To measure the concentration of serum amyloid A (SAA) protein in horses a sensitive and highly reproducible sandwich (ELISA) was established, using affinity purified SAA antibody. Results of the ELISA were found to have a high correlation (r = 0.95) with those of the single radial immunodiffusion test. Equine SAA concentration was measured by use of this ELISA. In clinically normal horses, the concentration of SAA was high immediately after birth to 2 weeks of age. After that, SAA concentration had periodic fluctuations in the range of approximately 10 to 30 microgram/ml. Mean (+/- SD) concentrations of SAA in foals (less than or equal to 12 months old) and adult horses (greater than or equal to 18 months old) were 21.23 +/- 12.20 and 14.93 +/- 9.07 microgram/ml, respectively. In mares during the perinatal period, SAA concentration remained stable within the reference range before parturition. It increased quickly after delivery, and reached a peak value of 101.29 +/- 98.82 microgram/ml on postpartum day 3, then began to decrease, at postpartum week 2, to the reference range by the end of postpartum month 1. In horses with experimentally induced inflammation, SAA concentration increased quickly and reached approximately four- to 40-fold increase over the pretreatment value on day 1 and remained high on days 2 to h after treatment. It then returned to the baseline value by 2 to 4 weeks in association with disappearance of local signs of inflammation. The SAA concentration was high in most horses with clinical signs of inflammation. It was concluded from these data that this ELISA is sensitive and reliable for measuring SAA in horses.

The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199 + FCS with various concentrations (0-20 mu-M) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 mu-M nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 mu-M nocodazole. Exposure to 10 mu-M nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 mu-M nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% O2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 mu-M nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.

Passive protection provided by sows inoculated with the virulent Miller strain of transmissible gastroenteritis virus (TGEV), or the ISU-1 strain of porcine respiratory coronavirus (PRCV), or both was evaluated in nursing pigs challenge exposed with virulent TGEV. Four sows (group B) were inoculated with PRCV oronasally twice at 4 and 2 weeks before parturition; 1 sow (group C) was inoculated similarly, but in 2 subsequent pregnancies; and 2 sows (group D) were oronasally primed with PRCV at 4 weeks before parturition, and 2 weeks later were administered a booster inoculation of virulent TGEV. Two additional sows (group E) remained uninoculated and served as seronegative controls, and 1 sow (group A) that had been naturally infected with TGEV served as a seropositive control. The degree of passive immunity transferred by these sows to their litters was assessed by challenge exposing the pigs of sows in groups BE (only the second litter of group C) with virulent TGEV at 3 to 5 days of age. After challenge exposure, clinical signs of infection and mortality were noted and fecal and nasal shedding of virus was assessed by ELISA. The IgA, IgG, and IgM antibody titers to TGEV were quantified in colostrum and milk of the sows by use of an isotype-specific monoclonal antibody-capture ELISA, using biotinylated monoclonal antibodies against each porcine isotype as detecting reagents. A plaque-reduction assay was used to quantify neutralizing antibody titers in serum, colostrum, milk, and fractionated whey (IgG and IgA/IgM). In the sow naturally infected with TGEV (group A), there was a pronounced decrease in IgG antibody titers to TGEV in the transition from colostrum to milk, and IgA TGEV antibodies became predominant, with high titers maintained throughout lactation. The 4 group-B sows partially protected their pigs after TGEV challenge exposure; mean mortality was 67%, compared with 100% in pigs suckling the 2 TGEV seronegative control sows (group-E litters). Although IgA TGEV antibodies were detected in colostrum and milk of group-B sows, IgG TGEV antibodies were the most abundant. The sow of group C had a marked increase in IgA TGEV antibody titers in colostrum and milk after reinoculation with PRCV during the second pregnancy, before TGEV challenge exposure of the litter. Its pigs were passively protected to a high degree after TGEV challenge exposure (27% litter mortality). The sows in group D, primed with PRCV and boosted with TGEV, provided the best passive protection after TGEV challenge exposure of their pigs. Not only litter mortality (27%) but also morbidity was reduced, compared with those factors for the other challenge exposed litters, and the sows did not become ill. In these swine, the high degree of passive protection observed could not be associated with the presence of only IgA TGEV antibodies in the milk, but high IgM TGEV antibody titers also were detected in colostrum and milk. Results of this study suggest that PRCV-inoculated sows are able to partially protect their pigs from TGEV challenge exposure and, on the basis of preliminary data, the degree of protection may increase after multiple PRCV exposures or after secondary exposure to TGEV during pregnancy. Also, an IgA respiratory tract-mammary gland link may exist as evident by the low titer of IgA TGEV antibodies in the milk of PRCV-inoculated sows, but may not be as efficient in inducing lactogenic IgA immunity as is the gastrointestinal tract-mammary gland link.

