Induction of prostaglandin endoperoxide synthase isoform-2 in ovine cotyledonary tissues during late gestation.

The present study was designed to characterize the developmental pattern of specific prostaglandin endoperoxide synthase (PGS) isoform immunoreactivity and activity in tissues of fetal origin during the last half of gestation in sheep. Fetal amnion, allantochorion, and cotyledons were collected under halothane general anesthesia on days 79-80, 105-108, 120-122, 128-131, and 140-145 (term) of pregnancy. Solubilized extracts were prepared and analyzed by immunoblots using anti-PGS antibodies previously shown to recognize PGS isoform-1 and -2 (PGS-1 and PGS-2). PGS activity from cotyledon microsomes was assayed by the measurement of prostaglandin E2 (PGE2) production under initial velocity conditions. All fetal tissues contained PGS-1 at each of the stages of gestation examined, with minimal regulation of this isoform from 79-144 days gestation. In contrast, PGS-2 increased gradually in the cotyledons from 120-139 days gestation, with the most marked expression observed at term. PGS-2 was not detected in amnion or allantochorion. PGS activity in cotyledons increased (P < 0.01) in parallel with immunoreactive PGS-2 levels; indicating that PGS-2, rather than PGS-1, is associated with increased PG synthesis in this tissue. Both the activity (n = 5/group) and the amount of PGS-2 increased significantly from 105-108 days gestation to term (P < 0.01). We conclude that the increase in PGS that occurs at term in sheep is predominantly active PGS-2 localized to fetal cotyledons.

Endocrine changes during 48 hours of food withdrawal in the pregnant rhesus monkey in the last third of gestation.

We studied the hormonal responses in four pregnant rhesus monkeys between 112 and 149 days gestation. After 2 days, during which the monkeys were fed ad libitum, their food was withdrawn at 1500 h for 48 h while allowing free access to water. The food then was returned, and the animals were studied for a further 2 days. The mean maternal whole blood glucose concentration significantly decreased, and plasma cortisol and dehydroepiandrosterone sulfate (DHEAS) concentrations significantly increased within 30 h of food withdrawal (P less than 0.05). The maternal plasma estradiol concentration increased significantly at 1000 h on the second day of food withdrawal (P less than 0.05), whereas the plasma progesterone concentration did not change. The maternal blood glucose and plasma cortisol, DHEAS and estradiol concentrations returned to baseline by the second day of food replacement. We conclude that the stress of hypoglycemia and/or the attendant inability to eat, together or separately, stimulate maternal adrenal glucocorticoid and androgen secretion during the period of food withdrawal. The increased maternal DHEAS and perhaps other adrenal androgen concentrations result in increased maternal estrogen production.

Relative glucose tolerance and subsequent development of hypertension in pregnancy.

OBJECTIVE: To test the hypothesis that relative carbohydrate tolerance, an indicator of insulin resistance, predicts subsequent risk for hypertension of pregnancy among previously normoglycemic, normotensive women. METHODS: We conducted a nested case-control study in women enrolled at a large Colorado urban health maintenance organization. Subjects were previously healthy pregnant women who tested abnormal on their initial 50-g glucose screens and subsequently completed 3-hour, 100-g oral glucose tolerance tests. Cases were 54 previously normotensive women who subsequently developed hypertension and controls were 51 subjects with normotensive pregnancies, matched to cases on parity. Subjects diagnosed with gestational diabetes (17 cases, six controls) were excluded from the main analyses. RESULTS: Among the 82 normoglycemic women (45 controls, 37 cases, 13 preeclampsia, 24 gestational hypertension), mean post-load glucose levels and total glucose area under the curve were significantly higher in cases than in controls (P < or =.04) and were positively correlated with peak mean arterial pressure. After adjustment for potential confounders, 2-hour post-load glucose levels remained strongly related to risk for hypertension (adjusted odds ratios = 1.48; 95% confidence interval 1.13, 1.92, per 10 mg/dL increase) and to peak mean arterial blood pressure (r =.23, P =.04), as did total glucose area under the curve (P < or =.04). Cases were also more likely to have had one abnormal glucose tolerance test (28% versus 5%, P =.004). Stratifying analyses by case severity (preeclampsia and gestational hypertension) yielded similar results. Among all subjects, more cases than controls were also diagnosed with gestational diabetes (31% versus 12%, P =.008). CONCLUSION: These findings are consistent with the hypothesis that insulin resistance precedes the clinical onset of hypertension in pregnancy, and may be important in the etiology of hypertension.

