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Effect of Actinomyces pyogenes and gram-negative anaerobic bacteria on the development of bovine pyometra
EFFECT
OF Actino~v=es DVogenes AND GRAM-NEGATIVE ANAEROBIC BACTERIA ON THE DEVELOPMENT OF BOVINE PYOMETRA P.W. Farin,’ L. Ball,’ J.D. Olson,’ R.G. Mortimer,’ R.L. Jones,2 W.S. AdneyZ and A.E. McChesney2 Laboratory, ‘Department of Clinical Sciences, ‘Diagnostic Colorado State University, Ft. Collins, CO 80523 Received
for publication Accepted
: April 11, 1988 21, 1989 : February
ABSTRACT Fifteen lactating Holstein cows were used in a trial designed to evaluate the effectiveness of intrauterine inoculation (challenge) of Actinomvces pvonenes (A) alone or in combination with Fusobacterium necroohorum (F) and (B) to induce pyometra. Cows were assigned to $acteroide$ melaninqaenicus one of five groups: A (n = 3), AB (n = 3). AF (n = 3), ABF (n = 3) or C (control, broth medium alone; n = 3). All cows exhibited estrus 12 or 13 d prior to challenge (Day Orfirst day of challenge). During the prechallenge period, the reproductive tract of each cow was palpated per rectum and uterine fluid aspirates for culture and uterine biopsies were also obtained. All cows received an intravenous injection of 5,000 IU of human chorionic gonadotropin (hCG; Day 5) and an intrauterine infusion of 40 ml of 0.7% iodine on Days 0, I and 2 of the solution (Day 1). Cows were then inoculated experiment. Sequential palpations of the reproductive tracts, samples of uterine fluid for culture and uterine biopsies were performed for a total of 30 A cow was diagnosed as having pyometra when d after the first inoculation. purulent uterine fluid and a corpus luteum were detected by palpation per rectum. The number of cows that developed pyometra in Group A was 2 of 3, in Group AB 3 of 3, in Group AF 3 of 3, in Group ABF 3 of 3 and in Group C Cows with pyometra did not exhibit estrus. In 7 of I I cows, 0 of 3. pyometra persisted for more than 21 d. In cows that developed pyometra, the same species of bacteria infused into the uterus were usually recovered one or more times during the postchallenge period. When clinical pyometra was diagnosed, histologic endometritis was invariably present. Histologic endometritis and concurrent isolation of A. pvoaenes alone or A. pvonenes with gram-negative anaerobic bacteria occurred in 91.7% of samples during the Regardless of bacterial treatment, postchallenge period. gram-negative anaerobic bacteria were frequently isolated with & pvoaenes during this period. Key words: pyometra,
experimental
Acknowledgment: Research supported
MAY 1989 VOL. 31 NO. 5
infection,
by USDA Competitive
A. pvoaenes,
Grant
anaerobes
No. 5-3211.
979
THERIOGENOLOGY
INTRODUCTION Cows with pyometra have variable amounts of mucopurulent exudate in the uterine lumen, they have a persistent corpus luteum, and they do not exhibit estrus (1). Bacteria recovered from the infected bovine uterus usually include Actinomvces ovoaenes (Corvnebacterium ovonenes) and gram-negative It has been proposed that these organisms act synergistically bacteria (2-4). to cause severe infections of the bovine uterus (2,4) as with other diseases the role of gram-negative However, anaerobic bacteria in the (0). pathogenesis of pyometra in dairy cattle has received only limited attention. Several studies have evaluated uterine and ovarian response to bacteria or purulent exudate infused into the uterus of the cow (7-10) and ewe (11,12). Although results of these studies have been variable, Rommel (10) successfully created uterine infection in dairy cows with intrauterine infusions of A. pvogenes during the luteal phase of the estrous cycle. Uterine samples obtained from postpartum dairy cows in Colorado and Wisconsin often yielded A. pvonenes and the gram-negative anaerobic bacteria, Fusobacterium necroohorum and Bacteroides spp. (2,3). Pyometra developed approximately IO d after the first postpartum ovulation in association with a high number of these bacteria (2). Thus, there appears to be a relationship between the presence of these bacteria during the luteal phase of the estrous The objectives of our experiment were cycle and the occurrence of pyometra. therefore, to produce pyometra in dairy cows during the luteal phase and to evaluate the role of A. pvoaenes alone or in combination with gram-negative anaerobic bacteria in its development.
MATERIALS
AND METHODS
Fifteen Holstein cows at least 3 yr of age were housed in a free-stall barn at the Colorado State University Dairy Center. All the cows were at least 100 d postpartum, they were not pregnant, and all were producing at least 30 lb of milk per day. Three cows were assigned to each of fiave groups medium and inoculated (challenged) with either prereduced Columbia broth (control group) or prereduced Columbia broth medium containing bacteria. (Group A); A. pvoaenes and Experimental treatments included A. pvoaenes E. necrophorum (Group AF); A. pvonenes and & melaninoaenicus (Group AB); A. pvoaenes, E. necroohorum and B. melaninoaenicus (Group AFB) and the control broth medium alone (Group C). Procedgres used during the pre- and post-challenge periods are outlined All cows, except those that were in natural estrus (No. 74 and in Figure I. 78), received a single intramuscular injection of 25 mg of prostaglandin F,, (PGF,,)C to synchronize estrus. Cows were in estrus either 12 or I3 d prior to the first day of treatment. These days were designated Days -13 and -12
:Pasco Laboratories Inc., Wheatridge, CO. Portions of these procedures were devised Campbell, Food and Drug Administration, Rockville, MD. ‘Lutalyse, The Upjohn Company, Kalamazoo,
980
in collaboration with Center for Veterinary
Dr. D.L. Medicine,
MI.
MAY 1989 VOL. 31 NO. 5
Figure
4
lP
HCG
t+++ -IO
SOLUTION
INFU6lON
CMORIONIC
= CHALLENGE
lODINE
:
3
-5 P
+
MC0
PERWD
SYNCHRONIZATION
GONAOOTROPIN
HUMAN
E9TRU6
= INTRAUTERINE
n
.
TREATMENT
;:
P
-13.4-12
CHALLEHGE
OF
2
!Jsm
1 9
6
9 P
G
A
P
13 P
-
-
-
20 P
PERIOD
BIOPSY
FOR
27 P
FOR
CULTURE
ASPIRATE
23 P
HlSTDPAlIiOLOGY
UTERINE
BACTERIAL
UTERINE
RUPATION
PROCEDURE:
*’ B
16
POST-CHALLENGE
i
;
3C P
1. Treatment and sampling schedule for both control and bacterial inocu’ation groups. All cows, except those that were in natural estrus (Cows 71 and 78) received a single injection of PGFzaon Day -15. All cows were in estrus on Day -12 or Day -13 prior to the first day of inoculation (Day 0).