Methods available for measurement of plasma lipoprotein-cholesterol concentrations and activities of lipoprotein lipase, hepatic lipase, lecithin:cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein were adapted for use in cats. A combined ultracentrifugation/precipitation procedure was used to isolate very low-density lipoproteins (VLDL), then to separate low-density lipoproteins (LDL) from high-density lipoproteins (HDL). The reagent used, 92 mM heparin-manganese chloride, provided complete precipitation of LDL with only trace and insignificant contamination by HDL. Efforts to selectively measure lipoprotein lipase activity in plasma, collected after IV injection of heparin, by inhibiting hepatic lipase with sodium dodecyl sulfate were unsuccessful, and the activity of this enzyme was calculated as the difference between total and hepatic lipase activities. The latter was measured in the presence of high salt concentration to inhibit lipoprotein lipase. Cholesterol esterifying activity was identified in feline plasma and was typical of LCAT, in that it was dependent on apolipoprotein A-I as a cofactor. The intra-assay and interassay coefficients of variation for measurement of lipoprotein lipase, hepatic lipase, and LCAT activities were 18.4, 4.6, and 7.2%, and 20.4, 10.7, and 5.3%, respectively. Appreciable cholesteryl ester transfer protein activity was not detected in either undiluted or diluted plasma. These methods were subsequently used to investigate the effects of pregnancy and lactation on lipoprotein metabolism in a group of 10 queens. Plasma concentrations of cholesterol and triglycerides were unaltered during pregnancy, but the concentrations of VLDL-cholesterol increased and those of HDL-cholesterol decreased. During lactation, the concentrations of cholesterol and triglycerides decreased owing to reductions in VLDL-cholesterol and LDL-cholesterol concentrations and continued suppression of HDL-cholesterol. These changes were associated with alterations in the activities of lipoprotein lipase, which increased after parturition, and hepatic lipase, which increased during pregnancy and lactation, that may help explain their metabolic origins. The activity of LCAT remained unchanged.

Fetal infectivity of Ehrlichia risticii was investigated in 19 ponies that were E risticii negative on the basis of results of an indirect fluorescent antibody (IFA) test. Thirteen pregnant ponies were infected by IV administration of E risticii between 90 and 180 days of gestation. Six pregnant ponies served as noninfected controls. Each infected pony had clinical signs of equine monocytic ehrlichiosis, was confirmed to be ehrlichemic, and developed an IFA titer to E risticii. Two infected ponies became recumbent, were unresponsive to supportive care, and were euthanatized. After recovery from clinical illness, the remaining ponies were observed throughout gestation for reproductive abnormalities. On abortion, each fetus was necropsied and tissue specimens from the liver, bone marrow, spleen, colon, and mesenteric lymph nodes were inoculated into canine monocyte cell cultures. Six infected ponies aborted at a mean 217 days of gestation, which was between postinoculation days 65 and 111. Five fetuses were recovered for evaluation, and E risticii was isolated from 4 of them. All 5 fetuses recovered had similar histologic findings, including enterocolitis, periportal hepatitis, and lymphoid hyperplasia with necrosis of the mesenteric lymph nodes and spleen. All fetuses tested negative for IgG to E risticii, although 3 had low IgM titer to E risticii. The remaining 5 infected ponies had normal parturition. Presuckle IFA titer to E risticii was measured in 4 of the term foals, and results for 3 were positive. Two foals from infected ponies were monitored for 6 months and daily gain in body weight was comparable to that of a control foal. None of the control ponies became ill or seroconverted during the clinical illness phase, and none aborted throughout gestation. Two control ponies seroconverted to E risticii 6 weeks before parturition. Results of this study indicate that E risticii is a primary abortifacient under experimental conditions.

