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Culture of bovine embryos after in vitro exposure to Brucella abortus
THERIOGENOLOGY
CULTURE OF BOVINE EMBRYOS AFTER IN VITRO EXPOSURE TO BRUCELLA ABORTUS (a,b,c) -__ D.A. Stringfrl$ow,l C.M. S anIan,’ R.R. Brown,1 G.B. Meadows,B W. Gray, s R.R. Yr,ung-White’
1 Department 3 Department Auburn
of Microbiology, 2Animal of Anatomy and Histology, University, AL 36849
Received
for
Health Research and Scho~ol of Veterinary
publication: Accepted:
Medicine
December 17. 1983 April 19, 1984
Abstract
Fifty-four day-6 through day-10 festrus=day 01 embryos were collected nonsurgically from 13 superovulated, brucellosis-free mixed breed cows. Forty-eight excellent and go
Keywords:
Bovine,
Embryos,
Brucella
abortus
(a)
School of Veterinary Published as Publication No. 1664, Alabama Agricultural Experiment Station Journal Series 5-83552.
(b)
This
(c)
study
was
funded
12-164-341
between
Mention of identification
trade
in
Auburn
names purposes
JUNE 1984 VOL. 21 NO. 6
part
under
Cooperative
University
does not only.
imply
Agreement
and USDA, APHIS, endorsement
Medicine. No.
but
number
VS. is
for
1005
THERIOGENOLOGY Introduction
The potential for extensive international trade in bovine embryos has resulted from development of efficient means for their collection, storage and transfer. To date, health certification of these embryos dams and their respective herds of has involved testing of sires, origin for proof that disease-producing agents including _B. abortus were not present (1, 2). can infect the bovine fetus, but It is well known that -~ B. abortus infection during the period of the ovum has not been proven or there has been cause for concern because B. disproven. In addi t ion, abortus has been associated with the bovine uterus for extended periods postpartum and at times when cows would be used for superovulation and embryo retrieval (3-7). Previous attempts to isolate field strain B. abortus from the uterine flushings of culture-positive and/or serologic-reactor cows have provided negative results, thus leading to the conclusion that transfer of embryos from infected donors might be achieved without We recently reported the results of a transfer of infection (8, 9). more intense study on the postabortion persistence of B. abortus in the uterine lumen of infected cows (10). In this study involving brucellae were found in uterine flushings artificially infected cows, collected from two cows 36 and 41 days after abortion and subsequent to All flushings collected after their initial postgestational estrus. their second estrous period were negative. Due to the limited number of cows sampled in this latter study, it was impossible to establish a minimum number of days postpartum beyond which the uterine lumen might Further, be expected to be free of brucellae. there is always the concern about an animal that may have brucellae contaminating the environment of an embryo many months postpartum. This was described by Hawk et al. (Ll) in a report on a bacteriological study of uteri in They isolated B. abortus from clinically normal cows of low fertility. -___ uterine flushings collected from a cow at the same time as a viable Recognizing that this situation may be day-16 embryo was recovered. to transfer embryos from infected donors or encountered during attempts we undertook the present study to determine donors of unknown status, if zona pellucida-intact embryos could be washed free of brucellae after exposure. --in vitro
Materials
and Methods
Embryo Recovery and Grouping. Embryos were recovered from 13 mixed breed beef and dairy cows shown to be free of B. abortus infection by testing sera from blood samples collected monthly for at Sera were tested for anti-brucella antibodies with least twodyears. the card, standard tube agglutination, 2-mercaptoethanol, and Each donor cow was programmed for superovulation rivanol tests (12).
(d)
1006
Brewer 21201.
