Association of Leptospira borgpetersenii serover hardjo type hardjobovis with bovine ova and embryos produced by in vitro fertilization

ELSEVIER

ASSOCIATION OF B TYPE HARDJOBOVIS

BORGPE!FERSENI~SEROVAR JiARDJO WITH BOVINE OVA AND EMBRYOS PRODUCED by IN VITRO FERTILIZATION

A. Bielanski and 0. Surujballi Agriculture and Agri-Food Canada, Animal Diseases Research Institute, Nepean, Ontario, Canada, K2H 8P9 Received

for publication: Accepted:

August 11, 1995 Jmuary 10, 1996

ABSTRACT The association of Lewtoswira borcmeterseni4 serovar hardio type hardjobovis with in vitro fertilized (IVF) embryos was investigated in in vivo and in vitro. The organism could not be isolated from IVF embryos that were generated from ova harvested from experimentally infected donor heifers and cultured in commercial INRA Menezo B2 medium containing low levels of penicillin and streptomycin. Recipient heifers (n=14) to which embrvos cultured in Menezo B2 medium were transferred. did not develop antibody titres to Lewtoswira, and this organism was not isolated from the resultina fetuses (n=5). Similarlv. the organism could not be isolated from IVF embryos that-were produced from in vitro-exposed oocytes to &ewtosw&R and cultured in Menezo B2 medium, or TCM-199 medium supplemented with penicillin and streptomycin. In contrast, leptospires were isolated from IVF embryos that were produced from in vitro- exposed oocytes and cultured in TCM199 medium free of antibiotics. The sequential washing procedure that was used with media free of antibiotics did not render infected embrvos free of the nathosenic microorsanism. Oocvtes exposed to Ukoswira in vitro were not transferred into the recipients. The presence of serovar bardiQ in the IVF system had no detrimental effect on fertilization rates or on embryonic development to the blastocyst stage. This study shows that it is possible to obtain transferable stage embryos from oocytes recovered from infected donors and from oocytes exposed in vitro to Gewtoswia. Such embryos may be associated with the microorganism, and the use of culture media supplemented with antibiotics for processing IVF embryos would be advisable in order to prevent any risk of transmission. Key

words:

fertilization,

leptospirosis,

embryos,

zona pellucida

Acknowledgments The authors extend appreciation to Mr. G. Raby and Mr. R. Marenger for their skilled technical assistance.

Theriogenology 46:4555. 1996 0 1996 by Elsevier Science Inc.

0093-691X/96/$15.00 PII SOO93-691X(96)00140-2

Theriogenology

46

INTRODUCTION Leotosnirosis is an infectious disease of animals and man caused by the spirochaete Leotosoira which is widespread throushout the world (1). These microorganisms are divided into serogroups based on antigenic relationships determined by crossagglutination reactions and are further subdivided into serovars by agglutinin-absorption patterns (2). So far, more than 200 serovars have been recognized; however, only a relatively small number of them are endemic in any one geographic region (17), and each serovar tends to be maintained in specific species hosts (12, 17). In the North American cattle population, serovar bardio has been identified as the major cause of leptospirosis (13, 18). Natural infection of cattle with serovar hardjo causes infertility and abortion (11). The organism has been found in the urinary and reproductive tract of infected female (9, 10) and male animals (8). In the available literature, there are no data on the possibility of transmission of A. &rpmterseni4 serovar hardio through embryo transfer (ET) in cattle. The aims of the present study were to determine 1) whether IVF embryos, from cows experimentally infected with serovar bardis type hardjobovis , or oocytes exposed to hardjobovis in vitro, are associated with the pathogenic agent when cultured (co-cultured) with oviductal cells ( bovine oviductal epithelial cells , BOEC) in medium with or without antibiotics, and 2) whether these embryos can develop to transferable stages . MATERIALS AND METHODS Procedure for In Vitro Production of Embryos Oocvte maturation. The maturation medium was TCM-199 with Earle's salts supplemented with 25 mM Hepes, 20% noninactivated estrous bovine serum (anti-Leotosnira antibody-free), 35 pg/ml porcine follicle stimulating hormone (Folltropin, Vetrepharm, London, Ontario, Canada), 10 IU/ml hCG (Chorionad, Vetcom, Quebec, Canada), 0.4 mM L-glutamine (Sigma, St Louis, MO), and 0.2 mM sodium pyruvate (Sigma). This medium was not supplemented with antibiotics. Oocytes in groups of 10 to 15 were matured in 50~1 drops of maturation medium overlaid with silicone oil (Dow Corning, Midland, MI) for 22 h in culture dishes (Nunc, InterMed, Kamstrup, Denmark) at 38.5OC in air with 5% CO, and 100% humidity. In vitro fertilization of oocvtes. oocvtes were washed 3 times in modified HEPES) and once in fertilization medium drops of IVF-TALP contained heparin (10 suspension.