Detection of Clostridium perfringens enterotoxin in stool specimens and culture supernatants by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to detect and quantitate Clostridium perfringens enterotoxin A in culture supernatants and in stool specimens from cases of diarrhea in which high numbers of enterotoxin-producing Clostridium perfringens were isolated. To analyze for enterotoxin A, polyvinyl chloride microtiter plates were coated with dilute immune whole rabbit serum. Enterotoxin A standards and samples were allowed to react with sensitized wells. The presence of the immobilized antigen in the wells was detected by the binding of immune rabbit immunoglobulin conjugated with peroxidase. Nanograms of enterotoxin were detectable. Four enterotoxin-positive and seven enterotoxin-negative cultures grown in Duncan-Strong medium gave expected results. Eighteen of 23 diarrheal stool specimens obtained after a food-poisoning outbreak at a state hospital were found to contain microgram quantities of enterotoxin per gram of stool, whereas five control diarrheal specimens contained less than 0.6 ng enterotoxin per gram of stool. These results indicate that the enzyme-linked immunosorbent assay technique is useful for differentiating enterotoxigenic strains and for diagnosing diarrhea caused by enterotoxigenic Clostridium perfringens.

Prostaglandin H synthase activity in the sheep placenta during cortisol-induced labor at 128-131 days of gestation and during spontaneous delivery at term.

This study investigated whether the prostaglandin H synthase (PGHS) enzyme activity of sheep fetal placental cotyledon can be induced by cortisol at 128-131 days of gestation (dga) as compared to gestational age matched controls, before PGHS's normal gestational increase would occur (experiment 1). This study also investigated whether active PGHS is diminished following prostanoid synthesis in the labor process (experiment 2). A PGHS activity assay was employed in which PGE2 product was measured under initial velocity conditions. Labor was induced before term by continuous infusion of 10 mg of cortisol succinate per day (day 1) followed by 15 mg per day (days 2-4, or until delivery) of cortisol succinate administered through the fetal saphenous vein. Cotyledonary tissue was collected from sheep at term (142-145 dga), as judged by the absence of labor-type myometrial electromyogram (EMG) activity, and during cortisol induction at 128-131 dga. Tissue was also collected from term laboring animals immediately after fetal delivery while the fetus was still attached to the umbilicus and before placental delivery. At 128-131 dga, cortisol had no significant effect on PGHS activity as compared to gestational age matched saline-infused controls; thus, it is unlikely that cortisol directly induces PGHS. In experiment 2, normal progression of active spontaneous labor led to a significant diminution of PGHS activity (p < 0.05) that may be partially explained, based on thin-layer chromatography (TLC) results, as a significant decrease in PGE2 (p < 0.05) production coincident with a lesser compensatory increase in PGD2 (p = 0.06) output.

Dietary vitamin D toxicity in a household of pot-bellied pigs (Sus scrofa).

Vitamin D intoxication developed in Vietnamese Pot-Bellied Pigs (Sus scrofa) fed 2 commercially available swine rations. Pronounced hypercalcemia and a history incompatible with other causes of hypercalcemia led to confirmation of this diagnosis by plasma vitamin D metabolite analysis in 2 affected animals as compared to a control animal. Feed sample analysis suggested the diet as the likely source of toxicity.

Serologic evaluation, efficacy, and safety of a commerical modified-live canine distemper vaccine in domestic ferrets.

