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Regular Article |
a Departments of Clinical Sciences
b Biomedical Sciences, Cornell University, Ithaca, New York 14853-6401
c Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, immunology, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Failure of normal placentation during the first trimester appears to be a common cause of early embryonic loss in cloned cattle and sheep [6, 7, 10]. Interestingly, current data from cloned goat pregnancies do not show the pattern of first trimester losses seen in other ruminants [11, 12]. First trimester bovine and ovine cloned fetuses display a wide variety of placental morphologies illustrated by variation in development of cotyledons, allantoic epithelium, and vascularization [6, 7, 13]. This is associated with developmentally retarded fetuses that are small for age and morphologically abnormal.
In naturally bred mammals, first trimester losses are generally thought to result from abnormalities of the embryo or its placenta, alterations in the maternal uterine environment, or fetomaternal interactions [14]. Abnormal embryonic or placental development in cloned pregnancies has been proposed to be associated with improper expression of critical imprinted genes that control placental development [15–18]. Variable expression of key placental imprinted genes has been found in both cloned and in vitro-produced pregnancies [19–22]. Fetomaternal interactions have not yet been studied in cloned pregnancies. As a high rate of embryonic loss in cloned pregnancies occurs concurrent with the initiation of intimate placental attachment, abnormalities in both placental development and fetomaternal interactions should be considered. Although maternal rejection of pregnancy is rare [23], it has been demonstrated in the mouse [24]. The possible role of the maternal response in causing pregnancy failure has been studied using interspecies pregnancies [25–27]. The current study investigated the expression of major histocompatibility complex class I (MHC-I) by trophoblast cells together with observation of the histologic response of the endometrium to cloned pregnancies.
All nucleated cells express MHC-I molecules, which present peptides produced by degradation of the cell's internal molecules for recognition by CD8+ lymphocytes. In second trimester bovine pregnancies, no trophoblast MHC-I expression is found in either placentomal or interplacentomal areas until the fifth month of pregnancy, when there is up-regulation of trophoblast MHC-I in the interplacentomal regions [28, 29]. As yet, the expression of MHC-I antigens on bovine trophoblast has not been documented in first trimester bovine pregnancies. MHC-I expression is down-regulated in sheep trophoblast cells during first and second trimesters of pregnancy [30]. Intimate contact between placental and maternal cells begins in the cow during the fourth or fifth week of gestation as illustrated by microvilli interdigitation in the interplacentomal areas and development of placentomes [31–33]. Thus, suppression of MHC-I during the first trimester of ruminant pregnancies is temporally associated with the initiation of intimate contact between endometrium and the chorioallantois.
If immunologic incompatibility is a factor in loss of cloned pregnancies, then it would be expected to be apparent around this period of placental attachment. This period is also when the highest rate of gestational loss is observed in cloned bovine pregnancies [6]. The goal of this study was therefore to characterize trophoblast MHC-I expression and investigate endometrial T-lymphocyte distribution in control and cloned first trimester bovine pregnancies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Donor cells A Day 45 male fetus produced by artificial insemination was recovered after slaughter and prepared for explant cell culture as previously described [34]. The cells used in the current experiment were derived from cells frozen at passage 2 (Day 10 of culture) then thawed and cultured in 4-well Nunc plates (Nunc International, Rochester, NY) containing Dulbecco modified Eagle medium-F12 plus 10% (v:v) fetal bovine serum (FBS) plus 1% (v:v) penicillin-streptomycin (P/S) at 37°C in air containing 5% CO2. At 50% confluence, the cells were serum starved (0.5% FBS) for 5 days before nuclear transfer (NT).
Oocyte preparation Recipient oocytes were slaughterhouse derived and matured for 17 h in Medium 199 (M199; Gibco Laboratories Inc.; Grand Island, NY) supplemented with 10% (v:v) fetal calf serum (FCS; Gibco) FSH 0.1 U/ml (Sioux Biochem, Sioux City, IA) LH 0.1 U/ml (Sioux Biochem), estradiol 1 µg/ml (Sigma, St. Louis, MO), 0.1 mM cysteamine (M 9768; Sigma), and 1% P/S. The cumulus-oocyte complexes were vortexed 17 h postmaturation for 3 min in Hepes-buffered M199 (H-M199; Gibco) plus 0.1% hyaluronidase (Sigma), washed, and then held in M199 plus 4 mg/ml BSA.