Diagnostic
Kit,
Hynson,
Westcott
and Dunning,
JUNE
Baltimore,
MD
1984 VOL. 21 NO. 6
THERIOGENOLOGY
and embryos were collected nonsurgically on days 6 through 10 The recovery medium was (estrus=day 0) as previously described (81. phosphate-buffered saline with 2% heat-inactivated fetal calf serum and no antibiotics (PBS-FCS) .e Embryos, recovered from uterine flush medium, were graded (131, and the stage of development was determined. The first 45 ZP-I embryos were grouped for washing, exposure, and culture procedures as follows: one 16-cell embryo, two morulas, and three blastocysts (two expanding) were processed individually. In addition, one group of two morulas, seven groups of three morulas, four and a group of four expanding groups of three expanding blastocysts, blastocysts were processed. The last nine embryos in the study were processed individually and were identified as follows: three ZP-I morulas, three ZP-D expanded-collapsed blastocysts and three hatched blastocysts. Trypticase agar slants with Preparation of Brucella Suspensions. 5% bovine serum were inoculated with B. abortus biotype I strain 2308 Each siant was harvested with 2 ml of and incubated for three days. PBS-FCS. Serial ten-fold dilutions of the brucella suspensions were made in PBS-FCS. The diluted suspensions (0.1 ml) were inoculated onto plates of trypticase agar with serum and antibiotics (TAS: trypticase agar plus 5% bovine serum; 100 mg cycloheximide; 6,000 units bacitracin; 60,000 units mycostatin per liter of medium) and spread for colony isolation with a sterile bent glass rod. After three days incubation, the colony-forming units (CFU) of brucellae were counted and concentrations of suspensions were recorded. A diluted suspension of brucellae was selected for the --in vitro exposure of embryos recovered from each cow. Two milliliters of PBS-FCS in each of Pre-exposure Wash Procedures. ten 35- x lo-mm sterile disposable tissue culture dishes constituted the sequential washes. Embryos were recovered from flush media and placed in the first wash dish in groups of one, two, three, or four. A 0.25-ml French straw was affixed to a tuberculin syringe and embryos were transferred from dish to dish as follows: each group was drawn into a straw (sequence of medium, air bubble, medium with embryos, and air) with a total volume of medium not exceeding 0.1 ml and expelled into the center of the next dish. The straw was rinsed twice by drawing approximately 0.2 ml of medium from the far edge of the dish Embryos were again drawn into the and expelling it into the near edge. straw as described above and carried to the next dish. The process was cant inued through ten dishes. In Vitro Exposure. After ten pre-exposure washes, each group of embryos was placed in 2 ml of a brucella suspension that contained from 2.0 x lo5 to- 1.8 x 1014 cells/ml and incubated for one to two hours at 37°C in a humidified atmosphere of air and 8% C02. Postexposure Wash Procedures. After exposure, the embryo --in vitro groups were transferred from the brucella suspensions to 2 ml of PBS-FCS and carried through ten washes using the same technique described above.
(e)
GIBCO Laboratories,
JUNE 1984 VOL. 21 NO. 6
Grand
Island,
NY
14072.
1007
THERIOGENOLOGY Bacterial Culture Procedures for Washes and Embryos. Three 0. l-ml samples from each wash dish were rnoculated on blood agar CBA), MacConkey agar (MA), and TAS plates and spread for colony isolation with a sterile bent glass rod. After three days’ incubation, the colony types and CFU on each medium were recorded. In addition, 1 ml of each of the postexposure washes for the last nine embryos (Table 2) was inoculated into tryptose broth and incubated. Each broth was subcultured (0.1 ml) on TAS plates after 3, 7, 10, 14, and 21 days or until a positive subculture was achieved. The TAS plates were incubated for seven days. After the tenth wash the groups of embryos were transferred to tryptose broth with 5% bovine serum (5 ml), frozen (-40°C) for 24 hours, thawed, and then incubated and subcultured as described above for broth cultures. All cultures were incubated in a humidified atmosphere of air and 8% CO2 at 37°C.
Results The grouping and culture results of 45 excellent and good ZP-I embryos and culture results of serial washes after in exposure of -- vitro the embryos to B. abortus are shown in Table 1. These embryos were recovered on days 6 through 8 (estrus=day 0). Stages of development included 16-cell through expanded blastocysts. Pre-exposure wash culture results are not shown, but Gram-positive bacteria were isolated from the first pre-exposure wash of embryo groups 4, 6 through 8 and 19. Brucellae were found in post-exposure washes 1 through 6 for two washes 1 through 5 for five groups, washes 1 through 4 for ten groups, and washes 1 through 3 for two groups. Brucellae were not groups, isolated from any of the 19 groups of embryos cultured. Culture results of the last nine embryos and their serial washes after exposure to B. abortus are presented in Table 2. --in vitro Brucellae were not isolated-from any of the three ZP-I day-7 embryos, and the washes were culture-negative after the third wash for two embryos and the fifth wash for one embryo. Embryos 4 and 5 had defects in their zonae pellucidae and were culture-negative. Brucellae were not found beyond the fifth and sixth wash, respectively, of these embryos. Embryo 6, also a ZP-D day-9 embryo, was culture-positive for brucel lae. The ninth wash for this embryo was the last culture-positive wash, but the sixth and eighth washes were negative. Embryo 7, a day-9 hatched blastocyst of excellent quality, was found to be free of brucellae after washing. Culture-positive washes from this embryo included washes 1 through 5, 7, 9, and 10. Embryos 8 and 9, both excellent day-10 blastocysts, also produced negative culture results. Brucellae were found in washes number 1 through 5, 7, 8, and 10 for embryo 8 and washes number 1 through 5, 7 and 8 for embryo 9.
Discussion
flora
1008
It was necessary or contaminants)
to pre-wash embryos to remove which would overgrow brucellae
bacteria (uterine in post-exposure
JUNE 1984 VOL. 21 NO. 6
THERIOGENOLOGY
Table
1.