OBJECTIVE: To determine efficacy and safety of a commercial modified-live canine distemper virus (CDV) vaccine used for prophylaxis in domestic ferrets. ANIMALS: Sixteen 16-week-old neutered male ferrets. PROCEDURES: Equal groups of ferrets were inoculated subcutaneously at 16 and 20 weeks of age with saline (0.9% NaCl) solution or a vaccine derived from the Onderstepoort CDV strain and attenuated in a primate cell line. Live virulent CDV was administered to all ferrets intranasally and orally 3 weeks after the second inoculation. Clinical signs and body weights were monitored regularly during the study. Blood samples for serologic examination were drawn prior to each inoculation, before challenge exposure, and 10, 15, and 21 days after exposure. Blood samples for reverse transcriptase polymerase chain reaction (RT-PCR) were obtained 5 days after the first vaccination, and 5, 10, 15, and 21 days after challenge exposure. RESULTS: After challenge exposure, control ferrets had significantly more clinical signs and weight loss, compared with vaccinates. All vaccinated ferrets survived, whereas all control ferrets died. The RT-PCR assay was successful in detecting CDV in blood and fresh or formalin-fixed tissues from infected ferrets. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that the vaccine when given SC to domestic ferrets as directed is safe and protective against challenge exposure with virulent CDV. The RT-PCR assay may simplify detection of CDV in fresh and fixed tissues.

Susceptibility of the Siberian polecat to subcutaneous and oral Yersinia pestis exposure.

To determine if the Siberian polecat (Mustela eversmannii) represents a suitable model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela nigripes), polecats were exposed to 10(3), 10(7), or 10(10) Yersinia pestis organisms by subcutaneous injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse. Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 10(3) group, 100% mortality in the 10(7) and 10(10) groups, and 83% mortality in the mouse-fed group). Within the challenged group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly longer than those receiving higher parenteral doses. Within challenged animals, mean survival time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal output; time to onset of lethargy and other signs was also related to risk of dying and/or plague dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while two seropositive animals later died. This study confirms that the Siberian polecat is susceptible to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets in future plague studies and related vaccine trials.

Pancreatic beta cell tumor in ferrets: 20 cases (1986-1994).

OBJECTIVE: To characterize survival times, disease-free intervals, and prognostic variables in ferrets with pancreatic beta cell neoplasia. DESIGN: Retrospective study. ANIMALS: 20 ferrets with histologically confirmed pancreatic beta cell tumors. PROCEDURE: Medical records of ferrets with pancreatic beta cell tumors were reviewed. Variables such as age, sex, duration of clinical signs, existence of other neoplasia, single versus multiple masses in the pancreas, benign or malignant histologic features, and the type of treatment used at redevelopment of clinical signs were examined to determine their relationship to survival times and disease-free intervals. RESULTS: Duration of clinical signs prior to diagnosis and initial surgery was inversely correlated with survival time and disease-free interval. Overall median survival time for all ferrets with pancreatic beta cell tumors was 483 days. Median disease-free interval for ferrets that had abdominal surgery for pancreatic beta cell tumor removal was 240 days. Distant tumor metastasis was not found in this group of ferrets, but local tumor recurrence was common. In 4 ferrets that had a second celiotomy, removal of recurrent pancreatic tumors did not prolong survival but did result in a second disease-free interval. CLINICAL IMPLICATIONS: Pancreatic beta cell tumors should be considered in ferrets over 2 years of age that have clinical signs of seizures, episodic lethargy, ptyalism, ataxia, and hind limb weakness. In the study reported here, complete surgical excision of the tumor resulted in temporary resolution of clinical signs in ferrets; however, redevelopment of clinical signs at a later time was common.

A comparison of sevoflurane and isoflurane for short-term anesthesia in polecats (Mustela eversmanni).

Twenty-four Siberian polecats (Mustela eversmanni) from 12 litters were anesthetized with either inhaled sevoflurane or isoflurane. With 7% delivered sevoflurane and 5% delivered isoflurane, time to loss of righting reflex (mean +/- SE) with sevoflurane (1.9 +/- 0.1 min) was significantly shorter compared with isoflurane (2.6 +/- 0.1 min). During maintenance at a light plane of anesthesia, systolic arterial pressure was significantly higher with sevoflurane (83 +/- 2 mm Hg) compared with isoflurane (66 +/- 2 mm Hg), and heart rate was significantly lower with sevoflurane (191 +/- 3 beats/min) compared with isoflurane (204 +/- 3 beats/min). There was no difference in respiratory rate jugular venous pH, pCO3, HCO3-, base excess, or recovery of righting reflex. Induction of anesthesia is more rapid and blood pressure is better maintained with sevoflurane compared with isoflurane; therefore, sevoflurane may be less stressful and safer. Inhaled sevoflurane should be an appropriate anesthetic for black-footed ferrets (Mustela nigripes) in laboratory and field conditions.

