Inflammation of the fetal ovine skin following in utero exposure to Ureaplasma parvum.

There is increasing evidence linking in utero infection and inflammation to preterm birth. Many commensal urogenital tract microorganisms, including the Mycoplasmas and Ureaplasmas, are commonly detected in association with preterm birth. Using an ovine model of sterile fetal inflammation, we demonstrated previously that the fetal skin generates a robust inflammatory response following in utero exposure to lipopolysaccharides from Escherichia coli. The fetal skin's response to colonization of the amniotic fluid by viable microorganisms remains unstudied. We hypothesised that in utero infection with Ureaplasma parvum serovar 3 would induce a proinflammatory response in the fetal skin. We found that (1) cultured fetal keratinocytes (the primary cellular constituent of the epidermis) respond to U. parvum exposure in vitro by increasing the expression of the chemotactant monocyte chemoattractant protein 1 (MCP-1) but not interleukin 1ß (IL-1ß), IL-6, IL-8, or tumor necrosis factor-α (TNF-α); (2) the fetal skin's response to 7 days of U. parvum exposure is characterized by elevated expression of MCP-1, TNF-α, and IL-10; and (3) the magnitude of inflammatory cytokine/chemokine expression in the fetal skin is dependent on the duration of U parvum exposure. These novel findings provide further support for the role of the fetal skin in the development of fetal inflammation and the preterm birth that may follow.

Toxoplasma gondii diagnosis in ovine aborted fetuses and stillborns in the State of Pernambuco, Brazil.

The aim of this research was to study the contribution of Toxoplasma gondii to reproductive failure using nested PCR and histopathological examination of fetuses, stillborns and placentas. We examined 245 organs of fetuses and 28 placentas from 35 abortions and stillborns from naturally occurring miscarriages in sheep in the State of Pernambuco, Brazil. At necropsy, fragments of brain, cerebellum, medulla, lung, heart, spleen, liver and placenta were taken for nested PCR and histopathological tests. Pathological examination revealed macroscopic lesions, suggesting T. gondii infection in 5/35 (14.3%) of the placentas. The histopathological examination revealed no lesions characteristic of toxoplasmosis in the organs investigated. In the five placentas, lesions consistent with toxoplasmosis were observed as an inflammatory non-suppurative infiltrate, along with multiple necrosis and mineralization. Nested PCR showed three aborted fetuses and two stillborns (14.3%) to test positive for T. gondii, with DNA amplification in all organs and the placenta, especially the heart and the placenta, which are the tissues of choice. This study substantiates the theory that T. gondii is involved in miscarriages and stillbirths and in the placentas of naturally infected sheep in Brazil. Such findings have not previously been described in the national literature.

Postnatal hypothalamic-pituitary-adrenal function in sheep is influenced by age and sex, but not by prenatal growth restriction.

The relationship between impaired fetal nutrient supply and postnatal hypothalamic-pituitary-adrenal (HPA) function was examined in ovine models of prenatal growth restriction (GR) caused by small placental size (SP) or by maternal undernutrition (UN). Adrenocorticotrophin (ACTH) and cortisol responses following corticotrophin-releasing hormone (CRH) plus arginine vasopressin (AVP) challenge were examined at 9, 18 and 24 months in growth-restricted (GR-SP) and normal birthweight (control) females (Experiment 1), and at 6 months in growth-restricted (GR-SP, GR-UN) and normal weight males and females (Experiment 2). In Experiment 1, GR-SP offspring were born early, were 40% lighter at birth and had higher fractional weight gains to weaning than control offspring. Baseline ACTH and cortisol were independent of GR and cortisol decreased with age. GR did not affect the HPA response to CRH+AVP challenge at any stage, but ACTH increased with age. In Experiment 2, birthweight was greater in control offspring than in GR-UN offspring, which had a higher birthweight again compared with GR-SP offspring. Only the latter group was born early and exhibited rapid catch-up growth to weaning. Neither nutritional route to GR altered HPA function at 6 months. Males grew faster than females and HPA responses after stimulation were lower in males. Together, the results of these studies demonstrate that postnatal HPA function in sheep is influenced by age and sex, but not by GR.

Maternal administration of erythromycin fails to eradicate intrauterine ureaplasma infection in an ovine model.

Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n = 16) or 10B medium (n = 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n = 4; 10B group, n = 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n = 7), medium and erythromycin (n = 8), ureaplasma only (Up; n = 6), or ureaplasma and erythromycin (Up/E; n = 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatography-mass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (<10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.

Myoblasts isolated from hypertrophy-responsive callipyge muscles show altered growth rates and increased resistance to serum deprivation-induced apoptosis.

Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. These observations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals.

Impact of maternal nutrition during pregnancy on pituitary gonadotrophin gene expression and ovarian development in growth-restricted and normally grown late gestation sheep fetuses.

The influence of maternal nutrition during pregnancy on anterior pituitary gonadotrophin gene expression and ovarian development in sheep fetuses during late gestation was investigated. Embryos recovered from superovulated adult ewes that had been inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After embryo transfer, adolescent ewes were offered a high or moderate amount of a complete diet. Pregnancies were terminated at day 131 +/- 0.6 of gestation and the fetal brain, anterior pituitary gland and gonads were collected. Gonadotrophin gene expression (LHbeta and FSHbeta subunits) in the fetal pituitary gland was examined using in situ hybridization. Ovarian follicular development was quantified in haematoxylin- and eosin-stained ovarian sections embedded in paraffin wax. Six dams that were offered a high nutrient intake carried normal-sized fetuses (weight within +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet) and 13 dams carried growth-restricted fetuses (weight

The effect of maternal undernutrition on ovine fetal growth.

Modifications in maternal nutrition during pregnancy can significantly disrupt fetal growth and subsequent post-natal health and survival. This study investigated the effects of undernutrition on fetal growth and the potential mechanisms involved. Tissue from pregnant ewes (n=27) was investigated on days 45, 90 and 135 of gestation (term = approximately 150 days). The thoracic girth (P<0.05) was greater in fetuses from nutrient restricted ewes on day 45 and there was also a trend towards an increased gut weight (P<0.08). By day 90, the fetal brain and thymus weight were lighter in underfed than in well-fed animals whilst the weight of the fetal ovaries was heavier (P<0.05). On day 135 the fetal heart, pancreas, thymus, gut and kidney weights were lighter in undernourished ewes (P<0.05). When expressed as a percentage of fetal body weight, significance was retained in the heart, pancreas and thymus (P<0.05). Bone growth was also affected. At day 90 the fetal femur and metatarsal were longer in underfed mothers (P<0.05). In contrast, the fetal humerus and scapula were shorter in underfed than in well-fed animals on day 135 (P<0.05) when the weight of the semitendinosus muscle (P<0.05) was also reduced. The fall in fetal glucose (P<0.1), insulin (P<0.01) and IGF-I (P<0.01) levels in underfed ewes on day 135 may have compromised fetal growth. Fetal plasma IGF binding protein-2 also increased between days 90 and 135 in underfed ewes (P<0.03), whilst levels were unaltered in well-fed animals. Although maternal and fetal plasma IGF-I levels increased with gestation (P<0.01) and the placentome morphology altered in all ewes (P<0.05), the fall in placental mass (P<0.05), amniotic and allantoic glucose concentrations (P<0.05) and maternal plasma glucose and insulin levels (P<0.05) in underfed ewes in late gestation may have compromised fetal substrate delivery. These perturbations in fetal development may have significant implications on adult health and carcass conformation, raising important health and economic issues in medical and agricultural sectors.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy.

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 x 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. At the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

In-utero overgrowth in ruminants following embryo culture: lessons from mice and a warning to men.

Unusually large offspring have been born in ruminants following the transfer to recipients of embryos that have either been subjected to some form of manipulation, e.g. nuclear transfer, or have been exposed to an unusual in-vivo or in-vitro environment. Overgrowth syndromes have been reported in other species, including humans and mice, but these have arisen from chromosomal abnormalities and spontaneous or experimentally induced genetic mutations. Overgrowth phenotypes across the species, however, exhibit many common features, including alterations in organ and tissue development, and placental anomalies. Our current working hypothesis is that the causative agent(s) alter(s) the expression of a gene or genes associated with growth and development. Imprinted genes have been implicated in this syndrome because: (i) similar phenotypes are observed in both humans and mice when the expression of such genes has been altered; and (ii) they may be more vulnerable to epigenetic modification during the period (oocyte to blastocyst) when embryos are cultured in vitro. Evidence supporting this hypothesis is reviewed and the implications for assisted reproduction in humans discussed.

A preliminary report on the effect of dietary energy on prostaglandin F2 alpha production in vitro, interferon-tau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes.