Culture Washes
Results of Zona Pellucida-Intact Embryos and Serial After In Vitro Exposure of Embryos to -B. ____abortus ____
Embryos/ wash group 1 1 1 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 4
Embryo group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 I
CFU = colony-forming 16C= 16-cell Mor = morula = blastocyst Bc
Table
2.
Embryo 1 2 3 4 5 6 7 8 9 CFU ZP-I ZP-D ZP-F
JUNE
= = = =
Culture Washes
Stage of embryos j day, 16C 7 day, Mor 7 day, Mor 7 day, Bc 8 day, Bc 8 day, Bc 7 day, Mor 7 day, Mar 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mar 8 day. Bc 8 day. Bc 8 day, Bc 8 day, Bc 8 day, Bc
xposure dose of abortus(CFU/ml) 2.0x10’ 3.6x106 6.4~10~~ 2.0x107 4.8~10~ 4.3x106 2 .4x1D6 2 .4x10b 2.5X107 2.6x107 2.5xlD7 d
2.fk11~7
6.4~10~~) 6.4~10~~ 3.0x109 3.0x109 3.0x109 2. 2x 106 2 .2x106
.ast losit
cultureive wash 5 4 4 6 5 6 3 5 4 3 4 4 4
units
ZP-D, Results of ZP-I, after In Vitro Exposure ____
Exposure dose of Condition B. abortus(CFU/ml) of embryo 7 day, ZP-I 4.6~108 7 day, ZP-I 4.6~108 7 day, ZP-I 4.6~10~ 9 day, ZP-D 1.8x1014 9 day, ZP-D 1.8x1014 9 day, ZP-D 1.8~10~~ 9 day, ZP-F 1 .8x1014 10 day, ZP-F 3.4x109 10 day, ZP-F 3.4x109 colony-forming units zona pellucida-intact zona pellucida-defect zona pellucida-free
1984 VOL. 21 NO. 6
and ZP-F Embryos to -B. abortus
and Serial
Last culture-pos. wash Broth _Plate 3 5 3 3 2 3 3 5 4 6 3 9 4 Ill 4 In 4 8
’
Embry-, culture Nei: Neg NI3$ NSF g
Nvg P(1s Nt’ t’ Ner.
NtL g
ive
1009
THERIOGENOLOGY wash and embryo cultures. The flush medium collected from 4 of the 13 donor cows was contaminated. The source of these bacteria (uterine flora or procedural contaminants) was not determined: however, the fact that all prewashes of embryos collected from nine donors were bacteriologically sterile is an indication of the efficiency of the “aseptic” nonsurgical recovery technique. It is not likely that brucellae will be retrieved with embryos collected from infected donor cows which have been allowed to cycle several times postcalving or postabortion (8, 9). However, based on information presently available, this possibility can not be excluded (10, 11). Washing and the use of antibiotics in medium are logical means of handling embryos to insure that brucellae will not be transferred. Embryos in this study were exposed in vitro to quantities of -brucellae that were probably in excess of what they would be exposed to in viva. Over one half the exposure doses approximated or exceeded the concentration of B. abortus found in grossly contaminated material such as fetal fluid and fetal placenta from infected cows (14). Exposure, washing, and subsequent negative culture of the 19 groups of embryos (Table 1) demonstrate a lack of affinity of brucellae for the intact zona pellucida. The numbers of brucellae, which were great in the initial washes, diminished rapidly, and the last positive washes often yielded fewer than five colonies per plate, indicating simple serial dilution. Care was taken to confirm the integrity of the zona pellucida of each embryo so far as was possible at 50x magnif icat ion, and all developmental stages of ZP-I embryos commonly recovered by nonsurgical means were represented up to and including the expanding blastocyst. The nine embryos listed in Table 2 were exposed, washed, and cultured as for previous embryos in this study. To increase the sensitivity of our methods for detecting brucellae in the washes, we supported our plate inoculations by inoculating broth cultures with larger volumes of the wash media. Embryos 1 through 3 were ZP-I. The last positive wash for embryo 2, detected by both plate and broth cultures, was the third. Broth cultures for the washing from this embryo were positive at the day-3 subculture. There was a total of 21 colonies on the BA and TAS plates inoculated from wash 2 (the last plate-positive wash) for embryo 3. Plates from wash 3 were negative but broth subcultures at day 3 were positive. Wash 3 was the last positive wash for embryo 1. A total of seven colonies was found on BA and TAS plates inoculated from this wash. Plates inoculated from the fourth and fifth washes for this embryo were negative; but broths, while negative at three days, were positive for both at seven days. It was apparent that the broth cultures were capable of detecting extremely small numbers of viable brucellae, yet the last positive wash, as determined by comparing broth to plate cultures, closely approximated each other. Embryos 4 through 6 (Table 2) were expanded-collapsed blastocysts. When examined at 50x magnification, embryos 4 and 5 were observed to have cracks in their zonae pellucidae. B. abortus was not isolated from either of these embryos and washes past the fifth and sixth, respectively, were negat ive. Results were not appreciably different at 50x magnification, from the ZP-I embryos. Embryo 6, when examined was observed to have a break and a gap in the zona pellucida. -8.