Enucleation Oocytes were enucleated beginning at 18 h postmaturation. Before enucleation, oocytes were incubated for 15 min in M199 with 4 mg/ml fatty acid-free BSA (Sigma) plus 7.5 µg/ml cytochalasin B (Sigma) and 5 µg/ml Hoechst 33342 (Sigma). Oocytes were enucleated in drops of H-M199 containing 7.5 µg/ml cytochalasin under sterile mineral oil at room temperature, and only oocytes with visible polar bodies were enucleated. Aspirated cytoplasm was visualized under UV light to ensure that the polar body and metaphase plate had been removed, and the enucleated oocyte was also briefly checked under UV light to ensure the removal of all DNA. After enucleation, oocytes were returned to M199 plus 4 mg/ml BSA at 39°C with 5% CO2.
Reconstruction and fusion Fibroblasts were combined with enucleated oocytes, and then the oocyte-fibroblast couplets were manually aligned with a pipette in groups of 4–6 and fused in a 0.5-mm fusion chamber (BTX, San Diego, CA) filled with 270 mM mannitol plus 0.05 mM magnesium chloride [35]. Fusion was performed with a direct current fusion pulse (1 x 40 µsec, 2.25 kV/cm) delivered by a BTX Electrocell Manipulator 830 (BTX). Oocytes were returned to M199 plus 4 mg/ml BSA, fusion was assessed 20–30 min later by light microscopy, and unfused couplets were fused a second time.
Oocyte activation and culture Oocyte activation was performed 3–5 h after fusion at 27 h postmaturation, by a 4-min incubation in Hepes-buffered M199 plus 5 µM ionomycin (Calbiochem, San Diego, CA), then 4 min in 30 mg/ml BSA in H-M199 followed by washing in 4 mg/ml BSA in H-M199 [36]. The fused oocytes were transferred into 2 mM 6-dimethylaminopurine in M199 plus 4 mg/ml BSA for 4 h followed by transfer into embryo culture medium for 7 days. Embryos were cultured in 50-µl drops of a synthetic oviductal fluid serum-free medium [35] under mineral oil (Sage Biopharma, Bedminster, NJ) in a 5% CO2, 5% O2, and 90% N2 atmosphere.
Embryo Transfer
Of 211 enucleated oocytes, 79.1% were successfully fused to fetal fibroblasts. Six days later, 38 grade 1 or 2 embryos were transferred into 19 recipient cows. Embryos were shipped from the laboratory by overnight courier to 1 of 2 commercial embryo transfer facilities (Trans Ova, Sioux Center, IA; EmTran, Elizabethtown, PA) in 1.5-ml cryotubes and transported in a portable incubator (Minitube, Verona, WI). Two embryos were nonsurgically transferred into each recipient. On Day 32 after NT, transrectal ultrasonography was performed on recipient cows to check for pregnancy, and the presence of a fetal heartbeat was used to determine fetal viability. Ten cows were pregnant on Day 32, each with a single viable fetus. Six of these 10 cows were slaughtered at 5 wk of gestation. Additional samples were recovered from 2 cows slaughtered on Days 49 and 63. The remaining 2 pregnancies failed before recovery could be attempted during the seventh week of gestation (Table 1).
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Control Fetuses
Control tissues (Holstein origin) were collected from commercial dairy cows sent to slaughter at a commercial slaughterhouse (Taylor Packing, Wyalusing PA). Tissues were recovered from 8 control pregnancies between 4 and 7 wk of gestation (Table 1). After the uterus was opened, the crown-rump length was measured, and the fetal age was estimated using a formula developed for purebred Holsteins [37].
Immunohistochemistry
Sampling For each cloned and control pregnancy, a minimum of 6 areas were sampled from the gravid horn adjacent to the fetus. Each sample contained both placentomal and interplacentomal tissue (endometrium and apposing placenta). At least 3 of these samples were frozen in O.C.T. Compound (Tissue-Tek, Sakura Inc., Torrance, CA) in isopentane chilled in liquid nitrogen and stored at -80°C, while 3 samples were fixed in 10% neutral buffered formalin.