PROCEDURE:
DAY:
PtW-
THERIOGENOLOGY on the time line (Figure I) and will be referred to hereafter as the entry date. Day 0 was defined as the first day of inoculation. All cows were observed for signs of estrus twice daily for the entire experiment. The uterus of each cow was biopsied for histopathological evaluation, and fluids were aspirllted from uterine lumen for bacteriological culture on the an intravenous entry date and on Days -5, 4 16 and 30. All cows received injection of 5,000 IU of hCG on Day -5 to extend the lifespan of the corpus luteum (13). The uterus of each cow was infused with 40 ml of 0.7% iodine solution (dilute Lugol’s iodine) on Day -I to extend the estrous cycle (14). and increase its susceptibility to invasion by damage the endometrium Uteri of all cows were inoculated on pathogens used in the treatment groups. Days 0, 1 and 2, which corresponded to the midluteal phase of the estrous cycle (Figure I). The reproductive tracts of all cows were palpated per rectum to evaluate the cervix, uterus and ovaries on the entry date, and on Days -5, 6, 9. 13, 16, 20, 23, 27 and 30. If a cow accumulated uterine exudate in association with a palpable corpus luteum, she was designated as having pyometra whether or not Although Day 30 the corpus luteum was retained longer than 21 d. postinoculation was designated the final day of the experiment, 9 of 15 cows were available for additional evaluation for periods of up to 82 d. Bacteria used in the challenge inocula were first obtained from uterine fluid of cows with pyometra using a sterilized infus?: pipette technique (2). The organisms were isolated on anaerobic blood agar, In a flexible anaerobic glove boxe with an atmosphere of 85% N,, 10% H,, 5% CO,. Bacterial isolates were identified (15,16) and maintained at 70°C in sterile horse blood, coated on sterile glass beads. The challenge inocula were prepared by streaking several anaerobic blood After 48 h the agar plates for confluent growth with each strain of bacteria. organisms were suspended in prereduced Columbia broth, and the turbidity was Viable bacterial counts in the adjusted to approximately 1.0 x IO9 bacteria/ml. The number of inocula were determined by triplicate spread plate counts. viable bacteria in the inocula were as follows: A. pvoaenes, 7.0 x IO’ to 8.4 x IO* colony forming units (cfu)/ml; E. necrophorum, I.1 x 10’ to 8.9 x IO* cfu/ml; B. melaninonenicus, I.7 x IO’ to 7.6 x IO8 cfu/ml. Total volume of inoculum for each cow was 30 ml. Inocula were transported under anaerobic conditions, withdrawn from their container, and infused into the uterus using standard artificial insemination techniques. To prevent contamination during the sterilized infusion pipette was protected with a uterine inoculation, sterilized outer sheath. Samples of uterine fluid taken during the postchallenge period (Figure Id MacConkey agar were inoculated onto blood agar plates AS% sheep blood), plates, Casman medium (5% horse blood) agar plates (incubated in 10% CO,, 35OC), anaerobic blood agara plates and into chopped meat broth3 (incubated Bacterial isolates were identified by standard methods anaerobically, 35°C). (15.16). d Sussex Drug Products, Edison, NJ. eCoy Laboratory Products, Ann Arbor,
Ml.
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
Uterine biopsy samples were obtained using a modified Folmer-Nielsen biopsy instrument similar to that described by McQueen (17). The instrument was inserted through the cervix to a plane near the bifurcation of the uterus. Biopsy samples were No attempt was made to preselect sites for biopsy. approximately 10 x 5 x 2 mm in size. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 urn and stained with hematoxylin and eosin. Sections were evaluated at the light microscopic level. all histologic evaluations were conducted by one To insure consistency, pathologist.
RESULTS None of the cows in the control group developed pyometra (Table I). In contrast, all cows receiving bacterial inoculations developed pyometra within 30 d with the exception of Cow 74 in Group A (Table I). A corpus luteum was palpable on one or both ovaries of all cows that developed pyometra. Seven of II cows had corpora lutea and purulent uterine fluid that persisted for more than 21 d during which time they were anestrous. Two cows in Group AF had purulent fluid accumulations for less than 21 d and spontaneously evacuated the fluid. Pyometra generated in Group AF was less persistent than in Groups A and AB. Persistence of pyometra in Group AFB was not determined beyond Day 30. When pyometra was diagnosed clinically a corpus luteum was consistently detected with one exception (Cow 70, Day 20). Three cows (No. 77, 73, 90) developed pyometra toward the end of the experimental period, and two of these went through a postchallenge follicular phase into a new luteal phase before developing pyometra. In contrast, the remaining cows maintained their prechallenge corpus luteum as they developed pyometra. Before challenge, several species of bacteria were isolated (Table 1). The majority were classified as insignificant bacteria (“other” bacteria) and included coagulase-negative Staphvlococcus spp. and Bacillus spp. SPP., aStrevtococcus When isolated, they were usually found in low numbers and were not accompanied by histological evidence of inflammation of the endometrium. Insignificant bacteria were the only bacteria recovered from cows in the control group. In addition, insignificant bacteria were recovered both before and after inoculation in the principals during times when they did not have pyometra. Only one cow (No. 70) in Group AF had pyometra associated with insignificant bacteria (Bacillus spp.) on Day 30 of the experiment; however, & pvoaenes and E. necrophorum were both isolated from samples taken earlier. This cow recovered spontaneously sometime between Day 30 and Day 51. The only treated cow (No. 74) that did not develop pyometra had a corpus luteum at the time of challenge, but it regressed 6 d later, and only insignificant bacteria were recovered in subsequent fluid samples. In most cases, the same species of bacteria that were infused into the uterus at the time of inoculation were recovered at least once during the postchallenge period (Table 1). Cows in Group AB did not yield B. melaninoeenicus on reculture during the formal experiment; however, A. pvoaenes, f. necrovhorum and B. melaninoaenicus were all present in the uteri of two cows and A. pvonenes was present alone in the third cow when uterine samples were cultured 51 days after initial inoculation. All three cows in
MAY 1989 VOL. 31 NO. 5
983
THERIOGENOLOGY
Table
1. Effect of intrauterine innoculation (challenge) on bacteriological, histopathological and clinical findings in dairy cows.
Days postchallenge
Days prechaliengeb Groupa
6
Cow No.