1010
JUNE 1984 VOL. 21 NO. 6
THERIOGENOLOGY
abortus was detected in the broth culture of this embryo at the day-7 ___The last positive wash (ninth) was preceded by two subculture. negative washes (sixth and eighth). We concluded that brucellae passed through the gap in the zona pellucida and in latter washes some were removed. This embryo provided a positive control for our culture system in addition to demonstrating the obvious hazard of trying to transfer such an embryo. Brucellae were Embryos 7 through 9 were hatched embryos (ZP-F). not cultured from these embryos, but small numbers of brucellae were carried along with them through the tenth washes for embryos 7 and 8 The intermittent presence of small and the eight wash for embryo 9. numbers of brucellae in the latter washes for these three embryos demonstrated that the brucellae were loosely attached. It remains to be seen if longer exposure will result in infection of embryos. We cannot say that ZP-F embryos can be safely transferred from infected animals. In summary, a comparison of our plate and broth cultures and the positive culture of one ZP-D embryo demonstrated the sensitivity of our culture systems. After short-term in vitro exposure to -B. abortus, -___ there was no tendency for brucellae to attach to ZP-I embryos, although they appeared to adhere loosely to ZP-F embryos. Results provide additional information that will aid in devising a scheme for safe transfer of embryos from brucellosis-infected donors or donors whose brucellosis infection status is unknown.
References
1.
Eaglesome, M.D., Hare, W.C.D. and Singh, E.L. Embryo transfer: discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. -21:106-112 (1980).
2.
Waters, H.A. Theriogenology
3.
Manthei, infection
4.
Hignett, P.G., Nagy, L.K. and Ironside, C.J.T. Bovine A study of an adult-vaccinated brucella-infected brucellosis: herd. Isolation of Brucella abortus from infected animals. Rec. -80:586-590 (1967).
Health certification (1981). -15:57-66
for
livestock
embryo
transfer.
Persistence of Brucella abortus C.A. and Carter, R.W. in cattle. Am. J. Vet. Res. (1950). -11:173-180
Studies in (1921).
abortion.
J.
Am. Vet.
Med.
Huddleson, I.F. Assoc. 58:524-531 -
6.
The agglutination test in relation Birch, R.R. and Gilman, H.L. to the persistence of Bacterium abortus in the body of the COW. Ann. Rept., N.Y. State Vet. Coll., Cornell Univ., 1929-1930:56-88 (1931).
1984 VOL. 21 NO. 6
infectious
Vet.
5.
JUNE
A
1011
THERIOGENOLOGY
7.
Fitch, C.P., Boyd, W.L., Localization of -Brucella Vet. -29:253-260 (1939).
Bishop, ~abortus
L.M. and Kelly. M.D. in the bovine uterus.
Cornell
8.
P.R. Stringfellow, D.A., Howell, V.L. and Schnurrenberger, Investigations into the potential for embryo transfer from Brucella ---abortus infected cows without transmission of infection. ____ Theriogenology 18:733-743 (1982).
9.
Voelkel, S.A., Stuckey, K.W., Looney, C.R., Enright, F.M., Humes, An attempt to isolate Brucella abortus from P.E. and Godke, R.A. uterine flushings of brucellosis-reactor donor cattle. Theriogenology -19:355-366 (1983).
10.
Stringfellow, D.A., Scanlan, C.M., Hannon, S.S., Panangala, V.S., Culture of uterine flushings, cervical Gray, B.W. and Galik, P.A. collected postabortion from heifers mucus, and udder secretions artificially exposed to Brucella abortus. 'Iheriogenology -20:77-83 (1983).
11.
M. and Winter, Hawk, H.W., Kiddy, C.A.. Wilson, J.B., Esposito, Bacteriological studies of uteri of clinically normal cows A.J. of low fertility. J. Dairy Sci. -41:120-128 (1958).
12.
USDA, APHIS, VS, Brucellosis Eradication Uniform Methods Effective April 1, 1982, APHIS 91-1 pp.20-22. Rules.
13.
Lindner, G.M. and Wright, R.W. Bovine embryo morphology and evaluation. Proceedings of the Workshop: IXth Annual Meeting 1983, pp. 21-32. the International Embryo Transfer Society.
14.
1012
and
of
Alexander, B., Schnurrenberger, P.R. and Brown,R.R. Numbers of Brucella abortus in the placenta, umbilicus and fetal fluid of two naturallywed cows. Vet. Rec. 108:500 (1981).