Two groups of ewes were fed to provide 1.70 x (high energy group; n = 15) or 0.56 x (low energy group; n = 15) energy requirements for maintenance of liveweight from 14 d before a synchronized mating in November until slaughter at 9 or 15 d after mating. We investigated the effects on interferon-tau (IFN tau) secretion by the conceptuses, prostaglandin F2 alpha (PG) production in vitro by endometrial tissue, and associated rates of embryo mortality, endometrial progesterone content and progesterone production by luteal tissue. No differences between groups in pregnancy rate were detected on Day 9 between the 2 groups. Proportionately (6/6 vs 2/5), there were more pregnant ewes in the high energy group on Day 15, although this difference did not reach significance (P = 0.06). The proportion of corpora lutea represented by embryos was significantly lower in undernourished ewes (P < 0.05). Secretion in vitro of PG was lower in the 2 pregnant ewes of the low energy group on Day 15, and it was accompanied by higher IFN tau secretion by conceptuses recovered from these ewes. However, the limited number of pregnant ewes recorded on Day 15 prevented any statistical comparison. Neither mean endometrial content of progesterone nor ovarian venous progesterone concentrations and production of progesterone by luteal were affected by nutrition. The provisional results of the present experiment indicate that undernutrition may induce a reduction in the rate of secretion of IFN tau and can therefore increase production of PG from the endometrium. This could initiate luteolysis. The lower pregnancy rates observed in underfed ewes could be mediated through this alteration in the signal of maternal recognition of pregnancy. However, these findings remain to be shown in further experiments including a larger number of animals, as they only represent data from 2 undernourished animals.

Ovine fetal malformations induced by in utero inoculation with Main Drain, San Angelo, and LaCrosse viruses.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

Variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of bovine-virus diarrhoea virus.

Pregnant Merino ewes were inoculated intravenously between days 63 and 65 of gestation with a non-cytopathogenic (ncp) bovine-virus diarrhoea-virus (BVDV) isolate (experiment A). The histomorphological findings and the distribution of viral antigen, as revealed by immunohistochemistry in brains of fetuses from experiment A, were compared with those seen in fetal brains from a previous study (experiment B), in which pregnant ewes had been intravenously infected between days 65 and 68 of gestation with the cytopathogenic (cp) BVDV strain Indiana. The two viruses showed remarkable variations concerning their pathogenicity for the developing fetal brain. The cp BVDV had a much higher neuropathogenic potential than the ncp BVDV and induced severe intracranial malformations in most fetuses. In experiment A, exclusively relatively mild leucoencephalomalacic lesions occurred. Between fetuses of the two experiments, significant differences concerning the distribution of viral antigen and the inflammatory response were found. In the majority of fetal brains from experiment B examined at days 10, 14 and 21 post inoculation (p.i.), antigen-containing differentiated brain cells (neurons, astrocytes, oligodendrocytes) and undifferentiated cells in the periventricular germinal zones were seen throughout the different zones of the developing telencephalon and cerebellum. At 21 days p.i., a marked inflammatory response consisting of brain macrophages and other mononuclear cells occurred in the meninges and in the brain parenchyma of fetuses from experiment B. In brain sections of fetuses infected with ncp BVDV, in contrast to fetuses infected with cp BVDV, viral antigen was not detectable during the early stages (days 10 and 20) p.i., and histopathological lesions were not seen at this stage. At days 41 and 47 p.i., antigen-positive astrocytes and oligodendrocytes were found in the developing white matter of the telencephalon and cerebellum. Furthermore, antigen-containing neurons were seen in the developing cerebral cortex. Cellular infiltrations in fetal brains from experiment A were limited to the leucoencephalomalacic areas in the developing cerebral and cerebellar white matter and consisted exclusively of brain macrophages. Immunohistochemical staining in brain sections of fetuses from both experiments revealed that numerous perivascular cells contained viral antigen, whilst positive endothelial cells were exclusively found in fetuses from experiment A. From the findings of this study it was concluded that the cp BVDV stain used in experiment B has a marked tropism for the fetal brain and both its already differentiated and undifferentiated cell populations, and that the resulting brain lesions primarily are the consequence of a direct cytolysis of these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Virological and pathological findings in sheep fetuses following experimental infection of pregnant ewes with cytopathogenic-bovine-virus diarrhoea virus.