-13/-l:
79 80 91
N~I’N/-~ Ng/N/Ng/N/-
O/N/O/N/Ng/N/-
O/N/O/N/O/N:-
O/N/Ng/N/Ng/N/-
O/ME/O/N/Ng/N/-
A
12 74 78
O/N/O/N/Ns/N/-
O/N/O/N/Ng/AFE/-
AF/A E/+ O/CE/A/CAcE/+
A/CAcE/+ O/N/AO/CAcE/+
AF/CAcE/+ O/N/AF/CAcE/+
AB
73 75 93
O/N:O/N:O/CME/-
Ng/N/O/N:N/O/-
Ng:N/A/AE/t A/AE/t
A/N/Ng/SCAcE/t A/CAcEjt
A/AFE/+ A/SCAcE/+ A/CAcE/t
AF
70 71 71
O/N/O/N/O/N/-
O/N/O/N/F/E/-
AF/AE/+ AF/Ns/t AO/N/-
AF/CAcE/+ A/SCAcE/O/N/-
O/SCAcE/+ O/N/AF/CAcE/+
ABF
76 90 92
Ng/CME/O/N/O/N/-
O/Ns/O/AE/Ng/Ns/-
AF/CAcE/+ O/N/ABFO/E/-
ABF/CAcE/+ Ng/CAcE/ABF/CAcE/t
ABFO/CAcE/+ Ng/Ns/t ABF/CAcE/t
Control
-5
16
30
“Cows were cha!lenged with broth medium alone (control group) or with bbacteria (groups A, AB. AF and ABF) as denoted in footnote c. Cows were challenged on day 0, I and 2. CBacteriological/histopathological/clinical findings: Bacteriology: A = A. pvonenes, B = B. melaninonenicus, F = E. necroohorum, 0 = Other bacteria, Ng = No growth, Ns = Not sampled. Histopathology: N = Normal, A = Acute, C = Chronic, F = Focal, M = Mild, S = Severe, AC = Active, E = Endometritis, Ns = Not sampled. Clinical findings: + = Pyometra and a corpus luteum present, - = Uterus empty. but one cow (No. 71) eliminated both Group AF developed pyometra, A. pvonenes and E. necroohorum by Day 30 when she was also normal based on All three cows in Group AFB clinical and histopathologic examination. developed pyometra, but the inoculated species of organisms were recovered from the uteri of only two cows (Table I). In Cow 90, insignificant organisms were recovered on Day 6, but no additional bacterial growth occurred for the remainder of the experiment; clinical pyometra was detected on Day 27. None of these cows were evaluated after Day 30; consequently, there was no opportunity to determine bacterial growth and persistence of pyometra beyond the course of the experiment.
during
984
Gram-negative anaerobic bacteria the postchallenge period (Table
were never present in the uterus alone I). In contrast, A. pvoaenes was often
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
isolated alone, even though gram-negative anaerobic bacteria were inoculated ms pvonenes alone or with gram-negative bacteria was with it. When insignificant bacteria recovered from 91.7% uteri having endometritis. were isolated from the uterus they were usually associated with normal endometrium (76.9%) and rarely with histologic endometritis (Table 2). Table
2.
Distribution postchallenge
of uterine perioda
bacteria
by biopsy
classification
during
Biopsy classification,
No. (96)
Normal
Endometritis
2 (16.7)
10 (83.0)
1A pvoaenes with gram-tre E ative anaerobic bacteria
0 (0.0)
12 (100)
Subtotal
2 (8.3)
22 (91.7)
Uterine 1A
bacteria
woaenesalone
Insignificant
b
bacteria
alone
Total
10 (76.9)
3 (23.0)
12 (32.4)
25 (67.6)
aIncludes all uterine samples that had bacterial bsamples were also obtained (n = 37). Present with or without insignificant bacteria. were not isolated in ‘Gram-negative bacteria during this period.
growth
the
and
absence
the
in which
of
A.
biopsy
pvoaenes
When pyometra was diagnosed clinically, inflammation of the endometrium with effusion of suppurative exudate into the uterine lumen was invariably present (Table I). With the exception of one uterine biopsy, pre- and postchallenge tissue samples were normal from cows in the control group. Most cows in the bacterial inoculation groups also had normal biopsy samples before challenge. When lesions were detected they were usually mild. A few neutrophils were observed between epithelial cells of one section, and scattered lymphocytes and hemosiderin bearing macrophages were present in two sections. In Group A, Cows 72 and 78 contracted pyometra and had sloughed much of their endometrial surface epithelium by 6 d after initial treatment. Bacteria, neutrophils and ulcerated endometrial surfaces coated with mucus Adjacent endometrial stroma was locally were seen in histologic sections. necrotic and contained many neutrophils. Lymphocytes, plasma cells, and/or neutrophils were both scattered and aggregated throughout the deep lamina These changes were classified as chronic active endometritis. At 13 d propria. after initial challenge, remaining epithelial cells were large, pleomorphic and were surrounded by neutrophils. Endometrial glands were markedly decreased in number and the endometrial stroma was locally necrotic and filled with lymphocytes, plasma cells ana neutrophils. Histologic changes were similar, but slightly more severe by Day 30 postchallenge. Tissue from Cow 74, who did
MAY 1989 VOL. 31 NO. 5
985
THERIOGENOLOGY not develop pyometra, had increased lymphocytes and postinoculation but was normal in subsequent evaluations.
plasma
cells
on Day 6
The combination of & pvoaenes with gram-negative anaerobic bacteria was associated with similar uterine histopathology. For example, two cows in Group AF (No. 70 and 71) had slightly swollen surface epithelial cells. Neutrophils were present both between and beneath the epithelial cells by day 6 after initial inoculation. Some areas contained sloughed epithelial cells. In addition, some endometrial glands were degenerated and surrounded by lymphocytes and neutrophils which extended throughout the lamina propria. By 13 d after challenge, many endometrial glands were degenerated, necrotic, or missing, and either contained or were surrounded by neutrophils. Plasma cells, neutrophils and lymphocytes were present in large numbers throughout these sections. By 30 d after challenge, most surface epithelium had sloughed, while mucus, bacteria, cell debris and neutrophils covered the luminal surface. Few normal endometrial glands were seen. In addition, the endometrial stroma was locally necrotic near the luminal surface and was packed with lymphocytes, plasma cells neutrophils and epithelioid-type cells. These findings were characteristic of active endometritis, usually chronic in nature.