Ultrasound therapy for the prevention and correction of contractures and bone mineral loss associated with wing bandaging in the domestic pigeon (Columba livia).

Figure-of-eight wing bandaging is widely used to treat wing injuries, to immobilize wings before and after fracture repair, and during transient wing paralysis. However, prolonged bandaging can lead to bone loss and to contractures and reduced range of joint motion. Studies evaluating the efficacy of therapeutic ultrasound to reverse and prevent bandaging-associated contractures in pigeons (Columba livia) showed a significant increase in elbow and carpal extension after 10 twice weekly ultrasound treatments when started either 4 or 11 days after bandage placement. In addition, after 42 days of wing bandaging, three ultrasound treatments stimulated a faster reversal of carpal wing rotation loss than removal of the bandage over the 10-day treatment period. Finally, bone loss in response to 28 days of bandaging was significant, progressed at 2.8% per week, and was not affected by ultrasound treatment twice weekly during this period. Therefore, therapeutic ultrasound prevented and reversed loss of wing extension associated with figure-of-eight bandaging but did not lessen the disuse osteoporosis created by bandaging in these birds.

Sevoflurane anesthesia in desert tortoises (Gopherus agassizii).

The effects of sevoflurane on anesthesia induction, recovery, ventricular pressures, heart rate, ventricular pH, blood gas values, and electrolytes were evaluated in desert tortoises (Gopherus agassizii). Tortoises were orotracheally intubated while awake and ventilated manually with 3-7% sevoflurane in oxygen (1 L/min) to achieve desired expired sevoflurane concentrations. Data, consisting of induction time, recovery time, systolic, diastolic, and mean ventricular pressures, heart rate, ventricular pH, blood gas values, and electrolytes, were collected prior to anesthesia and sequentially at 2.50% and 3.75% expired sevoflurane as measured at the junction of the endotracheal tube and the breathing circuit. Blood pressure was measured and blood samples were collected through a 25-ga needle passed through a cardiac access port that was placed while the tortoises were in dorsal recumbency. Mean (+/-SE) induction time was 2.55+/-0.55 min, recovery time was 27.58+/-7.55 min, and duration of anesthesia was 105+/-12 min. Mean (+/-SD) values for systolic, diastolic, and mean ventricular pressures in awake tortoises were 28+/-3 mm Hg, 22+/-2 mm Hg, and 24+/-2 mm Hg, respectively. Sevoflurane (2.5% expired) significantly decreased systolic (14+/-3 mm Hg), diastolic (12+/-1 mm Hg), and mean (13+/-1 mm Hg) ventricular pressures compared with those of awake tortoises. Ventricular pressures did not decrease further with increasing depth of anesthesia. Heart rate (32+/-4 beats/min) did not change significantly under sevoflurane anesthesia. Sevoflurane administration increased ventricular PO2 but did not change Na+, K+, or iCa++ concentrations. Sevoflurane appears to provide safe and effective anesthesia with rapid induction and recovery.

Clarithromycin pharmacokinetics in the desert tortoise (Gopherus agassizii).