Processing Formalin-fixed tissues were embedded in paraffin, sectioned at a thickness of 5 µm, and stained with hematoxylin and eosin. Frozen tissues were cryosectioned at a thickness of 8 µm and fixed for 10 min in chilled acetone. Sections were stained according to the immunoperoxidase procedure previously described [29]. This included blocking the tissue sections with normal goat serum and incubating them for 2 h at 37°C with IL A19 mouse monoclonal anti-bovine MHC-I primary antibody (provided by Jan Naessens, International Livestock Research Institute, Nairobi, Kenya) or MM1A mouse monoclonal anti-bovine CD3 monoclonal antibody (VMRD, Pullman, WA). Nonimmune mouse ascites (Sigma) and H42A anti-MHC-II monoclonal antibody (VMRD) were used as negative controls. These controls showed no binding to endogenous peroxidase or alkaline phosphatase. Incubations with biotinylated goat anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) and streptavidin peroxidase were followed by detection with 3-amino-9-ethylcarbazole (Zymed Laboratories, San Francisco, CA). Sections were counterstained with hematoxylin.
Quantification Microscopic assessment of the percentage of MHC-I-positive trophoblast cells and the number of CD3+ lymphocytes (both aggregate and interaggregate) was determined by visual estimation per 100x field using a 0.292-mm2 reticle to delineate linear boundaries.
The mean percentages of positive trophoblast cells per section and per case were calculated. A minimum of 3 tissue blocks per case that contained both interplacentomal and placentomal tissue were scored. The sections were coded, and observations were recorded without knowledge of the tissue origin.
Counting of CD3+ lymphocytes was performed in 2 ways because of the distribution of lymphocytes in the endometrium of cloned pregnancies. In control endometrium, lymphocytes were present as scattered individual cells. In contrast, in the endometrium from the clone pregnancies, lymphocytes were present both as individual cells and as large aggregates of closely adjacent cells (usually circular in cross-section). These aggregates were very rare in control sections. Thus, counting of CD3+ lymphocytes was modified to count individual CD3+ lymphocytes and to include separate counts of aggregates without individually counting the lymphocytes contained within them. This protocol was performed since the aggregates contained very large numbers of lymphocytes (several thousand). Mean counts per field were totaled per section, and means per case were calculated.
Statistics
Data were analyzed using SigmaStat statistical software (SPSS Inc., Chicago, IL). MHC-I expression in trophoblasts and interaggregate CD3+ lymphocyte counts from control and cloned tissue were compared using one-way ANOVA and the nonparametric Kruskal-Wallis test. To compare the percentages of fields that either were positive for MHC-I or contained CD3+ lymphocyte aggregates, the Fisher exact test was used. P values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major aim of this study was to recover placenta and uterus from 6 Day 35 pregnancies, as this is the period during which high rates of fetal loss have occurred in previous studies. Additional pregnancies were recovered at later stages of gestation (1 fetus at Day 49, 1 at Day 63) to gain information on later placental development (Table 1).
During the interval between confirmation of a viable pregnancy and slaughter (1–3 days), 3 of 6 of the cloned Day 35 fetuses died. In this study, fetal death was defined as the presence of hemorrhagic amniotic fluid that was evident grossly at embryo collection. These fetal deaths indicated that we had selected an ideal time period to investigate fetal loss in clones. One of the controls was dead at the time of sampling, and this case also showed signs of gross placental degeneration in addition to hemorrhagic amniotic fluid. The placental membranes were detached, and the caruncles were regressing. This was important, as it provided us with control data on the endometrial reactions to a naturally occurring pregnancy failure.
Of the 6 NT fetuses collected at Day 35 of gestation, only 2 had developed to within 1–2 days of the expected Day 35 crown-rump length. The remaining 4 were developmentally retarded at various developmental stages (2 at Days 31–32, 1 at Day 29, and 1 at Day 23). Of the additional cloned fetuses, the Day 49 cloned fetus showed a smaller than expected crown-rump length that indicated mild growth retardation, whereas the Day 63 cloned fetus showed normal growth. Hematoxylin and eosin-stained sections of the endometrial-trophoblast interface revealed apparently normal epithelial and trophoblast architecture (Fig. 1). There was no evidence of a hypoplastic trophoblast layer in any of the cloned placentas as suggested in a previous study [6].