DISCUSSION With the exception of one cow in Group A, all cows inoculated with A. with gram-negative anaerobic bacteria DVOP~~CS or & pyonenea in combination developed clinical pyometra within 30 d. Seven of the 11 cows that developed pyometra failed to exhibit estrus, had palpable corpora lutea and uterine fluid that persisted for more than 21 d thus fitting the classic description of pyometra in the cow (I). Rommel (IO) described induction of uterine infections in cows by an intrauterine infusion of A. pvonene$ alone during the Successful production of pyometra in dairy luteal phase of the estrous cycle. cows was also achieved with intrauterine infusions of either A. pvonenes alone or A. pvonenes with E. necroohorum (18). Sequential postinoculation uterine biopsies and cultures were not obtained in that study; thus the effects of these organisms on the endometrium was not discerned. Actinomvces pyonenes was frequently recovered from the uteri of cows with pyometra, which supports previous findings (2,19). However, in addition to & pvonenes, one or more of the gram-negative anaerobic bacteria were The concurrent isolation of these bacteria from the frequently isolated. Spontaneous infected bovine uterus augments previous observations (2-4). infection with gram-negative anaerobic bacteria occurred even when cows were inoculated with 4. pyoaeneg alone. Because no cows were inoculated with only B melaninonenicus or E. necronhorum in our study, we do not know if the -. However, Campbell (18) reported that intrauterine converse would occur. infusion of E. necroohorum alone during the luteal phase of the cycle failed to produce pyometra in postpartum cows. histologic findings and the There was a strong association between During the postchallenge species of bacteria present in the uterine lumen. period; histologic endometritis was detected 91.7% of the time when either & with gram-negative anaerobes were isolated pvoaenes alone or A. pvonenes Other investigators have observed that 97.4% (19,20) and from the uterus. 100% (21) of cows with histologic endometritis had concurrent isolation of A.
985
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY pyoae es from the uterine lumen. Studer and Morrow (22) also reported that associated with endometritis in postpartum dairy & DYnOPenCSwas frequently cows. In our study, bacteria classified as insignificant were considered to be contaminants of the uterus and were associated with few histopathologic changes, which is in agreement with earlier work (20). Pyometra has been defined as an accumulation of purulent exudate in the uterus associated with a retained corpus luteum (CL) and suppression of signs pyometra to exist regardless of CL of estrus (1). In this study, we considered lifespan although in most cases a CL was palpated in the same location on the ovary during sequential palpations. From a clinical viewpoint, the diagnosis of pyometra should not be predicated on the lifespan of the CL for several reasons. The lifespan of a corpus luteum cannot be determined by a single The necessity for return palpations and the resulting delay in palpation. diagnosis can lead to an increase in the number of days open and contribute to economic loss for the dairyman. Thus, we recommend a clinical definition in which we consider pyometra to exist if, upon palpation, a uterus is filled with fluid, lacks positive signs of pregnancy and may be thick walled, and a CL is present on one or both ovaries. In conclusion, pyometra was produced in cows by intrauterine infusion of with gram-negative anaerobic bacteria 1A Dvonenes alone or in combination during the luteal phase of the estrous cycle. Following inoculation, gramnegative anaerobic bacteria were frequently recovered concurrently with A. pvoeena from cows that developed pyometra; even from those inoculated with A. m alone. These results support the concept that anaerobic bacteria may act synergistically with & pvonen= in generating bovine pyomctra during the luteal phase of the cycle.
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Roberts, S.J. Veterinary Obstetrics and Genital Diseases SJ Roberts Publisher, Woodstock, VT, 1986, pp. 546-547.
2.
Olson, J.D., Ball, L., Mortimer, R.G., Farin, P.W., Adney, W.S. and Huffman, EM. Aspects of bacteriology and endocrinology of cows with pyometra and retained fetal membranes. Am. J. Vet. Res. 02251-2255 (1984).
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Peter, A.T. and Bosu, W.T.K. Effects of intrauterine infection on the function of the corpora lutea formed after first postpartum ovulations in dairy cows. Theriogenology 21:593-609 (1987).
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Ruder, C.A., Sasser, R.G., Williams, R.J., Ely, J.K., Bull, R.C. and Butler, J.E. Uterine infections in the postpartum cow II. Possible synergistic effect of Fusobacterium necroohorum and Corvnebacterium ovonenes. Theriogenology Li:573-580 (198 1).
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Roberts, D.S. The pathogenic synergy of Fusiformis Corvnebacterium ovoae es I. Influence of the leucocidal necroohorus. Br. J. ExpnPathol. =665-673 (1967).
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Effect of Lynn, J.E., McNutt, S.H. and Casida, L.E. bacterial inoculation and suckling on the bovine corpus uterus. Am. J. Vet. Res. 2:152l-1526 (1966).
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Rommel, W. Experimentelle von c. Pvonenes im genitale IV Int. Congr. Anim. Reprod.,
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Brinsfield, T.H., Higginbotham, D.K. and Hawk, W.H. induced uterine infection at various stages of the estrous corpus luteum of the ewe. J. Anim. Sci. 29:616-618 (1969).
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Coudert, S.P. and Short, R.V. Prolongation corpus luteum in sheep with experimental Fertil. 2579-582 (1966).
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and pregnancy Eduvie, L.O. and Seguin, B.E. Corpus luteum function rate in lactating dairy cows given human chorionic gonadotropin at middiestrus. Theriogenology a415-422 (1982).
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Seguin, B.E., Morrow, D.A. and Louis, T.M. Luteolysis, luteostasis, and irritation. the effect of prostaglandin F, o in cows after endometrial Am. J. Vet. Res. =57-61 (1974).
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Holdeman, L.V., Cato, E.P., and Moore, W.E.C. (ed.). Bacteriology Manual. Anaerobic Laboratory, Virginia Institute and State University, Blacksburg, 1977, pp. I-152.
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a model infection for drug evaluation. Campbell, D.L. Bovine pyometra Proc. of the Sot. for Theriogenology, Annual Mtg., Denver, CO. pp. 238239 abstr. (1984).
untersuchungen zur frage der pathogenitat Proc. des rindes wahrend des brustzyklus. The Hague, 1503-510 (1981).
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life of the J. Reprod.
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THERIOGENOLOGY
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Nonspecific uterine Griffin, J.F.T., Hartigan, P.J. and Nunn, W.R. infection and bovine fertility. 1. Infection patterns and endometritis during the first seven weeks postpartum. Theriogenology L:91-106 (1974).
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Miller, H.V., Kimsey, P.B., Kendrick, J.W., Darien, Et., Doering, I., Franti, C. and Horton, J. Endometritis of dairy cattle: Diagnosis, treatment and fertility. Bovine Practioner ul3-23 (1980).
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evaluation of bovine reproduStuder, E. and Morrow, D.A. Postpartum ctive potential: Comparison of findings from genital tract examination per rectum, uterine culture and endometrial biopsy. J. Am. Vet. Med. Assoc. 172:489-494 (1978).