After maturation, the Tvrode's solution (TALP(IVF-TALP; 4). The-50pl sg/ml) and 1~1 of BOEC

A swim-up method described by Parrish et al. (14) was used

Theriogenology

47

to prepare spermatozoa for IVF. Pooled frozen/thawed semen obtained from 6 commercialy available bulls that were listed above was used. The seronegative for the 6 serovars oocytes were inseminated in IVF-TALP drops (50~1) with approximately lx106/ml of motile sperm cells and then incubated for 18 h at 38.5OC in air with 5% COz. The fertilization and washing media were not supplemented with antibiotics. Preparation

of Bovine Oviductal

Epithelial

Cells

(BOEC)

Bovine oviductal epithelial cell suspension cocultures were prepared as described previously from oviducts of seronegative and Leotosoira free heifers (5). Suspensions of BOEC were added to the fertilization medium and to the zygote coculture medium. Embryo Co-culture Eighteen hours after fertilization, cumulus cells were removed from the zygotes by vortex agitation (2 min) in 1.5 ml of PBS (pH 7.2, 295 mOsm). The zygotes were then washed twice in PBS and once in co-culture medium. The co-culture was conducted in either TCW-199 medium with Earle's salts (with and without antibiotics) or in Menezo B2 medium. Both media were supplemented with 1 ~1 BOEC and 20% noninactivated estrus bovine serum (antiLedoswira antibody- free). After washing, 5 to 10 zygotes were placed in each droplet of co-culture medium and incubated under silicone oil for 8 d at 38.5OC in air with 5% CO,. The zygotes were examined for cleavage 72 h after fertilization and again at the end of the incubation period. The numbers of embryos that developed to morula and blastocyst stages were determined. Oocyte

and Embryo Washing

A semi-automatic micropipette fitted with a 5~1 plastic microtip was used to transfer the oocytes or embryos to a sterile dish (35x10 mm Falcon 3100) containing 2.5 ml of fresh medium. The dish was gently swirled for a few seconds, and the procedure repeated with a new pipette tip. Day 7 embryos were washed 10 times prior to bacterial isolation attempts or processing for scanning electron microscopy. The washing procedure was performed according to the methodology recommended by the International Embryo Transfer Society (IETS; (15), consisting of successive transfers of embryos into fresh aliquots of medium. In Vitro

Experiments

Bovine ovaries were collected from donors at a local abattoir and were transported to the laboratory in Dulbecco's phosphate-buffered saline (PBS) at 25OC, within 3 h of slaughter. The sanitary status of donor cows was unknown as well as the antibody titres to &ewtoswira serovars. Cumulus-oocyte complexes were retrieved from ovarian follicles by slicing the ovarian surface with a razor blade. After washing, oocytes were pooled

48

Theriogenology

and matured in vitro in the presence, or the absence of the serovar &u-d-lo.Five-day-old serovar bard-io was diluted in the maturation medium to give a final concentration approximately 10' bacteria/ml. Oocytes were matured and fertilized as described below. Embryos were cocultured in vitro to the morula/blastocyst stage in TCW-199 medium with Earle's salts (Gibco, Grand Island, NY) with 100 IU penicillin per ml and 100 mg streptomycin per ml (Gibco, Grand Island, NY) and also in similar medium without antibiotics. Alternatively, they were cultured in INEA Menezo B2 medium (Laboratoire C.C.D., Paris, France) which is only available supplemented with low levels of antibiotics (25 pg/ml penicillin and 40 pg/ml streptomycin). After 7 d, the samples of embryos that developed from oocytes which were either exposed or unexposed to hardjobovis and cultured in B2 or TCW-199 medium free of antibiotics were washed 10 times, as recommended by the IETS (15), and were examined by scanning electron microscopy (SEW). For SEW, specimens were fixed in 1.25% glutaraldehyde 1.0% paraformaldehyde, mounted on the holder, postfixed in tetroxide, dehydrated in an ethanol series, critical point and sputtert-coated with gold. Observations were made with Hitachi S-570 scanning microscope.

