction.
2. Materials and methods
2.1. Animals
21 adult Corriedale rams from a Brucellosis-free
¯ock were used. All animals were clinically normal,
and serologically and bacteriologically negative to B.
ovis, with normal seminal parameters determined two
weeks before the beginning of the experiment. Basal
level of ASab was determined in serum. Rams were
fed on natural pasture, alfalfa hay, maize-corn concentrate negative to mycotoxins or zearalenone, and
water ad-libitum.
2.2. Inoculum and experimental inoculation
The B. ovis strain 88E1 INTA, isolated from semen
of a ram with epididymitis, was used as inoculum. It
was cultivated on Columbia blood agar at 378C under
a 10% CO2 atmosphere for 6 days. The inoculum
concentration was adjusted to 3.5 1010 colony forming units (CFU) mlÿ1 using the technique described by
Miles and Misra (1938). The average CFU was calculated from the arithmetic means of duplicate plates
and the number of bacteria was expressed as log10.
Seven adult male rams were allocated at random to
any of three groups:
Group 1 (G1): conjunctival. The animals (n 7)
were each inoculated with 1 ml of inoculum
conjunctivally.
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Group 2 (G2): preputial. The rams (n 7) were
administered a 5 ml inoculum into the prepuce,
according to Webb et al. (1980).
Group 3 (G3): non-infected controls. Control rams
(n 7) were given the equivalent volume of sterile
PBS by both routes.
At week 50 four randomly selected animals from
each group (G1, G2 y G3) were treated with longacting oxytetracycline (Terramicina LA, P®zer Lab.,
Argentina) at a dose of 20 mg/kg every 72 h during 30
days to eliminate the B. ovis infection in the genital
tract. Each group was maintained in separated corrals
during the experimental period. All rams were killed
at week 80 PI for bacteriological and pathological
studies.
2.3. Clinical examination
All animals were clinically examined on inoculation day and sampled for blood and/or semen. Genital
organs were carefully observed and palpated.
2.4. Semen analysis
Semen samples were obtained through electroejaculation at weeks 4, 8, 12, 40, 60, 70, and 80 PI.
Smears were stained with May Grunwald±Giemsa
stain to evaluate sperm morphology, and also to detect
spermatic progeny cells and white blood cells (WBC).
The amount of WBC was determined in a 400
microscopic ®eld; it was quali®ed from 1 to 5 according to Kott et al. (1988). The smears were stained with
Gram and modi®ed Ziehl Neelsen (ZN) strain (Stamp
et al., 1950) to observe the presence of Brucella.
Aliquots of semen also were cultivated immediately
after collection. Each sample was cultivated on
Columbia blood agar, Skirrow agar, and modi®ed
Thayer Martin agar (Terzolo et al., 1991) at 378C
under a 10% CO2 atmosphere for 10 days. B. ovis was
identi®ed by the morphology of colonies and by Gram
and modi®ed ZN stains. Suspicious colonies were
referred to the Pan American Zoonosis Centre
(CEPANZO) in Buenos Aires for further typing by
standard procedures (Corbel and McHendry, 1985).
Aliquots of semen were centrifuged at 500G for
10 min. The sperm pellets obtained were stored at
ÿ208C; they were later employed as autologous
9
spermatic antigen in the cell mediated immunity test
(CMI).
2.5. Anti-Brucella serology
Blood samples were obtained at different PI periods: during the ®rst four weeks samples were taken
every 3 days, weekly from week 5 to 8, every 2 weeks
until week 16, and once a month until week 40. Three
additional blood samples were obtained from the
animals at 60, 70 and 80 weeks. Sera were tested
by complement ®xation (CF) and gel diffusion (GD)
tests for B. ovis. The antigen used was that described
by Myers et al. (1972).
2.6. Autoimmunity determinations
Antisperm antibodies (ASab): One drop of hemolysis-free inactivated serum (568C for 30 min) from
each animal was tested against one drop of semen
obtained from a normal donor ram [3 106 sperm/ml]
to identify antisperm immobilizing or agglutinating
antibodies as described by Mazzolli et al. (1983).
Those sera that agglutinated or immobilized 50% or
more of the present sperms were considered as positive at a dilution higher than 1 : 8, considering this as
basal ASab titer for all rams included in this study at
the beginning of the experiment.
Cell mediated immunity (CMI): This was evaluated
through the leukocyte migration inhibition test (MIT)
using heparinized peripheral blood samples (Mazzolli,
1971; Mancini et al., 1972). Three different antigens
were used: (a) autologous sperm (obtained from the
semen of each ram at the beginning of the experiment), (b) ovine testicular homogenate, and (c) bacterial antigen of the B. ovis 88E1 INTA strain.