MAY 1989 VOL. 31 NO. 5
989
OF Actino~v=es DVogenes AND GRAM-NEGATIVE ANAEROBIC BACTERIA ON THE DEVELOPMENT OF BOVINE PYOMETRA P.W. Farin,’ L. Ball,’ J.D. Olson,’ R.G. Mortimer,’ R.L. Jones,2 W.S. AdneyZ and A.E. McChesney2 Laboratory, ‘Department of Clinical Sciences, ‘Diagnostic Colorado State University, Ft. Collins, CO 80523 Received
for publication Accepted
: April 11, 1988 21, 1989 : February
ABSTRACT Fifteen lactating Holstein cows were used in a trial designed to evaluate the effectiveness of intrauterine inoculation (challenge) of Actinomvces pvonenes (A) alone or in combination with Fusobacterium necroohorum (F) and (B) to induce pyometra. Cows were assigned to $acteroide$ melaninqaenicus one of five groups: A (n = 3), AB (n = 3). AF (n = 3), ABF (n = 3) or C (control, broth medium alone; n = 3). All cows exhibited estrus 12 or 13 d prior to challenge (Day Orfirst day of challenge). During the prechallenge period, the reproductive tract of each cow was palpated per rectum and uterine fluid aspirates for culture and uterine biopsies were also obtained. All cows received an intravenous injection of 5,000 IU of human chorionic gonadotropin (hCG; Day 5) and an intrauterine infusion of 40 ml of 0.7% iodine on Days 0, I and 2 of the solution (Day 1). Cows were then inoculated experiment. Sequential palpations of the reproductive tracts, samples of uterine fluid for culture and uterine biopsies were performed for a total of 30 A cow was diagnosed as having pyometra when d after the first inoculation. purulent uterine fluid and a corpus luteum were detected by palpation per rectum. The number of cows that developed pyometra in Group A was 2 of 3, in Group AB 3 of 3, in Group AF 3 of 3, in Group ABF 3 of 3 and in Group C Cows with pyometra did not exhibit estrus. In 7 of I I cows, 0 of 3. pyometra persisted for more than 21 d. In cows that developed pyometra, the same species of bacteria infused into the uterus were usually recovered one or more times during the postchallenge period. When clinical pyometra was diagnosed, histologic endometritis was invariably present. Histologic endometritis and concurrent isolation of A. pvoaenes alone or A. pvonenes with gram-negative anaerobic bacteria occurred in 91.7% of samples during the Regardless of bacterial treatment, postchallenge period. gram-negative anaerobic bacteria were frequently isolated with & pvoaenes during this period. Key words: pyometra,
experimental
Acknowledgment: Research supported
MAY 1989 VOL. 31 NO. 5
infection,
by USDA Competitive
A. pvoaenes,
Grant
anaerobes
No. 5-3211.
979
THERIOGENOLOGY
INTRODUCTION Cows with pyometra have variable amounts of mucopurulent exudate in the uterine lumen, they have a persistent corpus luteum, and they do not exhibit estrus (1). Bacteria recovered from the infected bovine uterus usually include Actinomvces ovoaenes (Corvnebacterium ovonenes) and gram-negative It has been proposed that these organisms act synergistically bacteria (2-4). to cause severe infections of the bovine uterus (2,4) as with other diseases the role of gram-negative However, anaerobic bacteria in the (0). pathogenesis of pyometra in dairy cattle has received only limited attention. Several studies have evaluated uterine and ovarian response to bacteria or purulent exudate infused into the uterus of the cow (7-10) and ewe (11,12). Although results of these studies have been variable, Rommel (10) successfully created uterine infection in dairy cows with intrauterine infusions of A. pvogenes during the luteal phase of the estrous cycle. Uterine samples obtained from postpartum dairy cows in Colorado and Wisconsin often yielded A. pvonenes and the gram-negative anaerobic bacteria, Fusobacterium necroohorum and Bacteroides spp. (2,3). Pyometra developed approximately IO d after the first postpartum ovulation in association with a high number of these bacteria (2). Thus, there appears to be a relationship between the presence of these bacteria during the luteal phase of the estrous The objectives of our experiment were cycle and the occurrence of pyometra. therefore, to produce pyometra in dairy cows during the luteal phase and to evaluate the role of A. pvoaenes alone or in combination with gram-negative anaerobic bacteria in its development.
MATERIALS
AND METHODS
Fifteen Holstein cows at least 3 yr of age were housed in a free-stall barn at the Colorado State University Dairy Center. All the cows were at least 100 d postpartum, they were not pregnant, and all were producing at least 30 lb of milk per day. Three cows were assigned to each of fiave groups medium and inoculated (challenged) with either prereduced Columbia broth (control group) or prereduced Columbia broth medium containing bacteria. (Group A); A. pvoaenes and Experimental treatments included A. pvoaenes E. necrophorum (Group AF); A. pvonenes and & melaninoaenicus (Group AB); A. pvoaenes, E. necroohorum and B. melaninoaenicus (Group AFB) and the control broth medium alone (Group C). Procedgres used during the pre- and post-challenge periods are outlined All cows, except those that were in natural estrus (No. 74 and in Figure I. 78), received a single intramuscular injection of 25 mg of prostaglandin F,, (PGF,,)C to synchronize estrus. Cows were in estrus either 12 or I3 d prior to the first day of treatment. These days were designated Days -13 and -12
:Pasco Laboratories Inc., Wheatridge, CO. Portions of these procedures were devised Campbell, Food and Drug Administration, Rockville, MD. ‘Lutalyse, The Upjohn Company, Kalamazoo,
980
in collaboration with Center for Veterinary
Dr. D.L. Medicine,
MI.
MAY 1989 VOL. 31 NO. 5
Figure
4
lP
HCG
t+++ -IO
SOLUTION
INFU6lON
CMORIONIC
= CHALLENGE
lODINE
:
3
-5 P
+
MC0
PERWD
SYNCHRONIZATION
GONAOOTROPIN
HUMAN
E9TRU6
= INTRAUTERINE
n
.
TREATMENT
;:
P
-13.4-12
CHALLEHGE
OF
2
!Jsm
1 9
6
9 P
G
A
P
13 P
-
-
-
20 P
PERIOD
BIOPSY
FOR
27 P
FOR
CULTURE
ASPIRATE
23 P
HlSTDPAlIiOLOGY
UTERINE
BACTERIAL
UTERINE
RUPATION
PROCEDURE:
*’ B
16
POST-CHALLENGE
i
;
3C P
1. Treatment and sampling schedule for both control and bacterial inocu’ation groups. All cows, except those that were in natural estrus (Cows 71 and 78) received a single injection of PGFzaon Day -15. All cows were in estrus on Day -12 or Day -13 prior to the first day of inoculation (Day 0).