and osmium dried a

In Vivo Experiments Nineteen heifers (15 to 18 months) seronegative for serovars bardjo, pomona, sefroe, cowenhaceni, canicola and criwwotvwhosa were infected by conjunctival instillation to both eyes (1 ml/eye) and intravenous injection (5 ml) of a culture of serovar hard_iotype hardjo-bovis (10' organisms/ml). One animal was approximately 4 mo pregnant when infected. The first group of animals (n-9) was euthanized at 4 d postinfection, and the remainder (n-10) at 11 d postinfection. Five presumed uninfected heifers that were seronegative to LeDtoswira were used as controls for each experimental group of animals. Immediately after slaughter, the reproductive tracts were harvested aseptically, and cumulus-oocyte complexes were aspirated from the ovarian follicles and processed for IVF. Oocytes were matured and fertilized as described below. Embryos were cocultured in vitro with BOEC to the morula/blastocyst stage in Menezo B2 medium. Eight days after IVF, groups of 2 to 4 embryos which developed to the morula or blastocyst stage were washed 10 times in PBS supplemented with 10% estrus bovine serum (anti&ewtoswfEL2antibody-free) but without antibiotics and were transferred nonsurgically into one of the uterine horns of recipient heifers (seronegative for the 6 serovars listed above; n=14) which were 5 to 10 d postestrus. Forty-five days after embryo transfer, the animals were injected with a prostaglandin analogue (Estrumate, Coopers, Willowdale, Ontario, Canada), and 48 h later uterine swabs and aborted fetuses were collected.

Theriogenology

49

Blood samples for serological testing were obtained from all experimentally infected heifers before infection and then every second day until slaughter. Similarly, blood samples for serological testing were obtained from the recipient animals before the embryos were transferred and weekly after transfer for 5 mo. Microscopic Agglutination Test (MAT) The MAT was used to measure antibody titres to Lewtospira serovars. This test was performed in microtitre plates as described (7) with 4 day-old cultures of &ewtoswira. The sera were diluted two-fold in saline, starting at a 1:50 dilution. The MAT titre was the reciprocal of the highest dilution of the serum in which 150% of the organisms were agglutinated. Bacteriological Culture The &. boruwetersenil serovar hardio type hardjobovis was a field isolate obtained from Dr. C. Bolin of the USDA (Ames, IA). The organism was grown in reconstituted SPL 5X Leptospira Medium (Scientific Protein Laboratories, Waunakee, WI) at 29OC. Cell counts were determined with a Petroff-Hausser bacteria counter using dark ground illumination (Canadawide Scientific, Ottawa, Ontario). Bacterial isolation was attempted from each animal used in the in vivo experiment. Samples of urine, kidney tissue, ova/embryos, fetuses, follicular fluids, uterine swabs, oviductal cells and IVF medium were cultured in SPL 5X medium supplemented with 5-fluorouracil (100 ).&g/ml; Sigma). Tubes containing 7 ml of medium were inoculated with aliguots (1 ml or 0.1 ml) of urine and other fluid samples. Tissue (approximately 1 g) from the cortex and medulla regions of the kidney was macerated in a petri dish with 1 ml SPL medium supplemented with 5-fluorouracil. The resulting fluid was added to tubes of medium which were then serially diluted ten-fold. Pooled follicular fluid and cumulus complexes were collected from each animal used in the in vitro experiment, and cultured in SPL medium supplemented with 5-fluorouracil. Oocytes and embryos from both experiments were cultured in groups of 4-10 without sonication. All culture tubes were incubated at 29OC for 6 months and examined weekly by darkfield microscopy for the presence of leptospires. Statistical Method Chi-square analysis was used to evaluate differences in cleavage rates of oocytes and development of embryos between the experimental and control groups. RESULTS In experimental samples, motile leptospires were observed by