Clarithromycin is a new, safe orally administered macrolide antibiotic active against Mycoplasma sp. in humans. Single-dose and multidose pharmacokinetic parameters were determined for clarithromycin in wild-caught desert tortoises (Gopherus agassizii) seropositive for M. agassizii. Clarithromycin blood levels were measured in three tortoises for up to 72 hr after a single oral dose of 7.5 mg/kg. In a second group of six tortoises, levels were measured after a dose of 15 mg/kg. Noncompartmental iterative two-stage Bayesian and nonparametric expectation maximization pharmacokinetic parameters were determined for each animal assuming first order rate constants. At 15 mg/kg, the maximum concentration was 1.37 microg/ml, the time to maximum concentration was 8.0 hr, and a plasma half-life of 11.69 hr was derived from the latter method. The absorption constant was 0.08/hr, the absorption half-life was 8.47 hr, and the weight-normalized volume of distribution was 5.30 L/kg. Predictions derived by the latter method suggested a dosage of 15 mg/kg p.o. every 24 hr to achieve maximal blood levels of > or =1 microg/ml for multiple dosing. However, results from a preliminary multidose study with three tortoises indicate that the drug is accumulated; therefore, the predicted dose may be closer to 15 mg/kg p.o. every 2-3 days to maintain blood levels of 2-7.5 microg/ml. (For n = 3, 2-point linear regression median estimates for the apparent elimination rate constant (K) and half-life are 0.0227/hr and 30.52 hr, respectively.) This multidose accumulation reflects a slower apparent elimination than that predicted in the eight single-dose tortoises (i.e., K = 0.0593/hr, t1/2 = 11.69 hr). This study highlights a potential pitfall of depending solely on single-dose studies and the potential value of oral administration in reptiles.

Noninvasive monitoring of fetal growth and development in the Siberian polecat (Mustela eversmanni).

The Siberian polecat (Mustela eversmanni) is the preferred species to assess procedures and establish normative values for application in the related and endangered black-footed ferret (Mustela nigripes). This study was undertaken to physically, ultrasonographically, and radiographically evaluate fetal development in a spontaneously breeding captive Siberian polecat population. Ultrasonographically, fetal sac enlargement allowed presumptive pregnancy detection as early as 12 days of gestation, the fetal pole was the first definitive sign of pregnancy at about 18 days of gestation, when the fetal heart beat also appeared, and definitive pregnancy detection by ultrasound was essentially 100% accurate after 18 days. The estimation of fetal number by ultrasound was less reliable than by radiography, as it is in other litter-bearing species. Crown-rump growth, organ differentiation, and calcification patterns resembled those of domestic carnivores except that comparable developmental stages in polecats occurred at disproportionately later times, suggesting that young Siberian polecats are delivered in a less developed state. Careful palpation permitted detection of pregnancy after day 17 but with less certainty than with ultrasound. Radiographic evaluation was insensitive and of limited value for pregnancy detection until near term. Litter number and fetal detail were difficult to assess until ossification could be observed, 3-6 days before parturition.

Pregnancy-induced hypertension and insulin resistance: evidence for a connection.

PIH, the most common complication of pregnancy, remains a major source of maternal-child morbidity and mortality. Yet the etiology of this disorder is still little understood. There is now a growing body of evidence linking PIH and insulin resistance. Both proteinuric and non-proteinuric PIH predict future essential hypertension, and to a lesser extent, diabetes, disorders strongly related to glucose intolerance and insulin resistance. PIH is associated with diabetes, occurring in up to 50% of diabetic pregnancies. PIH is characterized by the same features that define IRS, including hypertension, dyslipidemia, disruption of endothelial and platelet function and related disturbances of prostanoid synthesis, coagulation and fibrinolytic abnormalities, hyperuricemia, atherosclerotic changes, and obesity. During the last decade, controlled studies by at least 11 different research groups in nine countries have established significant positive associations between both proteinuric and nonproteinuric PIH and various measures of insulin resistance. In particular, prospective investigations by at least five groups of investigators have indicated that relative hyperinsulinemia, glucose intolerance, and insulin insensitivity predict the subsequent development of PIH. These and other studies suggest that insulin resistance may play a causal role in the pathogenesis of PIH, and that some aspects of PIH may represent an early manifestation of IRS, precipitated by the profound metabolic and hemostatic challenges of gestation.

Morbidity from anterior ilium bone harvest. A comparative study of lateral versus medial surgical approach.