JUNE 1984 VOL. 21 NO. 6
CULTURE OF BOVINE EMBRYOS AFTER IN VITRO EXPOSURE TO BRUCELLA ABORTUS (a,b,c) -__ D.A. Stringfrl$ow,l C.M. S anIan,’ R.R. Brown,1 G.B. Meadows,B W. Gray, s R.R. Yr,ung-White’
1 Department 3 Department Auburn
of Microbiology, 2Animal of Anatomy and Histology, University, AL 36849
Received
for
Health Research and Scho~ol of Veterinary
publication: Accepted:
Medicine
December 17. 1983 April 19, 1984
Abstract
Fifty-four day-6 through day-10 festrus=day 01 embryos were collected nonsurgically from 13 superovulated, brucellosis-free mixed breed cows. Forty-eight excellent and go
Keywords:
Bovine,
Embryos,
Brucella
abortus
(a)
School of Veterinary Published as Publication No. 1664, Alabama Agricultural Experiment Station Journal Series 5-83552.
(b)
This
(c)
study
was
funded
12-164-341
between
Mention of identification
trade
in
Auburn
names purposes
JUNE 1984 VOL. 21 NO. 6
part
under
Cooperative
University
does not only.
imply
Agreement
and USDA, APHIS, endorsement
Medicine. No.
but
number
VS. is
for
1005
THERIOGENOLOGY Introduction
The potential for extensive international trade in bovine embryos has resulted from development of efficient means for their collection, storage and transfer. To date, health certification of these embryos dams and their respective herds of has involved testing of sires, origin for proof that disease-producing agents including _B. abortus were not present (1, 2). can infect the bovine fetus, but It is well known that -~ B. abortus infection during the period of the ovum has not been proven or there has been cause for concern because B. disproven. In addi t ion, abortus has been associated with the bovine uterus for extended periods postpartum and at times when cows would be used for superovulation and embryo retrieval (3-7). Previous attempts to isolate field strain B. abortus from the uterine flushings of culture-positive and/or serologic-reactor cows have provided negative results, thus leading to the conclusion that transfer of embryos from infected donors might be achieved without We recently reported the results of a transfer of infection (8, 9). more intense study on the postabortion persistence of B. abortus in the uterine lumen of infected cows (10). In this study involving brucellae were found in uterine flushings artificially infected cows, collected from two cows 36 and 41 days after abortion and subsequent to All flushings collected after their initial postgestational estrus. their second estrous period were negative. Due to the limited number of cows sampled in this latter study, it was impossible to establish a minimum number of days postpartum beyond which the uterine lumen might Further, be expected to be free of brucellae. there is always the concern about an animal that may have brucellae contaminating the environment of an embryo many months postpartum. This was described by Hawk et al. (Ll) in a report on a bacteriological study of uteri in They isolated B. abortus from clinically normal cows of low fertility. -___ uterine flushings collected from a cow at the same time as a viable Recognizing that this situation may be day-16 embryo was recovered. to transfer embryos from infected donors or encountered during attempts we undertook the present study to determine donors of unknown status, if zona pellucida-intact embryos could be washed free of brucellae after exposure. --in vitro
Materials
and Methods
Embryo Recovery and Grouping. Embryos were recovered from 13 mixed breed beef and dairy cows shown to be free of B. abortus infection by testing sera from blood samples collected monthly for at Sera were tested for anti-brucella antibodies with least twodyears. the card, standard tube agglutination, 2-mercaptoethanol, and Each donor cow was programmed for superovulation rivanol tests (12).
(d)
1006
Brewer 21201.