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Trophoblast MHC-I Expression
MHC-I was expressed in the trophoblast of each of the eight cloned pregnancies, whereas there was no expression in any region of the control placentas. Furthermore, no MHC-I expression was observed in the placenta from the control pregnancy in which the fetus had died. In placentas from cloned pregnancies, the level of MHC-I expression within individual 100x fields varied considerably (Fig. 2). The highest MHC-I expression was seen in the trophectoderm surrounding the smallest cloned fetus that was dead at the time of retrieval (NT1). The mean percentage of MHC-I-positive trophoblasts in cloned pregnancies per 100x field varied from 17.9% to 56.5%. The percentage of fields that contained MHC-I-positive trophoblasts varied from 52.5% to 93.8% (Fig. 3). The percentage of positive fields was also significantly less in NT6, a Day 35 fetus with normal crown-rump length and morphology. Conversely, MHC-I expression was high in NT2 and NT3, which were both alive and had only marginal developmental retardation. Thus, there was variability between the level of MHC-I expression and fetal viability.
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Maternal endometrial MHC-I expression appeared to be similar between control and cloned pregnancies, although this was not carefully assessed. One difference noted in the cloned pregnancies was a greater abundance of columnar endometrial epithelium (which was MHC-I positive).
Endometrial CD3+ Lymphocytes
The most striking difference between cloned and control pregnancies was the increased occurrence in the former of T lymphocyte aggregates located in either the superficial or deep stroma of both the intercotyledonary and cotyledonary areas of endometrium (Fig. 4, C and D). Interspersed between these aggregates were increased numbers of T lymphocytes mainly distributed immediately beneath the epithelium and adjacent to the endometrial glands. These lymphocyte aggregates were present in each of the 8 clones and in total were found in over one third of the fields examined (87 aggregates from 236 fields; 36.9% of fields). In contrast, aggregates were far less common in controls and found in only 2 of 8 control pregnancies (total of 5 aggregates from 310 fields; 1.6% of fields; P < 0.001).
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The greatest number of CD3+ lymphocytes was found in NT1, which also had the greatest expression of MHC-I. Significantly more aggregates of CD3+ lymphocytes were found in the endometrium of NT1 compared with that of other cloned or control pregnancies. The fetus from this pregnancy was the least well developed, with a crown-rump length matching that of a Day 23 fetus. This finding indicated either that the fetus was not capable of normal growth or that the uterine environment/placental transfer of nutrients did not allow normal growth to occur.
The lymphocyte aggregates were subjectively much larger in the clones and contained from 200 to several thousand CD3+ lymphocytes (Figs. 1 and 4). In contrast, the largest aggregates in the controls contained fewer than 200 lymphocytes. As the lymphocytes were tightly clumped and present in such large numbers, it was difficult to count these cells in the 8-µm sections with any accuracy. In 6 of 8 cloned pregnancies, the percentage of fields that contained CD3+ lymphocyte aggregates was significantly greater than that in all of the controls (P < 0.05; Fig. 5). In the remaining 2 cloned pregnancies (NT5, NT7), the percentage of fields containing lymphocyte aggregates was not significantly greater than that in 1 of the controls (C7). Lymphocyte aggregates were thus present in each of the cloned pregnancies regardless of observed viability or morphology.
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In 4 cloned pregnancies (NT1, NT2, NT4, NT5), the number of CD3+ lymphocytes present in the tissue between these aggregates (i.e., interaggregate CD3+ cells) was significantly greater than that in each of the controls (P < 0.05). Each of the fetuses from these cloned pregnancies showed growth retardation (Table 1). In the remaining 4 cloned pregnancies, interaggregate CD3+ lymphocytes were not significantly greater than that in at least 1 of the 8 controls. In the cloned pregnancies in which interaggregate CD3+ numbers were not significantly different from that in at least 1 control (NT3, NT6, NT7, NT8), relatively normal morphology and fetal growth were observed.