Objective--To determine the relative role of endogenous prostaglandin F2 alpha (PGF2 alpha) secretion in cloprostenol-induced abortion in mares that no longer require luteal progesterone secretion for maintenance of pregnancy, and to evaluate the ability of a prostaglandin cyclooxygenase inhibitor (flunixin meglumine) to prevent cloprostenol-induced abortion. Design--The effect of flunixin meglumine on PGF2 alpha secretion and outcome of pregnancy was compared between mares treated with cloprostenol only and mares treated with cloprostenol plus flunixin meglumine. Animals--Five pregnant mares, aged 4 to 15 years, of light-horse type. Procedure--Cloprostenol (250 micrograms) was administered at 24-hour intervals to 5 pregnant mares. Flunixin meglumine (500 mg, IV) was administered at 8-hour intervals starting 15 minutes before the first cloprostenol administration. Hourly blood samples were analyzed for 15-ketodihydro-PGF2 alpha, progesterone, and estrogen concentrations. Previously reported data on cloprostenol-induced abortion in 6 pregnant mares treated daily with cloprostenol only were used as historic controls. Results--The mean (+/- SEM) interval from first cloprostenol administration to fetal expulsion 56.4 (+/- 13.7) hours and number of cloprostenol administrations 3.2 (+/- 0.6) in the 5 flunixin meglumine-treated mares were not significantly different, compared with values for 6 pregnant mares treated daily with cloprostenol only, 48.6 (+/- 5.6) hours and 2.8 (+/- 0.2) cloprostenol administrations. Flunixin meglumine did not inhibit endogenous PGF2 alpha secretion. Prostaglandin F2 alpha secretion rates on the day before and day of fetal expulsion were similar in both groups. Conclusion--Flunixin meglumine at a dosage of 500 mg/animal, administered IV every 8 hours, is ineffective in modulating uterine PGF2 alpha secretion during cloprostenol-induced abortion. Clinical Relevance--Flunixin meglumine is ineffective in the modulation of prostaglandin-induced uterine PGF2 alpha secretion and, therefore, does not offer a viable alternative for the prevention of abortion in mares at risk of abortion because of systemic illness.

Newborn foals of mares grazing on Acremonium coenophialum-infected fescue pasture throughout gestation or from gestation day 300 to parturition had increased gestation duration and decreased serum triiodothyronine concentration. Pregnant mares were allotted to 4 treatments: grazing continuously on endophyte-free (E-) fescue, grazing continuously on endophyte-infected (E+) fescue, grazing on E+ fescue from gestation day 300 to parturition, and grazing on E+ fescue from conception to gestation day 300. Morphometric studies indicated that foals born to mares exposed to endophyte late in gestation had large, distended thyroid follicles lined by flat cuboidal epithelial cells. Mean triiodothyronine concentration in foals exposed to endophyte (395.2 ng/dl) was decreased (P < 0.01), compared with mean values in control foals (778.0 ng/dl). Thyroxine and reverse triiodothyronine concentrations were not significantly different among groups. Foal organ weight as a percentage of foal body weight was not significantly different among experimental groups.

A field trial was conducted on a commercial swine farm quarantined because of infection with pseudorabies virus. The purpose was to investigate, in growing pigs born to hyperimmunized sows, the immunogenicity of a vaccine with a glycoprotein I (gE) deletion. One hundred twenty pigs were assigned at random to 1 of 3 vaccination schedules at ages: 8 and 12 weeks; 8, 12, and 14 weeks; and 8, 12, and 16 weeks. Immune response was measured at 8, 12, 14, 16, and 18 weeks, using the serum neutralization test, a screening ELISA, and assays of IgG and IgA in serum and nasal secretions. Results of the serum neutralization test and the screening ELISA indicated that, for pigs vaccinated only at 8 and 12 weeks, the percentage of pigs with pseudorabies virus serum antibodies decreased substantially by 18 weeks; for pigs given a booster at 14 or 16 weeks, the prevalence of serum antibodies at 18 weeks was higher, with 16-week booster vaccination eliciting the best response. At each age, nasal IgA and IgG values were highly correlated (r greater than or equal to 0.70), as were serum IgA and IgG values; correlations of serum with nasal IgA and IgG values were somewhat lower (approx range, r = 0.40 to 0.70). Nevertheless, an increase in serum IgA or IgG values on vaccination was no guarantee of an increase in nasal IgA or IgG values. For serum and nasal mucosal antibodies, a poor immune response was associated with high quantities of maternally derived antibodies. Vaccination at 16 weeks was necessary to ensure eliciting of an immune response in almost all pigs.