Inhibition of migration was expressed as a percentage;
it was the mean value of several determinations per
animal (eight tubes). Inhibition levels of 25% or more
were considered signi®cant in our experimental conditions and indicated the presence of positive cellular
immunity.
2.7. Post-mortem examination
Samples for bacteriological determinations were
taken from testes, epididymides, seminal vesicles,
ampullae, bulbourethral, and prostatic glands. Each
10
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
tissue sample was ground separately and cultivated as
described above. Specimens from the same tissues
were ®xed in Bouin's, solution embedded in paraf®n,
cut at 5 mm, and stained using haematoxylin and eosin.
3.2. Anti-Brucella serology
All inoculated rams were positive to B. ovis for both
tests, i.e., GD and CF (1/10 or higher). All G1 rams
presented positive titers from week 3 PI onwards. The
maximum percentage of rams positive to CF was
reached from week 8 PI onwards, then descended
and became negative in some rams, or retained low
titers in others until week 80 PI.
57% of G2 animals had positive CF titers from week
3 PI onwards and all (100%) the G2 rams from week 6
PI onwards. The maximum percentage of positive CF
was reached at week 12 PI onwards. The percentage of
seropositive animals in G1 and G2 groups decreased
progressively until week 40 and 30 PI, respectively.
Disappearance of antibody titers coincided with
remission of epididymal lesions in 4 out of 10 rams
from both groups. After antibiotic treatments all
rams in G1 (n 4) and G2 (n 4) were CF negative,
(Fig. 1a and b).
2.8. Statistical evaluation
The geometric means of CF titers were calculated
for each group of rams. The simple linear correlation
was calculated at different moments of the experimental period between the percentage of rams with CF
and with ASab positive titers for each sampling time.
3. Results
3.1. Bacteriological and serological ®ndings
The results corresponding to G1 and G2 are shown
in Tables 1 and 2. Control rams (G3) were clinically
normal, serologically negative for GD and CF antiBrucella, ASab and CMI. They had neither macroscopic nor histological lesions in their genital organs.
All the bacteriological cultures of semen and tissues
were negative.
3.3. ASab and cell mediated immunity
Positive immobilizing ASab were detected in
85.7% and 71.4% of ram serum from groups G1
Table 1
Serologic anti-Brucella titers (CF), presence of ASab, and isolates of B.ovis of genital tissues in rams inoculated via conjunctival (G1) or
preputial route (G2)a
Inoculation
route
Ram
No.
Conjunctival
Preputial
a
Serology
Brucella ovis isolation
CFb
ASab
(>1/8)
Epididymal
tail
Epidiymal
head
Seminal
vesicle
Ampullae
Testicle
Bulbouretral
gland
1
2
3
4c
5c
6c
7c
1/320
1/80
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
8
9
10
11c
12c
13c
14c
1/320
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Results at the time of necropsy, 80 weeks post-inoculation.
Complement ®xation reciprocal titers (>1/10).
c
Rams treated with oxytetracycline (20 mg/kg) during weeks 50±60 post-inoculation.
b
ÿ
ÿ
ÿ
11
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Table 2
Serologic anti-Brucella titers (CF), presence of ASab, and isolates of B. ovis of semen (BoS) or genital tissues (BoGT) in rams inoculated via
conjunctival (G1) or preputial route (G2)
Inoculation route
Conjunctival
Preputial
a
b
Ram No.
During experimental period
At necropsy
CFa
ASab (>1/8)
BoS
CF
ASab (>1/8)
BoGT
1
2
3
4b
5b
6b
7b
1/320
1/320
1/320
1/80
1/80
1/40
1/320
ÿ
1
ÿ
ÿ
1/320
1/80
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
8
9
10
11b
12b
13b
14b
1/320
1/320
1/10
1/80
1/40
1/160
1/320
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
1/320
1/40
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Complement ®xation, maximal reciprocal titers.
Rams treated with oxytetracycline (20 mg/kg) during weeks 50±60 post-inoculation.
and G2 respectively, starting from week 3 PI onwards.
In G1 rams the positive ASab decreased to 42.8% from
week 6 to 15 PI, whereas in animals from G2 ASab
diminished in week 30 PI and kept their positive titers
until week 80 PI. Simultaneously, the percentage of
rams with positive anti-Brucella antibodies decreased
until all were negative. In all rams from both groups
G1 and G2 a negative correlation between number of
rams with positive CF titers and positive ASab titers
was determined at the beginning (10±20 weeks postinoculation) and at the end of the experimental time
(60±80 weeks post-inoculation). In 7/8 of the inoculated animals treated with antibiotics, the cellular
immunity was positive (25% or more CMI) for spermatic autologous, testicular, and bacterial B. ovis
antigens until week 80 PI. These rams showed ASab
positive, but negative anti-Brucella titers and negative
isolation of B. ovis.