PROCEDURE:
DAY:
PtW-
THERIOGENOLOGY on the time line (Figure I) and will be referred to hereafter as the entry date. Day 0 was defined as the first day of inoculation. All cows were observed for signs of estrus twice daily for the entire experiment. The uterus of each cow was biopsied for histopathological evaluation, and fluids were aspirllted from uterine lumen for bacteriological culture on the an intravenous entry date and on Days -5, 4 16 and 30. All cows received injection of 5,000 IU of hCG on Day -5 to extend the lifespan of the corpus luteum (13). The uterus of each cow was infused with 40 ml of 0.7% iodine solution (dilute Lugol’s iodine) on Day -I to extend the estrous cycle (14). and increase its susceptibility to invasion by damage the endometrium Uteri of all cows were inoculated on pathogens used in the treatment groups. Days 0, 1 and 2, which corresponded to the midluteal phase of the estrous cycle (Figure I). The reproductive tracts of all cows were palpated per rectum to evaluate the cervix, uterus and ovaries on the entry date, and on Days -5, 6, 9. 13, 16, 20, 23, 27 and 30. If a cow accumulated uterine exudate in association with a palpable corpus luteum, she was designated as having pyometra whether or not Although Day 30 the corpus luteum was retained longer than 21 d. postinoculation was designated the final day of the experiment, 9 of 15 cows were available for additional evaluation for periods of up to 82 d. Bacteria used in the challenge inocula were first obtained from uterine fluid of cows with pyometra using a sterilized infus?: pipette technique (2). The organisms were isolated on anaerobic blood agar, In a flexible anaerobic glove boxe with an atmosphere of 85% N,, 10% H,, 5% CO,. Bacterial isolates were identified (15,16) and maintained at 70°C in sterile horse blood, coated on sterile glass beads. The challenge inocula were prepared by streaking several anaerobic blood After 48 h the agar plates for confluent growth with each strain of bacteria. organisms were suspended in prereduced Columbia broth, and the turbidity was Viable bacterial counts in the adjusted to approximately 1.0 x IO9 bacteria/ml. The number of inocula were determined by triplicate spread plate counts. viable bacteria in the inocula were as follows: A. pvoaenes, 7.0 x IO’ to 8.4 x IO* colony forming units (cfu)/ml; E. necrophorum, I.1 x 10’ to 8.9 x IO* cfu/ml; B. melaninonenicus, I.7 x IO’ to 7.6 x IO8 cfu/ml. Total volume of inoculum for each cow was 30 ml. Inocula were transported under anaerobic conditions, withdrawn from their container, and infused into the uterus using standard artificial insemination techniques. To prevent contamination during the sterilized infusion pipette was protected with a uterine inoculation, sterilized outer sheath. Samples of uterine fluid taken during the postchallenge period (Figure Id MacConkey agar were inoculated onto blood agar plates AS% sheep blood), plates, Casman medium (5% horse blood) agar plates (incubated in 10% CO,, 35OC), anaerobic blood agara plates and into chopped meat broth3 (incubated Bacterial isolates were identified by standard methods anaerobically, 35°C). (15.16). d Sussex Drug Products, Edison, NJ. eCoy Laboratory Products, Ann Arbor,
Ml.
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
Uterine biopsy samples were obtained using a modified Folmer-Nielsen biopsy instrument similar to that described by McQueen (17). The instrument was inserted through the cervix to a plane near the bifurcation of the uterus. Biopsy samples were No attempt was made to preselect sites for biopsy. approximately 10 x 5 x 2 mm in size. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 urn and stained with hematoxylin and eosin. Sections were evaluated at the light microscopic level. all histologic evaluations were conducted by one To insure consistency, pathologist.
RESULTS None of the cows in the control group developed pyometra (Table I). In contrast, all cows receiving bacterial inoculations developed pyometra within 30 d with the exception of Cow 74 in Group A (Table I). A corpus luteum was palpable on one or both ovaries of all cows that developed pyometra. Seven of II cows had corpora lutea and purulent uterine fluid that persisted for more than 21 d during which time they were anestrous. Two cows in Group AF had purulent fluid accumulations for less than 21 d and spontaneously evacuated the fluid. Pyometra generated in Group AF was less persistent than in Groups A and AB. Persistence of pyometra in Group AFB was not determined beyond Day 30. When pyometra was diagnosed clinically a corpus luteum was consistently detected with one exception (Cow 70, Day 20). Three cows (No. 77, 73, 90) developed pyometra toward the end of the experimental period, and two of these went through a postchallenge follicular phase into a new luteal phase before developing pyometra. In contrast, the remaining cows maintained their prechallenge corpus luteum as they developed pyometra. Before challenge, several species of bacteria were isolated (Table 1). The majority were classified as insignificant bacteria (“other” bacteria) and included coagulase-negative Staphvlococcus spp. and Bacillus spp. SPP., aStrevtococcus When isolated, they were usually found in low numbers and were not accompanied by histological evidence of inflammation of the endometrium. Insignificant bacteria were the only bacteria recovered from cows in the control group. In addition, insignificant bacteria were recovered both before and after inoculation in the principals during times when they did not have pyometra. Only one cow (No. 70) in Group AF had pyometra associated with insignificant bacteria (Bacillus spp.) on Day 30 of the experiment; however, & pvoaenes and E. necrophorum were both isolated from samples taken earlier. This cow recovered spontaneously sometime between Day 30 and Day 51. The only treated cow (No. 74) that did not develop pyometra had a corpus luteum at the time of challenge, but it regressed 6 d later, and only insignificant bacteria were recovered in subsequent fluid samples. In most cases, the same species of bacteria that were infused into the uterus at the time of inoculation were recovered at least once during the postchallenge period (Table 1). Cows in Group AB did not yield B. melaninoeenicus on reculture during the formal experiment; however, A. pvoaenes, f. necrovhorum and B. melaninoaenicus were all present in the uteri of two cows and A. pvonenes was present alone in the third cow when uterine samples were cultured 51 days after initial inoculation. All three cows in
MAY 1989 VOL. 31 NO. 5
983
THERIOGENOLOGY
Table
1. Effect of intrauterine innoculation (challenge) on bacteriological, histopathological and clinical findings in dairy cows.
Days postchallenge
Days prechaliengeb Groupa
6
Cow No.