Neospora caninum-induced abortion is a major production problem in the daily cattle industry in the United States and worldwide. Abortions attributable to naturally acquired N caninum infection also have been observed in pygmy goats. We studied experimentally induced infections with N caninum in pregnant pygmy does to determine whether abortions attributable to N caninum infection would occur after inoculation. Seven pregnant pygmy does (1 control doe and 6 inoculated with N caninum) were studied. The control doe remained clinically normal throughout the study and delivered 2 healthy kids. Abortion, fetal death, and stillbirths were observed in some pregnant does inoculated with N caninum. Two pregnant pygmy does inoculated with N caninum early in gestation (day 51) had fetuses that died and were aborted, or died and were reabsorbed. Neospora caninum tachyzoites and lesions were observed in the brain, spinal cord, and heart of aborted fetuses; parasites also were isolated from the placenta. Four additional pregnant pygmy does (2 inoculated at mid-gestation [day 85], and 2 at late gestation [day 127]) did not abort after inoculation. However, 1 doe inoculated during mid-gestation delivered a stillborn fetus that had died about 1 week prior to parturition. This kid was congenitally infected with N caninum. Neospora caninum was isolated from the placentas of all inoculated does examined. Neonatal neosporosis was not observed in live-born kids, nor were stages of N caninum isolated from any live-born kid. Does did not undergo abortion or have congenitally infected kids when they were rebred and evaluated for neosporosis.

Plasma and milk concentrations of parathyroid hormone-related protein (PTHrP) at various stages of pregnancy and lactation were determined in thirty-nine 3- to 16-vear-old Brown Swiss and Red Holstein X Simmental dairy cows originating from 4 herds. Eighteen of the cows were separated into 2 groups: low-parity (LP, n = 8) cows if they were in their first or second pregnancy and high-parity (HP, n = 10) cows if they were in their third or greater pregnancy. Blood samples were collected from each cow on 1 occasion, 15 to 5 days before calving, and blood and milk samples were collected daily during 6 days after calving. Serum total and ionized calcium (Catot and Ca2+, respectively) and milk Catot concentrations were also quantified. A transient postpartum decrease of serum Catot and Ca2+ concentrations was observed, whereas milk Catot concentration was constant. Plasma concentration of PTHrP was detected in 11 of 21 cows by use of an immunoradiometric assay (range, 0.45 to 1.82 pmol/L). Daily mean (+/- SD) colostrum and milk PTHrP concentrations ranged from 3.25 (+/- 3.23) to 4.69 (+/- 1.36) nmol/L in LP cows and 2.74 (+/- 0.5) to 5.95 (+/- 0.33) nmol/L in HP cows. In all cows of the HP group and most cows of the LP group, milk PTHrP concentration was highest in the day-1 sample. Milk PTHrP concentration correlated positively with milk Catot concentration in HP cows (r = 0.5959, P < 0.0001). In contrast, there was a negative relation between milk PTHrP and milk Catot concentrations in LP cows (r = -0.3285, P < 0.02). Milk PTHrP concentration was not correlated with serum Ca2+ concentration at postpartum days 5 and 6, when serum Catot and Ca2+ concentrations had returned to prepartum values. Because correlation of the corresponding day, milk PTHrP concentration most likely is not a major determinant of Ca transport into milk and the PTHrP released into the blood stream is most likely not a major determinant of the endocrine regulation of serum Catot and Ca2+. Thus, although it is involved, PTHrP is not a major factor in the integrative endocrine, paracrine, and autocrine regulation of Ca homeostasis in lactating cows. It is hypothesized that Ca may be actively transported from blood into milk with a process modulated by PTHrP. These data suggest that PTHrP produced by the mammary gland is most likely not involved in the pathogenesis of parturient paresis (milk fever) in dairy cows.