Eighteen pregnant Merino ewes were inoculated intravenously between days 65 and 68 of gestation with the unpurified cytopathogenic (cp) bovine virus diarrhoea virus (BVDV) strain Indiana (experiment I). In experiment II, three ewes were inoculated with the same virus after two successive plaque isolations in order to compare its pathogenicity for the fetus with special regard to lesions in the fetal brain. In experiment I, fetal blood and tissue samples, allantoic fluids and placentomes were collected sequentially between 10 and 80 days post-inoculation (p.i.). BVDV was recovered from 6 of 19 fetuses examined during the first 3 weeks after inoculation. From fetuses sampled between 30 and 50 days p.i. virus was isolated from three cases only, and from 60 days p.i. onwards virus was no longer recovered. BVDV was longer detected in the allantoic fluid than in fetal tissues and continued to be present until 80 days post-inoculation. From tissue samples of two fetuses of experiment I, only non-cytopathogenic BVDV was isolated, whilst samples from seven fetuses contained the cp BVDV biotype as revealed by an immunoplaque assay. The cp biotype was also isolated from placentomes. In experiment II, virus was not isolated from any of the tissue samples of two living fetuses collected at 67 days post-inoculation. In both experiments, cp BVDV was recovered from allantoic fluid samples. In contrast to the developing fetal brain, other tissues or organs seemed to be less vulnerable to the cp BVDV strain Indiana. The partial purification of this virus strain did not affect its pathogenicity for the brains of the developing fetuses.

Maturation of immunological reactivity in the fetal lamb infected with Akabane virus.

The development of cell-mediated immunological reactivity was studied in fetal lambs infected with Akabane virus. Examination of hepatic cells from fetuses between 40 and 75 days' gestation that had been infected via the transplacental route revealed inconsistent responses to Akabane, together with a uniform failure to respond to non-specific mitogens which contrasted with the behaviour of control, uninfected lambs. Following direct inoculation of fetal lambs with virus between 50 and 120 days' gestation, specific proliferative responses were observed on the part of the spleen cells from some. Direct challenge of fetal lambs of 4 months' gestation evoked cellular responses in lymph draining from the site of virus inoculation similar to those produced by challenge of adult sheep. The proliferative response of lymph-borne cells was substantially better if live, rather than inactivated, virus had been used.

In utero adenoviral infection of sheep.

The occurrence of natural in utero adenovirus infection in sheep was examined. Isolation of three adenovirus strains from the kidneys of 174 sheep foetuses is reported; all three isolates belonged to Type 2 bovine adenovirus (BAV 2) Subtype B. Five of 25 blood samples from sheep foetuses contained virus-neutralizing antibodies (1:4-1:24) against BAV 2. These data prove that transplacental transfer of adenovirus infection can occur naturally in sheep.

Simple technique for the direct isolation of toxoplasma tissue cysts from fetal ovine brain.

A simple technique is described for the isolation of Toxoplasma gondii tissue cysts from fetal ovine brain by centrifugation on a discontinuous density gradient of 30 per cent and 90 per cent Percoll (colloidal silica solution). Brain samples from 51 aborted ovine fetuses were examined by both the Percoll and mouse inoculation techniques; eight infections were detected by the Percoll technique compared to 12 by mouse inoculation. Possible reasons for this discrepancy and the scope for improving the Percoll technique are discussed.

Copper deficiency in pregnant ewes: its influence on the course of pregnancy and the health status of the offspring

Swayback disease (SD) which affects young sheep and a number of other animals is caused by the unavailability of metabolizable copper during the last half of pregnancy. In an effort to increase the present understanding of the development of SD and related human disorders we induced copper deficiency in pregnant ewes and studied its effects on pregnancy and the offspring. Nineteen pregnant ewes were rendered copper deficient by injection of ammonium tetrathiomolybdate (ATM), at 1mg/Kg during (I)the last 8-11 weeks, (II)the last 4-6 weeks and (III)the last 1-2 weeks of pregnancy. Treatment was continued until the lambs were weaned and sacrificed. Atomic absorption spectroscopy revealed that prolonged ATM treatment causes unpredictable changes in serum copper levels in the pregnant ewes, varying from large increases of 133 percent to large decreases of 65 percent. An unusually high number of treated ewes (29 percent) died during pregnancy. Neonates from ewes of the 1-2 week treatment group had normal serum copper levels, while 86 percent of the lambs from the 4-8 week group displayed subclinical serum copper levels (<0.5 ppm). Lambs from ewes in the 8-11 week group had serum copper levels ranging from moderate, 0.6 ppm to very high, 3.4 ppm. Nine of the 22 offspring showed signs of SD: none of these were from ewes receiving ATM treatment for the shortest period: 44 percent of the lambs from the intermediate group and 63 percent of those from the group receiving the longest ATM treatment were diseased. We conclude that while copper deficiency occuring in the last two weeks of pregnancy appears to have no effect, acute deficiency occuring at earlier stages, particularly the last 8-11 weeks, is likely to precipitate swayback disease in the offspring. Finally, it has been suggested that the early postnatal development stage of the human, corresponds to the final prenatal stage of the lamb, the present findings therefore suggest that the human foetus might be susceptible to copper deficiency even during the final weeks of pregnancy (AU)