Diagnostic
Kit,
Hynson,
Westcott
and Dunning,
JUNE
Baltimore,
MD
1984 VOL. 21 NO. 6
THERIOGENOLOGY
and embryos were collected nonsurgically on days 6 through 10 The recovery medium was (estrus=day 0) as previously described (81. phosphate-buffered saline with 2% heat-inactivated fetal calf serum and no antibiotics (PBS-FCS) .e Embryos, recovered from uterine flush medium, were graded (131, and the stage of development was determined. The first 45 ZP-I embryos were grouped for washing, exposure, and culture procedures as follows: one 16-cell embryo, two morulas, and three blastocysts (two expanding) were processed individually. In addition, one group of two morulas, seven groups of three morulas, four and a group of four expanding groups of three expanding blastocysts, blastocysts were processed. The last nine embryos in the study were processed individually and were identified as follows: three ZP-I morulas, three ZP-D expanded-collapsed blastocysts and three hatched blastocysts. Trypticase agar slants with Preparation of Brucella Suspensions. 5% bovine serum were inoculated with B. abortus biotype I strain 2308 Each siant was harvested with 2 ml of and incubated for three days. PBS-FCS. Serial ten-fold dilutions of the brucella suspensions were made in PBS-FCS. The diluted suspensions (0.1 ml) were inoculated onto plates of trypticase agar with serum and antibiotics (TAS: trypticase agar plus 5% bovine serum; 100 mg cycloheximide; 6,000 units bacitracin; 60,000 units mycostatin per liter of medium) and spread for colony isolation with a sterile bent glass rod. After three days incubation, the colony-forming units (CFU) of brucellae were counted and concentrations of suspensions were recorded. A diluted suspension of brucellae was selected for the --in vitro exposure of embryos recovered from each cow. Two milliliters of PBS-FCS in each of Pre-exposure Wash Procedures. ten 35- x lo-mm sterile disposable tissue culture dishes constituted the sequential washes. Embryos were recovered from flush media and placed in the first wash dish in groups of one, two, three, or four. A 0.25-ml French straw was affixed to a tuberculin syringe and embryos were transferred from dish to dish as follows: each group was drawn into a straw (sequence of medium, air bubble, medium with embryos, and air) with a total volume of medium not exceeding 0.1 ml and expelled into the center of the next dish. The straw was rinsed twice by drawing approximately 0.2 ml of medium from the far edge of the dish Embryos were again drawn into the and expelling it into the near edge. straw as described above and carried to the next dish. The process was cant inued through ten dishes. In Vitro Exposure. After ten pre-exposure washes, each group of embryos was placed in 2 ml of a brucella suspension that contained from 2.0 x lo5 to- 1.8 x 1014 cells/ml and incubated for one to two hours at 37°C in a humidified atmosphere of air and 8% C02. Postexposure Wash Procedures. After exposure, the embryo --in vitro groups were transferred from the brucella suspensions to 2 ml of PBS-FCS and carried through ten washes using the same technique described above.
(e)
GIBCO Laboratories,
JUNE 1984 VOL. 21 NO. 6
Grand
Island,
NY
14072.
1007
THERIOGENOLOGY Bacterial Culture Procedures for Washes and Embryos. Three 0. l-ml samples from each wash dish were rnoculated on blood agar CBA), MacConkey agar (MA), and TAS plates and spread for colony isolation with a sterile bent glass rod. After three days’ incubation, the colony types and CFU on each medium were recorded. In addition, 1 ml of each of the postexposure washes for the last nine embryos (Table 2) was inoculated into tryptose broth and incubated. Each broth was subcultured (0.1 ml) on TAS plates after 3, 7, 10, 14, and 21 days or until a positive subculture was achieved. The TAS plates were incubated for seven days. After the tenth wash the groups of embryos were transferred to tryptose broth with 5% bovine serum (5 ml), frozen (-40°C) for 24 hours, thawed, and then incubated and subcultured as described above for broth cultures. All cultures were incubated in a humidified atmosphere of air and 8% CO2 at 37°C.
Results The grouping and culture results of 45 excellent and good ZP-I embryos and culture results of serial washes after in exposure of -- vitro the embryos to B. abortus are shown in Table 1. These embryos were recovered on days 6 through 8 (estrus=day 0). Stages of development included 16-cell through expanded blastocysts. Pre-exposure wash culture results are not shown, but Gram-positive bacteria were isolated from the first pre-exposure wash of embryo groups 4, 6 through 8 and 19. Brucellae were found in post-exposure washes 1 through 6 for two washes 1 through 5 for five groups, washes 1 through 4 for ten groups, and washes 1 through 3 for two groups. Brucellae were not groups, isolated from any of the 19 groups of embryos cultured. Culture results of the last nine embryos and their serial washes after exposure to B. abortus are presented in Table 2. --in vitro Brucellae were not isolated-from any of the three ZP-I day-7 embryos, and the washes were culture-negative after the third wash for two embryos and the fifth wash for one embryo. Embryos 4 and 5 had defects in their zonae pellucidae and were culture-negative. Brucellae were not found beyond the fifth and sixth wash, respectively, of these embryos. Embryo 6, also a ZP-D day-9 embryo, was culture-positive for brucel lae. The ninth wash for this embryo was the last culture-positive wash, but the sixth and eighth washes were negative. Embryo 7, a day-9 hatched blastocyst of excellent quality, was found to be free of brucellae after washing. Culture-positive washes from this embryo included washes 1 through 5, 7, 9, and 10. Embryos 8 and 9, both excellent day-10 blastocysts, also produced negative culture results. Brucellae were found in washes number 1 through 5, 7, 8, and 10 for embryo 8 and washes number 1 through 5, 7 and 8 for embryo 9.
Discussion
flora
1008
It was necessary or contaminants)
to pre-wash embryos to remove which would overgrow brucellae
bacteria (uterine in post-exposure
JUNE 1984 VOL. 21 NO. 6
THERIOGENOLOGY
Table
1.