The possibility that the endometrial inflammatory reaction (increased numbers of CD3+ cells) in the cloned pregnancies was caused by fetal death seemed unlikely when the CD3+ numbers were compared from the 3 dead clones (NT1, NT4, NT5) and the 1 dead control fetus (C6) in this data set. The dead control fetus (C6) had no aggregates and a normal number of interaggregate cells (23.2 ± 5.5 cells per field).
Day 63 Cloned Pregnancy
The Day 63 cloned fetus showed normal morphology and possessed a well-vascularized placenta with >20 placentomes visible. The MHC-I expression in the Day 63 cloned placenta differed from that of the Day 35 clones (Fig. 6). It is possible that MHC-I expression was beginning to be down-regulated in the trophoblast cells by Day 63; expression was mainly found in the mesoderm beneath the trophoblast cells. The number of interaggregate T lymphocytes expressing CD3 (Fig. 6B) was slightly elevated, although not significantly elevated, in this pregnancy. This finding indicates either that MHC-I expression did not affect this pregnancy or that it did not incite a significant maternal response because of MHC-I compatibility of the fetal and maternal tissue types.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abnormal T lymphocyte numbers and distribution may be a cause or symptom of the high early embryonic mortality seen in cloned pregnancies. This problem is very significant and, together with low initial pregnancy rates, represents the major cause of the low efficiency in producing cloned offspring. In control pregnancies, lymphocyte distribution was diffuse throughout the intercaruncular stratum compactum. In cloned pregnancies, lymphocytes accumulated immediately beneath the epithelium or in large periglandular or subepithelial aggregates. The increased numbers of lymphocytes in cloned pregnancies and their distribution in the endometrium suggest active recruitment in response to a strong antigenic stimulus. This temporal relationship of lymphocytic infiltration with pregnancy failure is highly suggestive of maternal rejection of the pregnancy.
Endometrial lymphocyte populations have been documented in ruminant and porcine pregnancies. Lymphocyte numbers decrease during early pregnancy in sheep, pigs, and cattle [38–41]. During early pregnancy, the nongravid horn contains greater numbers of lymphocytes than the gravid horn, particularly in areas where the allantochorion has failed to attach to the endometrium [41]. In this study, we analyzed the lymphocyte populations from only the gravid horns of cloned and control pregnancies. The CD3 antibody used in this experiment is specific for the signaling complex of the T-lymphocyte receptor and does not react with B cells, macrophages, or natural killer cells. The role of uterine leukocytes during pregnancy is not yet established, and it has been proposed that successful pregnancy involves effective fetomaternal communication, with leukocytes playing a prominent role [42]. The immune system is thus capable of both destroying a fetus and assisting its development through the action of different types of lymphocytes and their cytokines.
The 6 Day 35 fetuses showed variation in morphology. Some were dead at the time of collection, as evidenced by either hemorrhagic amniotic fluid or morphology. The greatest numbers of lymphocytes were present in the endometrium of cloned pregnancies that had very recently failed (NT1, NT4). This could be interpreted either as inflammation after fetal death or as inflammation that caused fetal death. The latter is a more credible explanation for several reasons. Increased numbers of lymphocytes were present not only in the endometrium adjacent to dead clones but also in that adjacent to obviously viable fetuses. Furthermore, there was no increase in the number of lymphocytes in a control pregnancy with a dead fetus. The time between death of the cloned fetus and sample retrieval after slaughter was not long enough (less than 3 days) to generate a chronic lymphocytic response.
The lack of trophoblast MHC-I expression in first trimester control pregnancies is in agreement with that reported in sheep and pigs [43], and to the best of our knowledge, this is the first time that it has been reported in cattle. The noninvasive trophoblasts of horses also do not express MHC-I, although there is expression in the transient chorionic girdle cells during the second month of gestation [44]. It is believed that MHC-I expression is down-regulated during early pregnancy to facilitate placental attachment. Expression of MHC-I is expected to occur in second and third trimester mammalian pregnancies [28–30, 45].