3.4. Semen examination
In¯ammatory WBC were detected in the semen of
71.4% of the rams in both G1 and G2 and coincided
with clinical detection of testicular±epididymal
lesions. The score reached 4 and 5 (5±10 and >10
cells values for microscopic ®eld respectively) in all
cases and remained unchanged during the assay. The
semen presented oligospermy and spermatic progeny
cells increased in 71.4% and 66%, in G1 and G2 rams
respectively, at week 9 PI. B. ovis was initially recovered from semen of two G2 rams (29%) at week 4 PI,
but from week 8 PI onwards it was recovered from
57% of the animals in each group. In both groups, B.
ovis was recovered from the genital tissues of 43% and
29% in G1 and G2 rams, respectively. Occasionally,
the ZN technique applied to seminal smears showed B.
ovis in phagocytic cells or around them. Neither WBC
nor bacteria were detected in the semen of antibiotic
treated rams.
3.5. Clinical and pathological ®ndings
Genital alterations were detected in 71.4% of the
inoculated rams. Two rams belonging to G1 showed
bilateral epididymal lesions and another two animals
presented unilateral lesions from the 6th week PI
onwards. The other rams manifested epididymitis
from week 8 onwards. Two animals from group G2
with clinical lesions from week 3 onwards were
detected while the others showed lesions from week
6 onwards. The lesions comprised testicular adherences, deformation and increase in epididymal tone.
12
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Fig. 1. Percentage of seropositive rams for anti-Brucella ovis (CF 1/10) and antispermatic antibodies (ASab 1/8) in eight rams after: (a)
conjunctival (G1) and (b) preputial (G2) experimental inoculation with B. ovis during 80 weeks. Antibiotic treatment was administered on
week 50 in four rams in both G1 and G2.
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Multiple caseous masses and cysts with purulent
contents were observed in the sectioned epididymis.
Epididymitis, seminal vesiculitis, and chronic ampullitis were observed histologically in 71.4% of the
animals of G1 and G2 in coincidence with the presence of macroscopic lesions. In those rams that
presented clinical lesions, numerous spermatic granulomas, 80% of them in the tail of the epididymis,
were observed as a mass of neutrophils, macrophages
with phagocyted spermatozoa, and giant cells in direct
contact with mononuclear cells. Degeneration, atrophy, and mineralization were frequently found in
testicles. The histological ®ndings were similar to
those presented by other authors in cases of epididymitis due to B. ovis (Biberstein et al., 1964; Shott and
Young, 1971; Searson, 1987; Foster, 1987; Blasco,
1990; Ladds, 1993).
4. Discussion
The results of the present study indicate that genital
brucellosis in rams is associated with a long-standing
antispermatic response that could be con®rmed by the
presence of ASab and CMI against autologous sperm
and testicular tissue. The inoculations via mucosal
routes was very effective is establishing the disease.
This was con®rmed by a high percentage of rams
with speci®c positive titers to B. ovis, the isolation
of the agent from semen and genital tissues, as well
as by the development of typical lesions. Similar
®ndings were reported by other authors in both
naturally and experimentally B. ovis infected rams
(Shott and Young, 1971; Afzal et al., 1986; Foster
et al., 1988).
Allergic orchiepididymitis has been induced in
bulls and guinea pigs (Parsonson et al., 1971;
Mazzolli, 1971) after inoculation of testicular
homogenates, with lesions similar to those of our
study. The exposure of the immune system to
spermatic antigens, as a result of trauma or infection
in the genital tract, stimulates a high titer of ASab
production that could persist for many years
(Alexander and Anderson, 1987; Purvis and
Christiansen, 1993; Hinting et al., 1996).
The presence of severe macroscopic and histological lesions in the genital tissues of rams infected with
B. ovis indicates a possible break in the immune
13
tolerance and a response to spermatic autosensitization. Infection does not necessarily initiate an autoimmune phenomenon, but it is known that infections
in the genital tract are often associated with positive
cellular immunity, and ASab in animals as well as in
men (Afzal et al., 1986; Foster, 1987; Ladds, 1993;
Hinting et al., 1996). Dogs chronically infected with
B. canis with a bacteremia lasting for more than four
months had cutaneous delayed-type hypersensitivity
reactions when tested with soluble canine testicular
extracts. Those dogs with testicular atrophy had the
most severe skin test responses. Seemingly, isoimmune responses to sperm antigens are involved in
infertility caused by B. canis infection of male dogs
(George and Carmichael, 1984).