Culture Washes
Results of Zona Pellucida-Intact Embryos and Serial After In Vitro Exposure of Embryos to -B. ____abortus ____
Embryos/ wash group 1 1 1 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 4
Embryo group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 I
CFU = colony-forming 16C= 16-cell Mor = morula = blastocyst Bc
Table
2.
Embryo 1 2 3 4 5 6 7 8 9 CFU ZP-I ZP-D ZP-F
JUNE
= = = =
Culture Washes
Stage of embryos j day, 16C 7 day, Mor 7 day, Mor 7 day, Bc 8 day, Bc 8 day, Bc 7 day, Mor 7 day, Mar 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mor 7 day, Mar 8 day. Bc 8 day. Bc 8 day, Bc 8 day, Bc 8 day, Bc
xposure dose of abortus(CFU/ml) 2.0x10’ 3.6x106 6.4~10~~ 2.0x107 4.8~10~ 4.3x106 2 .4x1D6 2 .4x10b 2.5X107 2.6x107 2.5xlD7 d
2.fk11~7
6.4~10~~) 6.4~10~~ 3.0x109 3.0x109 3.0x109 2. 2x 106 2 .2x106
.ast losit
cultureive wash 5 4 4 6 5 6 3 5 4 3 4 4 4
units
ZP-D, Results of ZP-I, after In Vitro Exposure ____
Exposure dose of Condition B. abortus(CFU/ml) of embryo 7 day, ZP-I 4.6~108 7 day, ZP-I 4.6~108 7 day, ZP-I 4.6~10~ 9 day, ZP-D 1.8x1014 9 day, ZP-D 1.8x1014 9 day, ZP-D 1.8~10~~ 9 day, ZP-F 1 .8x1014 10 day, ZP-F 3.4x109 10 day, ZP-F 3.4x109 colony-forming units zona pellucida-intact zona pellucida-defect zona pellucida-free
1984 VOL. 21 NO. 6
and ZP-F Embryos to -B. abortus
and Serial
Last culture-pos. wash Broth _Plate 3 5 3 3 2 3 3 5 4 6 3 9 4 Ill 4 In 4 8
’
Embry-, culture Nei: Neg NI3$ NSF g
Nvg P(1s Nt’ t’ Ner.
NtL g
ive
1009
THERIOGENOLOGY wash and embryo cultures. The flush medium collected from 4 of the 13 donor cows was contaminated. The source of these bacteria (uterine flora or procedural contaminants) was not determined: however, the fact that all prewashes of embryos collected from nine donors were bacteriologically sterile is an indication of the efficiency of the “aseptic” nonsurgical recovery technique. It is not likely that brucellae will be retrieved with embryos collected from infected donor cows which have been allowed to cycle several times postcalving or postabortion (8, 9). However, based on information presently available, this possibility can not be excluded (10, 11). Washing and the use of antibiotics in medium are logical means of handling embryos to insure that brucellae will not be transferred. Embryos in this study were exposed in vitro to quantities of -brucellae that were probably in excess of what they would be exposed to in viva. Over one half the exposure doses approximated or exceeded the concentration of B. abortus found in grossly contaminated material such as fetal fluid and fetal placenta from infected cows (14). Exposure, washing, and subsequent negative culture of the 19 groups of embryos (Table 1) demonstrate a lack of affinity of brucellae for the intact zona pellucida. The numbers of brucellae, which were great in the initial washes, diminished rapidly, and the last positive washes often yielded fewer than five colonies per plate, indicating simple serial dilution. Care was taken to confirm the integrity of the zona pellucida of each embryo so far as was possible at 50x magnif icat ion, and all developmental stages of ZP-I embryos commonly recovered by nonsurgical means were represented up to and including the expanding blastocyst. The nine embryos listed in Table 2 were exposed, washed, and cultured as for previous embryos in this study. To increase the sensitivity of our methods for detecting brucellae in the washes, we supported our plate inoculations by inoculating broth cultures with larger volumes of the wash media. Embryos 1 through 3 were ZP-I. The last positive wash for embryo 2, detected by both plate and broth cultures, was the third. Broth cultures for the washing from this embryo were positive at the day-3 subculture. There was a total of 21 colonies on the BA and TAS plates inoculated from wash 2 (the last plate-positive wash) for embryo 3. Plates from wash 3 were negative but broth subcultures at day 3 were positive. Wash 3 was the last positive wash for embryo 1. A total of seven colonies was found on BA and TAS plates inoculated from this wash. Plates inoculated from the fourth and fifth washes for this embryo were negative; but broths, while negative at three days, were positive for both at seven days. It was apparent that the broth cultures were capable of detecting extremely small numbers of viable brucellae, yet the last positive wash, as determined by comparing broth to plate cultures, closely approximated each other. Embryos 4 through 6 (Table 2) were expanded-collapsed blastocysts. When examined at 50x magnification, embryos 4 and 5 were observed to have cracks in their zonae pellucidae. B. abortus was not isolated from either of these embryos and washes past the fifth and sixth, respectively, were negat ive. Results were not appreciably different at 50x magnification, from the ZP-I embryos. Embryo 6, when examined was observed to have a break and a gap in the zona pellucida. -8.