-13/-l:
79 80 91
N~I’N/-~ Ng/N/Ng/N/-
O/N/O/N/Ng/N/-
O/N/O/N/O/N:-
O/N/Ng/N/Ng/N/-
O/ME/O/N/Ng/N/-
A
12 74 78
O/N/O/N/Ns/N/-
O/N/O/N/Ng/AFE/-
AF/A E/+ O/CE/A/CAcE/+
A/CAcE/+ O/N/AO/CAcE/+
AF/CAcE/+ O/N/AF/CAcE/+
AB
73 75 93
O/N:O/N:O/CME/-
Ng/N/O/N:N/O/-
Ng:N/A/AE/t A/AE/t
A/N/Ng/SCAcE/t A/CAcEjt
A/AFE/+ A/SCAcE/+ A/CAcE/t
AF
70 71 71
O/N/O/N/O/N/-
O/N/O/N/F/E/-
AF/AE/+ AF/Ns/t AO/N/-
AF/CAcE/+ A/SCAcE/O/N/-
O/SCAcE/+ O/N/AF/CAcE/+
ABF
76 90 92
Ng/CME/O/N/O/N/-
O/Ns/O/AE/Ng/Ns/-
AF/CAcE/+ O/N/ABFO/E/-
ABF/CAcE/+ Ng/CAcE/ABF/CAcE/t
ABFO/CAcE/+ Ng/Ns/t ABF/CAcE/t
Control
-5
16
30
“Cows were cha!lenged with broth medium alone (control group) or with bbacteria (groups A, AB. AF and ABF) as denoted in footnote c. Cows were challenged on day 0, I and 2. CBacteriological/histopathological/clinical findings: Bacteriology: A = A. pvonenes, B = B. melaninonenicus, F = E. necroohorum, 0 = Other bacteria, Ng = No growth, Ns = Not sampled. Histopathology: N = Normal, A = Acute, C = Chronic, F = Focal, M = Mild, S = Severe, AC = Active, E = Endometritis, Ns = Not sampled. Clinical findings: + = Pyometra and a corpus luteum present, - = Uterus empty. but one cow (No. 71) eliminated both Group AF developed pyometra, A. pvonenes and E. necroohorum by Day 30 when she was also normal based on All three cows in Group AFB clinical and histopathologic examination. developed pyometra, but the inoculated species of organisms were recovered from the uteri of only two cows (Table I). In Cow 90, insignificant organisms were recovered on Day 6, but no additional bacterial growth occurred for the remainder of the experiment; clinical pyometra was detected on Day 27. None of these cows were evaluated after Day 30; consequently, there was no opportunity to determine bacterial growth and persistence of pyometra beyond the course of the experiment.
during
984
Gram-negative anaerobic bacteria the postchallenge period (Table
were never present in the uterus alone I). In contrast, A. pvoaenes was often
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY
isolated alone, even though gram-negative anaerobic bacteria were inoculated ms pvonenes alone or with gram-negative bacteria was with it. When insignificant bacteria recovered from 91.7% uteri having endometritis. were isolated from the uterus they were usually associated with normal endometrium (76.9%) and rarely with histologic endometritis (Table 2). Table
2.
Distribution postchallenge
of uterine perioda
bacteria
by biopsy
classification
during
Biopsy classification,
No. (96)
Normal
Endometritis
2 (16.7)
10 (83.0)
1A pvoaenes with gram-tre E ative anaerobic bacteria
0 (0.0)
12 (100)
Subtotal
2 (8.3)
22 (91.7)
Uterine 1A
bacteria
woaenesalone
Insignificant
b
bacteria
alone
Total
10 (76.9)
3 (23.0)
12 (32.4)
25 (67.6)
aIncludes all uterine samples that had bacterial bsamples were also obtained (n = 37). Present with or without insignificant bacteria. were not isolated in ‘Gram-negative bacteria during this period.
growth
the
and
absence
the
in which
of
A.
biopsy
pvoaenes
When pyometra was diagnosed clinically, inflammation of the endometrium with effusion of suppurative exudate into the uterine lumen was invariably present (Table I). With the exception of one uterine biopsy, pre- and postchallenge tissue samples were normal from cows in the control group. Most cows in the bacterial inoculation groups also had normal biopsy samples before challenge. When lesions were detected they were usually mild. A few neutrophils were observed between epithelial cells of one section, and scattered lymphocytes and hemosiderin bearing macrophages were present in two sections. In Group A, Cows 72 and 78 contracted pyometra and had sloughed much of their endometrial surface epithelium by 6 d after initial treatment. Bacteria, neutrophils and ulcerated endometrial surfaces coated with mucus Adjacent endometrial stroma was locally were seen in histologic sections. necrotic and contained many neutrophils. Lymphocytes, plasma cells, and/or neutrophils were both scattered and aggregated throughout the deep lamina These changes were classified as chronic active endometritis. At 13 d propria. after initial challenge, remaining epithelial cells were large, pleomorphic and were surrounded by neutrophils. Endometrial glands were markedly decreased in number and the endometrial stroma was locally necrotic and filled with lymphocytes, plasma cells ana neutrophils. Histologic changes were similar, but slightly more severe by Day 30 postchallenge. Tissue from Cow 74, who did
MAY 1989 VOL. 31 NO. 5
985
THERIOGENOLOGY not develop pyometra, had increased lymphocytes and postinoculation but was normal in subsequent evaluations.
plasma
cells
on Day 6
The combination of & pvoaenes with gram-negative anaerobic bacteria was associated with similar uterine histopathology. For example, two cows in Group AF (No. 70 and 71) had slightly swollen surface epithelial cells. Neutrophils were present both between and beneath the epithelial cells by day 6 after initial inoculation. Some areas contained sloughed epithelial cells. In addition, some endometrial glands were degenerated and surrounded by lymphocytes and neutrophils which extended throughout the lamina propria. By 13 d after challenge, many endometrial glands were degenerated, necrotic, or missing, and either contained or were surrounded by neutrophils. Plasma cells, neutrophils and lymphocytes were present in large numbers throughout these sections. By 30 d after challenge, most surface epithelium had sloughed, while mucus, bacteria, cell debris and neutrophils covered the luminal surface. Few normal endometrial glands were seen. In addition, the endometrial stroma was locally necrotic near the luminal surface and was packed with lymphocytes, plasma cells neutrophils and epithelioid-type cells. These findings were characteristic of active endometritis, usually chronic in nature.
DISCUSSION With the exception of one cow in Group A, all cows inoculated with A. with gram-negative anaerobic bacteria DVOP~~CS or & pyonenea in combination developed clinical pyometra within 30 d. Seven of the 11 cows that developed pyometra failed to exhibit estrus, had palpable corpora lutea and uterine fluid that persisted for more than 21 d thus fitting the classic description of pyometra in the cow (I). Rommel (IO) described induction of uterine infections in cows by an intrauterine infusion of A. pvonene$ alone during the Successful production of pyometra in dairy luteal phase of the estrous cycle. cows was also achieved with intrauterine infusions of either A. pvonenes alone or A. pvonenes with E. necroohorum (18). Sequential postinoculation uterine biopsies and cultures were not obtained in that study; thus the effects of these organisms on the endometrium was not discerned. Actinomvces pyonenes was frequently recovered from the uteri of cows with pyometra, which supports previous findings (2,19). However, in addition to & pvonenes, one or more of the gram-negative anaerobic bacteria were The concurrent isolation of these bacteria from the frequently isolated. Spontaneous infected bovine uterus augments previous observations (2-4). infection with gram-negative anaerobic bacteria occurred even when cows were inoculated with 4. pyoaeneg alone. Because no cows were inoculated with only B melaninonenicus or E. necronhorum in our study, we do not know if the -. However, Campbell (18) reported that intrauterine converse would occur. infusion of E. necroohorum alone during the luteal phase of the cycle failed to produce pyometra in postpartum cows. histologic findings and the There was a strong association between During the postchallenge species of bacteria present in the uterine lumen. period; histologic endometritis was detected 91.7% of the time when either & with gram-negative anaerobes were isolated pvoaenes alone or A. pvonenes Other investigators have observed that 97.4% (19,20) and from the uterus. 100% (21) of cows with histologic endometritis had concurrent isolation of A.