Although antibodies do not necessarily mean infertility, many cases showed a close correlation between
reproductive tract infection and autosensitization with
positive cellular immunity and ASab in serum, seminal plasma or both (Mazzolli and Barrera, 1989).
Moreover, ASab in infertile men were inversely correlated with gestation percentage (Rumke et al.,
1974), conception rate (Fuchs and Alexander,
1983), poor initial spermatic motility, and decreased
cervical mucus penetration (Mathur et al., 1986). The
ASab were demonstrated in men with prostatitis,
positive seminal cultures, and testicular obstruction
(Hendry, 1989). Obstruction of the male genital tract,
whatever the cause, is often associated with antisperm
immune reaction and infertility (Hinting et al., 1996;
Yamamoto et al., 1996).
GonzaÂlez et al., 1992 found that 50% of men with
ASab in the seminal plasma showed astenozoospermia, teratozoospermia, leukocytospermia, and hypofunction of seminal vesicles with high prevalence of
IgA in the seminal plasma. Also, chronic genital
infection could have an adjuvant role in spermatic
autoimmunity (Purvis and Christiansen, 1993). Shott
and Young (1971) attempted to demonstrate the
contribution made to the pathological process by
addition of testicular±epididymal homogenates in
rams inoculated with B. ovis, but they did not ®nd
a correlation between the pathological and the
immunological processes. However, other authors
communicate that B. canis infection in dogs induced
an immune response of variable intensity against
spermatozoa due to absorption of sperm antigens
(Serikawa et al., 1984).
14
F.A. Paolicchi et al. / Small Ruminant Research 36 (2000) 7±15
Male Beagles infected with B. canis for 3 months or
more developed serum antibodies that agglutinated
normal canine spermatozoa, and antibodies were
observed in seminal plasma of chronically infected
dogs (George and Carmichael, 1984). In our work, the
antispermatic response in rams had a similar behaviour in both groups (G1 and G2) and coincided with
the presentation of epididymal lesions. Moreover, with
the antibiotic treatment a gradual decrease of titers
against B. ovis as well as the elimination of the
bacterium in semen and genital tissues allowed the
identi®cation of the rams with positive ASab and
cellular immune responses. The ASab were detected
early after infection, remaining higher than the basal
titer during a long period of time, as could be observed
for 80 weeks PI. In men presenting obstruction and/or
infection of genital organs, ASab have been considered the cause of prolonged sterility and the period of
infertility was estimated to be of 5 years in average
(Hinting et al., 1996).
The relevant leukocytospermia together with the
alterations in seminal quality observed in our study
coincides with the ®ndings of other authors in similar
experimental studies (Kott et al., 1988; Kortebani
et al., 1992). It was demonstrated that leukocytes
produce cytokines, tumor necrosis factor-a and free
radicals, and also reduce motility and the fertilizing
potential of spermatozoa (Tomlinson et al., 1992). In
fact, polymorphonuclear leukocytes are a major
source of harmful oxygen-derived free radicals, especially after interaction with bacterial products and
cytokines (Wang et al., 1997). Additionally, B. ovis
infection in rams modi®ed the carbohydrate composition of organs participating in maturation, transport,
and storage of spermatozoa. Glycoconjugates are
instrumental in receptor±ligand interactions. These
alterations have been postulated to be involved in
the impaired fertility observed in B. ovis infected rams
(Paolicchi et al., 1995).
Today, ASab detection is one of the most important
steps in the evaluation of infertility in men (Dondero et
al., 1997; Lenzi et al., 1997), and ASab could become
an important tool in the diagnosis of animal infertility.
Continued investigation of infertility in rams will lead
to novel treatment regimens as well as to the development of new prophylactic measures. Finally, this
ovine disease could be a useful model in comparative
pathology and biomedical research.
Acknowledgements
The authors would like to thank Dr. C. GarcõÂaCarrillo and Dr. N. Lucero for biotyping of the Brucella strains. We also thank Dr. C. Campero, Dr. H.
Terzolo, and the staff of the histology and bacteriology
laboratories for their collaboration with our work. We
wish to thank Mr. R. Gregoret and Ms. E. Piazza
(INTA Castelar, Argentina) for their assistance in GD
and CF serologic diagnostics. We thank Dr. Silvina
Viviana Macnie for skilled assistance in language
revision. This work was partially supported by a grant
from the National Research Council (CONICET),
Argentina to FAP. EJG is a Research Career Member
of CONICET.
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