Inappropriate expression of MHC-I as seen in the current study represents another example of altered gene expression due to the NT process. Trophoblast MHC-I expression is hypothesized to be suppressed by the endometrium and therefore requires intimate contact between placenta and endometrium [44]. In the current study, the signal to suppress MHC-I expression may not have been received or may not have been sent, or this signal may have been temporally altered. Alterations in gene expression have been postulated to be likely causes of pregnancy failure in cloned pregnancies because imprinted genes are very susceptible to reprogramming failure after NT or in vitro embryo culture [15, 20, 46]. Imprinted genes are also crucial for correct placental development [18]. The expression of genes shown to be important for implantation in individual NT embryos (genes for interleukin 6, fibroblast growth factor 4, and fibroblast growth factor receptor 2), show variable profiles [19]. There is also likely to be an added effect of embryo culture conditions on imprinted gene expression profiles [21, 22].
Cloned pregnancies in mid to late gestation show a higher than normal abortion rate and signs of abnormal placentation such as edema or reduced placentome numbers [5, 47, 48]. It is not known how first trimester trophoblast MHC-I expression would affect those pregnancies that survive to the second trimester. Cattle normally express MHC-I antigens on interplacentomal and arcade region trophoblast cells during the second half of pregnancy [28, 29]. Consequently, if cloned fetuses with inappropriate first trimester MHC-I expression survive, MHC-I expression restricted to the interplacentomal placenta would not be expected to affect placental development or function during the second half of pregnancy. However, if during the first trimester MHC-I expression partially inhibited placental development, for example by reduction in placentome number or in crypt formation, then placental efficiency would be compromised. Furthermore, if there was MHC-I expression on placentomal villous trophoblast cells, this could result in an immune response in the stroma of the placentomes that would interfere with nutrient transfer. If MHC-I expression is found to be widespread among cloned fetuses, the use of recipient animals with the same MHC-I tissue type as the clone may reduce the uterine response to trophoblast cells and improve the viability of first trimester cloned fetuses.
To minimize variation, we used a single fibroblast cell line to produce each of the cloned fetuses. Although the techniques used for cell culture, NT, and embryo culture were not novel, it is possible that we have presented a group of fetuses that due to cell line, culture conditions, or NT technique are unique in producing MHC-I expression. Recently, we examined a Day 50 cloned pregnancy produced by another laboratory using a different NT technique and a different cell line from those used in the current study (data not shown). In that pregnancy, no increase in the number of endometrial lymphocytes was found. Additionally, no expression of MHC-I in trophoblasts was observed (unpublished observations). We intend to determine the MHC-I expression of fetuses generated from different cell lines and laboratory techniques in future experiments.
It is interesting to compare the stage of fetal losses and endometrial morphology in cloned bovine pregnancies with that of interspecies pregnancies such as donkey-in-horse and goat-in-sheep pregnancies. In donkey-in-horse pregnancies, pregnancies can readily be established, although only a small proportion of donkey-in-horse pregnancies will progress beyond the first trimester. The gross vascularity of these interspecies placentas is reduced, villous and crypt formation is rudimentary, and there is widespread accumulation of lymphocytes in the endometrium [25]. One intriguing aspect of these donkey-in-horse pregnancies is that the female that has carried one cross-species pregnancy to term is highly likely to do so in future pregnancies. It may be that some females are inherently more able to maintain interspecies or perhaps even cloned pregnancies. In goat-to-sheep embryo transfers, placental attachment either fails to be established or fails to be maintained [27, 49]. Underdeveloped cotyledons and lack of villous formation are characteristic findings, and the histologic findings are suggestive of maternal immune rejection of the placental tissue [50]. These observations detail intriguing similarities in placental pathology between interspecies and NT fetuses.
We conclude that MHC-I expression in the trophoblast of cloned fetuses is abnormal and is likely to have caused the observed elevations in maternal lymphocyte numbers, which would be detrimental to the maintenance of pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by funding from the Department of Clinical Sciences and the College of Veterinary Medicine, Cornell University. 
2 Correspondence: Jonathan Hill, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Box 34, Ithaca, NY 14853-6401. FAX: 607 253 3531; jrh35{at}cornell.edu
Accepted: January 29, 2002.
Received: December 21, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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