985
MAY 1989 VOL. 31 NO. 5
THERIOGENOLOGY pyoae es from the uterine lumen. Studer and Morrow (22) also reported that associated with endometritis in postpartum dairy & DYnOPenCSwas frequently cows. In our study, bacteria classified as insignificant were considered to be contaminants of the uterus and were associated with few histopathologic changes, which is in agreement with earlier work (20). Pyometra has been defined as an accumulation of purulent exudate in the uterus associated with a retained corpus luteum (CL) and suppression of signs pyometra to exist regardless of CL of estrus (1). In this study, we considered lifespan although in most cases a CL was palpated in the same location on the ovary during sequential palpations. From a clinical viewpoint, the diagnosis of pyometra should not be predicated on the lifespan of the CL for several reasons. The lifespan of a corpus luteum cannot be determined by a single The necessity for return palpations and the resulting delay in palpation. diagnosis can lead to an increase in the number of days open and contribute to economic loss for the dairyman. Thus, we recommend a clinical definition in which we consider pyometra to exist if, upon palpation, a uterus is filled with fluid, lacks positive signs of pregnancy and may be thick walled, and a CL is present on one or both ovaries. In conclusion, pyometra was produced in cows by intrauterine infusion of with gram-negative anaerobic bacteria 1A Dvonenes alone or in combination during the luteal phase of the estrous cycle. Following inoculation, gramnegative anaerobic bacteria were frequently recovered concurrently with A. pvoeena from cows that developed pyometra; even from those inoculated with A. m alone. These results support the concept that anaerobic bacteria may act synergistically with & pvonen= in generating bovine pyomctra during the luteal phase of the cycle.
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Roberts, S.J. Veterinary Obstetrics and Genital Diseases SJ Roberts Publisher, Woodstock, VT, 1986, pp. 546-547.
2.
Olson, J.D., Ball, L., Mortimer, R.G., Farin, P.W., Adney, W.S. and Huffman, EM. Aspects of bacteriology and endocrinology of cows with pyometra and retained fetal membranes. Am. J. Vet. Res. 02251-2255 (1984).
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Peter, A.T. and Bosu, W.T.K. Effects of intrauterine infection on the function of the corpora lutea formed after first postpartum ovulations in dairy cows. Theriogenology 21:593-609 (1987).
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Ruder, C.A., Sasser, R.G., Williams, R.J., Ely, J.K., Bull, R.C. and Butler, J.E. Uterine infections in the postpartum cow II. Possible synergistic effect of Fusobacterium necroohorum and Corvnebacterium ovonenes. Theriogenology Li:573-580 (198 1).
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Roberts, D.S. The pathogenic synergy of Fusiformis Corvnebacterium ovoae es I. Influence of the leucocidal necroohorus. Br. J. ExpnPathol. =665-673 (1967).
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(Theriogenology).
necroohorus endotoxin
and of E.
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Gallagher. T.J. and Ball, L. exudate into the bovine uterus.
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Hawk, H.W., Brinsfield, T.H., Turner, G.D., Whitmore, Norcross, M.A. Effect of ovarian status on induced acute responses in cattle uteri. Am. J. Vet. Res. a362-366 (1964).
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Effect of Lynn, J.E., McNutt, S.H. and Casida, L.E. bacterial inoculation and suckling on the bovine corpus uterus. Am. J. Vet. Res. 2:152l-1526 (1966).
Effect of infusion of uterine purulent Theriogenology 13:31 I-319 (1980). G. W. and inflammatory
intrauterine luteum and
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Rommel, W. Experimentelle von c. Pvonenes im genitale IV Int. Congr. Anim. Reprod.,
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Brinsfield, T.H., Higginbotham, D.K. and Hawk, W.H. induced uterine infection at various stages of the estrous corpus luteum of the ewe. J. Anim. Sci. 29:616-618 (1969).
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Coudert, S.P. and Short, R.V. Prolongation corpus luteum in sheep with experimental Fertil. 2579-582 (1966).
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and pregnancy Eduvie, L.O. and Seguin, B.E. Corpus luteum function rate in lactating dairy cows given human chorionic gonadotropin at middiestrus. Theriogenology a415-422 (1982).
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Seguin, B.E., Morrow, D.A. and Louis, T.M. Luteolysis, luteostasis, and irritation. the effect of prostaglandin F, o in cows after endometrial Am. J. Vet. Res. =57-61 (1974).
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Holdeman, L.V., Cato, E.P., and Moore, W.E.C. (ed.). Bacteriology Manual. Anaerobic Laboratory, Virginia Institute and State University, Blacksburg, 1977, pp. I-152.
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Koneman, E.W., Allen, S.D., Dowell, Jr., V.R., and Sommers, H.M. Atlas and Textbook of Diagnostic Microbiology. JB Lippincott Philadelphia, 1983, pp. l-689.
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A biopsy instrument McQueen, R.D. samples for histologic and metabolic al881-1885 (1967).
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a model infection for drug evaluation. Campbell, D.L. Bovine pyometra Proc. of the Sot. for Theriogenology, Annual Mtg., Denver, CO. pp. 238239 abstr. (1984).
untersuchungen zur frage der pathogenitat Proc. des rindes wahrend des brustzyklus. The Hague, 1503-510 (1981).
of the functional uterine infections,
for obtaining investigations.
bovine Am.
Influence of cycle on the
life of the J. Reprod.
Anaerobe Polytechnic
Color Co.,
endometrial J. Vet. Res.
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Some observations on Hartigan, P.J., Griffin, J.F.T. and Nunn, W.R. Q v ebacte ‘u ovoaeneq infection of the bovine uterus. Theriogenology $3!167 (1;;4?
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Nonspecific uterine Griffin, J.F.T., Hartigan, P.J. and Nunn, W.R. infection and bovine fertility. 1. Infection patterns and endometritis during the first seven weeks postpartum. Theriogenology L:91-106 (1974).
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Miller, H.V., Kimsey, P.B., Kendrick, J.W., Darien, Et., Doering, I., Franti, C. and Horton, J. Endometritis of dairy cattle: Diagnosis, treatment and fertility. Bovine Practioner ul3-23 (1980).
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evaluation of bovine reproduStuder, E. and Morrow, D.A. Postpartum ctive potential: Comparison of findings from genital tract examination per rectum, uterine culture and endometrial biopsy. J. Am. Vet. Med. Assoc. 172:489-494 (1978).
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