The morbidity of bone harvest was compared between anterior lateral and medial surgical approaches in a randomized prospective study. Forty consecutive patients, each requiring a minimum 40 cc of loose corticocancellous bone for maxillofacial reconstruction, were randomly placed into two equal groups. Morbidity vectors assessed included bone volume, blood loss, length of surgery, length of hospital stay, incidence of seroma, incidence of anterior thigh paresthesia, postoperative pain, and gait disturbance. The results demonstrated no significant difference in morbidity between these two approaches; therefore selection of either approach is the surgeon's personal preference. A thorough understanding of the osseous anatomy of the anterior ilium and its muscular attachments, a good surgical technique, an efficient surgical team, and a continuous flow of required surgical instruments are essential to reduce the morbidity of bone harvest.

Effect of a 48-hour intravenous delta 4-androstenedione infusion on the pregnant rhesus monkey in the last third of gestation: changes in maternal plasma estradiol concentrations and myometrial contractility.

Increased myometrial activity in the pregnant rhesus monkey occurs in situations in which plasma estrogen concentrations are elevated. Examples of such situations are after laparotomy, with or without hysterotomy, and before delivery. The increased activity occurs primarily in the hours of darkness. To investigate the possibility of a causal relationship between the rise in estrogens and myometrial activity, we infused androstenedione intravenously for 48 hours to the rhesus monkey in the last third of gestation. Myometrial activity was quantified either as an increase in the number of individual contraction events or as a change in the power spectrum at high frequencies characteristic of contractions. Androstenedione infusion was followed by increased myometrial activity. Maternal plasma 17 beta-estradiol concentration was significantly elevated at 10 AM on the second day of androstenedione infusion. We conclude that, in the rhesus monkey late in gestation, estradiol may play a role in the regulation of the contraction activity observed during the hours of darkness in several different situations.

Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons.

Recent identification of a second, inducible form of prostaglandin H synthase (PGHS-2) led to the hypothesis that constitutively expressed PGHS (PGHS-1) is involved in the homeostatic role of eicosanoids, whereas the inducible enzyme is responsible for their inflammatory actions. We report here the purification of PGHS-2 from near-term sheep placental cotyledons. The PGHS-2 from this tissue was purified in multimilligram quantities by a combination of anion-exchange, size-exclusion, and affinity chromatography. This enzyme is different from ovine seminal vesicle PGHS-1 and was characterized as PGHS-2 based on (a) chromatographic properties, (b) immunochemical reactivities with isoenzyme-specific antibodies, (c) amino acid microsequencing, (d) kinetics of reaction with arachidonic acid (Km = 2.1 +/- 0.2 microM vs 8.3 +/- 0.2 microM for ovine PGHS-1), and (e) different sensitivities for several non-steroidal antiinflammatory drugs. Since the first identification of PGHS, ram seminal vesicles served as a rich source of the enzyme (PGHS-1). Our studies establish the sheep placental cotyledons as a rich natural source of PGHS-2.

Prostaglandin synthase activity of fetal sheep cotyledons at 122 days of gestation and term: expression of prostaglandin synthetic capacity in fetal cotyledonary tissue near labor is location-dependent.

An activity assay is described quantification of prostaglandin synthase (PGHS) in sheep placental cotyledon under initial velocity conditions through measurement of the stable product prostaglandin E2 (PGE2). The effects of temperature, time, and substrate concentration on initial reaction velocity, and lipoxygenase and PGHS product formation in cotyledonary tissue, were examined in detail. We used this activity assay to determine whether or not an increase of active PGHS by placental location within the uterus might contribute selected prostaglandins (PG) for the directed initiation of parturition. Sheep cotyledon tissue was collected (n = 6 animals) from the ventral aspect of the uterine body, mid-horn, and horn tip at 122 days of gestation (dga), and from the same locations in the ventral body and horn tip at 142-145 dga (in animals at term but not in labor; n = 4). At 122 dga, there was no increase in active PGHS in cotyledonary tissue from the horn tip, mid-horn, or uterine body. By 142-145 dga, the horn showed significantly (p < 0.01) more enzyme activity than the body. At the same time, production of PGE2, expressed as a percentage of total eicosanoids, had not changed significantly. The development of an increase in PGHS toward the uterine tip implies that variations in regional PG production may contribute to the progression of labor.