1010
JUNE 1984 VOL. 21 NO. 6
THERIOGENOLOGY
abortus was detected in the broth culture of this embryo at the day-7 ___The last positive wash (ninth) was preceded by two subculture. negative washes (sixth and eighth). We concluded that brucellae passed through the gap in the zona pellucida and in latter washes some were removed. This embryo provided a positive control for our culture system in addition to demonstrating the obvious hazard of trying to transfer such an embryo. Brucellae were Embryos 7 through 9 were hatched embryos (ZP-F). not cultured from these embryos, but small numbers of brucellae were carried along with them through the tenth washes for embryos 7 and 8 The intermittent presence of small and the eight wash for embryo 9. numbers of brucellae in the latter washes for these three embryos demonstrated that the brucellae were loosely attached. It remains to be seen if longer exposure will result in infection of embryos. We cannot say that ZP-F embryos can be safely transferred from infected animals. In summary, a comparison of our plate and broth cultures and the positive culture of one ZP-D embryo demonstrated the sensitivity of our culture systems. After short-term in vitro exposure to -B. abortus, -___ there was no tendency for brucellae to attach to ZP-I embryos, although they appeared to adhere loosely to ZP-F embryos. Results provide additional information that will aid in devising a scheme for safe transfer of embryos from brucellosis-infected donors or donors whose brucellosis infection status is unknown.
References
1.
Eaglesome, M.D., Hare, W.C.D. and Singh, E.L. Embryo transfer: discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. -21:106-112 (1980).
2.
Waters, H.A. Theriogenology
3.
Manthei, infection
4.
Hignett, P.G., Nagy, L.K. and Ironside, C.J.T. Bovine A study of an adult-vaccinated brucella-infected brucellosis: herd. Isolation of Brucella abortus from infected animals. Rec. -80:586-590 (1967).
Health certification (1981). -15:57-66
for
livestock
embryo
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Persistence of Brucella abortus C.A. and Carter, R.W. in cattle. Am. J. Vet. Res. (1950). -11:173-180
Studies in (1921).
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Am. Vet.
Med.
Huddleson, I.F. Assoc. 58:524-531 -
6.
The agglutination test in relation Birch, R.R. and Gilman, H.L. to the persistence of Bacterium abortus in the body of the COW. Ann. Rept., N.Y. State Vet. Coll., Cornell Univ., 1929-1930:56-88 (1931).
1984 VOL. 21 NO. 6
infectious
Vet.
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JUNE
A
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THERIOGENOLOGY
7.
Fitch, C.P., Boyd, W.L., Localization of -Brucella Vet. -29:253-260 (1939).
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L.M. and Kelly. M.D. in the bovine uterus.
Cornell
8.
P.R. Stringfellow, D.A., Howell, V.L. and Schnurrenberger, Investigations into the potential for embryo transfer from Brucella ---abortus infected cows without transmission of infection. ____ Theriogenology 18:733-743 (1982).
9.
Voelkel, S.A., Stuckey, K.W., Looney, C.R., Enright, F.M., Humes, An attempt to isolate Brucella abortus from P.E. and Godke, R.A. uterine flushings of brucellosis-reactor donor cattle. Theriogenology -19:355-366 (1983).
10.
Stringfellow, D.A., Scanlan, C.M., Hannon, S.S., Panangala, V.S., Culture of uterine flushings, cervical Gray, B.W. and Galik, P.A. collected postabortion from heifers mucus, and udder secretions artificially exposed to Brucella abortus. 'Iheriogenology -20:77-83 (1983).
11.
M. and Winter, Hawk, H.W., Kiddy, C.A.. Wilson, J.B., Esposito, Bacteriological studies of uteri of clinically normal cows A.J. of low fertility. J. Dairy Sci. -41:120-128 (1958).
12.
USDA, APHIS, VS, Brucellosis Eradication Uniform Methods Effective April 1, 1982, APHIS 91-1 pp.20-22. Rules.
13.
Lindner, G.M. and Wright, R.W. Bovine embryo morphology and evaluation. Proceedings of the Workshop: IXth Annual Meeting 1983, pp. 21-32. the International Embryo Transfer Society.
14.
1012
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Alexander, B., Schnurrenberger, P.R. and Brown,R.R. Numbers of Brucella abortus in the placenta, umbilicus and fetal fluid of two naturallywed cows. Vet. Rec. 108:500 (1981).
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