50

Theriogenology

dark field microscopy in drops of oocyte culture medium 24 h after maturation and in fertilization medium 24 h after insemination. Leptospires were isolated from all cultured samples (n-18) of Day 7 post-fertilization zygotes and embryos (n=56) obtained from oocytes exposed to hardjobovis and cocultured in TCM-199 medium not supplemented with antibiotics and then washed 10 times. In contrast, no leptospires were isolated from samples of Day 7 post-fertilization uncleaved ova/embryos that were exposed to hardjobovis and cultured either in CM-199 medium containing antibiotics (n=32) or in Menezo B2 medium (n=71) and subsequently washed. The control samples of media and embryos not exposed to hardjobovis tested negative. However, leptospires were observed in high numbers by SEM on the surface of the sona pellucida of Day 7 embryos co-cultured in media with and without antibiotics (Figure 1). However, the microorganisms which were seen in association with the embryos that were cocultured in the presence of antibiotics appeared fragmented. Leptospires were not seen in uninfected control embryos. Embryonic development in the different groups is shown in Table 1. All of the experimentally infected animals showed an elevation in body temperature (up to 4O'C) and anorexia on Day 3 and Day 4 postinfection. No other signs of clinical disease were observed prior to slaughter. Of the 9 animals slaughtered on Day 4 postinfection, Uwtoswti-like organisms were observed by dark field microscopy in the urine samples of 5 animals, in the kidneys of 2 animals, and in the follicular fluid and cumulusoocyte complexes of 3 of these animals. Leptospires were also observed in the amniotic fluid of the pregnant heifer. Leptospires were isolated from the kidneys of 2 animals. Embryos (n=36) produced by IVF and culture of oocytes from these donors tested negative for the presence of leptospires by dark field examination and culture. Of the 10 animals slaughtered on Day 11 postinfection, leptospires were observed by darkfield microscopy in the urine, kidneys, follicular fluid and cumulus-oocyte complexes of 3 animals and in the oviduct washing fluid of these animals. In addition, 2 other heifers had visible leptospires in follicular fluids and cumulus-oocyte complexes. No leptospires were isolated from any Day 11 samples. Embryos produced (n=25) by IVF/IVC of the oocytes from these donors also tested negative for leptospires. All samples that were cultured from the control uninfected donors tested negative. On Day 4 post-infection, all of the 9 animals slaughtered and 8 of the 10 remaining animals showed MAT antibody titers of >lOO to serovar bardio. The remaining 2 animals developed MAT antibody titers of >lOO to serovar hardio by Day 11 oostinfection. None of the control animals develoued antibodv titers to serovar bardio or any signs of clinical*disease p&or to slauahter. None of the 14 recivients develoved MAT antibodv titers fo serovar hardio, nor was this organism*isolated from iny of the uterine swabs or fetuses (n=5).

51

Figure 1. Surface of the zona pellucida of a cow embryo showing the presence of adherent LeDtosKdra (SEM).

52

Theriogenology

Development of oocytes shown in Table 1.

following

in vitro fertilization

is

Table 1. Cleavage and embryonic development of oocytes exposed in vitro to serovar bardjo, and oocytes collected from animals experimentally infected with serovar bardio. Experimental group In Vitro Controla Infected Controlb Infected ControlC Infected In Vivo Controld Infected Controle Infected

Ratio Cleavage

(%) Blastocyst

43/80

(54)

17/43

(40)

63/107 56/96 62/120 39/72 42/80

(58) (59) (52) (54) (53)

29/63 14/56 14/62 S/39 lo/42

(46) (25) (23) (21) (24)

36/62 88/147 30/54 75/135

(58) (60) (56) (56)

13/36 27/88 10/30 24/75

(36) (31) (33) (32)

a Embryos co-ultured in Menezo B2 medium b Embryos co-cultured in TCM-199 medium without antibiotics c Embryos co-cultured in TCM-199 with antibiotics d Oocytes fertilized 7 d after infection of donor heifers e Ooocytes fertilized 14 d after infection of donor heifers. All control groups refer to uninfected oocytes. Values in the same column within a given experiment are not significantly different from the control uninfected group (P>O.O5). DISCUSSION In the in vivo experiment, we infected animals by simultaneous conjunctival instillation and IV inoculation of hardjobovis. The 2 routes of infection and high concentration of microorganism used was intended to induce acute leptospiremia in order to increase the probability of obtaining infected ova and not to mimic natural infection. In our opinion, this is a useful approach, since there is a lack of data regarding 1) the levels to which the embryos may be exposed in vivo and 2) the minimal infective dose required for transmission of the dissease. All of the animals infected as part of the in vivo experiment developed MAT antibody titres of >lOO to serovar hardio at the time of slaughter, confirming that these heifers were exposed to the pathogenic agent. However, the microorganism isolated in culture from only a small proportion of the was urine, kidney and follicular fluid samples and from oocytes that

Theriogenology

were obtained from the animals slaughtered on Days 4 and 11 postinfection. Levtosvira is often difficult to isolate from body tissues and fluids by standard culture methods (6, 17), and it is possible that the microorganism was not detected in the remaining animals due to the low sensitivity of the assay and cultural technique used. Nevertheless, the detection of the mlike microorganisms in the follicular fluid and cumulus-oocyte of some of the animals and in 1 of the oviductal complexes washes is important, since this may indicate that ma can infect the ovary and presumably be transmitted with ova to the oviduct after ovulation. The presence of L;evtogg,&gin organs such as the ovary, oviduct and uterus of nonpregnant cows slaughtered at a commercial abattoir was reported previously, but the source of infection was not determined (10). It is not known whether treatment of infected donors with antibiotics in accordance with the International Animal Heath Code (OIE; 3) prior to ova collection, would render follicular fluid and ova free of these microorganisms. To determine the survivability of serovar bardjo in the IVF system, and the association of this microorganism with embryos, the oocyte maturation medium, the fertilization medium, and 1 of the 3 embryo co-culture media were not supplemented with antibiotics. Levtosvira was isolated from embryos which were cocultured for 8 d in the antibiotic free medium. Ten supplementary sequential washes that were performed subsequent to the 7 already done during the processing of IVF embryos were not effective in the removal of this microorganism since it was isolated from the embryos. However, Levtosvira was not isolated from embryos that were co-cultured for 8 d in either of the 2 media which contained low levels antibiotics. One of these media, Menezo B2, contains of penicillin and streptomycin, and has been proven to support early embryonic development of IVF embryos (19). However, in preliminary experiments (unpublished observations), we found that serovar hardio can survive in this medium under IVF conditions (39OC, 8 d) for up to 3 d. The effectiveness of the antibiotics in this medium and/or high temperature of incubation of embryos for killing serovar bardio, was confirmed by the lack of seroconversion of the recipients after embryo transfer. However, this result must be interpreted with caution, since it could be possible that the number of microorganisms associated with the embryos was lower than that required for seroconversion of the heifers. Although viable organisms were recovered only from the embryos cultured in medium without antibiotics, the &evtosvira was detected by SEM on the zona pellucida of embryos that were cultured in media with and without antibiotics. It is possible that the Levtospires observed with the embryos cultured in the presence of antibiotics were dead since they appeared fragmented and could not be cultured. The observation of Levtosvira on the zona pellucida of bovine embryos is similar to the finding of Shisong and Wrathall (16), who reported that this microorganism has the ability to attach to the zona pellucida of porcine

Theriogenology

54 embryos and can not be removed by washing.

In this experiment, the embryo transfer technique was used as an in vivo test of the transmission of serovar mdio to recipients, rather than an evaluation of the developmental capacity of IVF embryos. For this reason, embryos were transferred in groups of 2 to 4 to increase the chance of transmission of the microorganism to the recipients. However, these experiments do show that the presence of serovar &CdiQ has no detrimental effect on fertilization and the development of embryos in vitro, and that pregnancies may be achieved after transfer of these embryos. To our knowledge, this is the first published report on the presence of &eDtosob in an IVF system and on the use of IVF embryos from infected donors for ET in cows. Separate studies are needed to establish in detail the effect of this microorganism on pregnancy rates and fetal development. In conclusion, from a practical viewpoint, this study showed that it is possible to obtain transferable stage embryos from cows infected with hardjobovis and oocytes exposed in vitro to hardjobovis. Such embryos may be associated with the microorganism, so the use of culture and washing media supplemented with suitable levels of appropriate antibiotics for processing IVF embryos is advisable to prevent carriage and transmission of this infection. More detailed experiments are needed to determined whether embryos produced by IVF from infected donors have a potential for disease transmission by embryo transfer. REFERENCES 1. 2. 3. 4. 5. 6.

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