Carol E. Kyle

Maternal undernutrition and the ovine acute phase response to vaccination

BackgroundThe acute phase response is the immediate host response to infection, inflammation and trauma and can be monitored by measuring the acute phase proteins (APP) such as haptoglobin (Hp) or serum amyloid A (SAA). The plane of nutrition during pregnancy is known to affect many mechanisms including the neuroendocrine and neuroimmune systems in neonatal animals but effects on the APP are unknown. To investigate this phenomenon the serum concentration of Hp and SAA was initially determined in non-stimulated lambs from 3 groups (n = 10/group). The dams of the lambs of the respective groups were fed 100% of requirements throughout gestation (High/High; HH); 100% of requirements for the first 65 d of gestation followed by 70% of requirements until 125 d from when they were fed 100% of requirements (High/Low; HL); 65% of liveweight maintenance requirements for the first 65 d gestation followed by 100% of requirements for the remainder of pregnancy (Low/High; LH). The dynamic APP response in the lambs was estimated by measuring the concentration of Hp and SAA following routine vaccination with a multivalent clostridial vaccine with a Pasteurella component, Heptavac P™ following primary and secondary vaccination.ResultsThe Hp and SAA concentrations were significantly lower at the time of vaccination (day 8–14) than on the day of birth. Vaccination stimulated the acute phase response in lambs with increases found in both Hp and SAA. Maternal undernutrition led to the SAA response to vaccination being significantly lower in the HL group than in the HH group. The LH group did not differ significantly from either the HH or HL groups. No significant effects of maternal undernutrition were found on the Hp concentrations. A significant reduction was found in all groups in the response of SAA following the second vaccination compared to the response after the primary vaccination but no change occurred in the Hp response.ConclusionDecreased SAA concentrations, post-vaccination, in lambs born to ewes on the HL diet shows that maternal undernutrition prior to parturition affects the innate immune system of the offspring. The differences in response of Hp and SAA to primary and secondary vaccinations indicate that the cytokine driven APP response mechanisms vary with individual APP.

Cellular and hormonal disruption of fetal testis development in sheep reared on pasture treated with sewage sludge

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood.

Alkyl phenols and diethylhexyl phthalate in tissues of sheep grazing pastures fertilized with sewage sludge or inorganic fertilizer.

We studied selected tissues from ewes and their lambs that were grazing pastures fertilized with either sewage sludge (treated) or inorganic fertilizer (control) and determined concentrations of alkylphenols and phthalates in these tissues. Mean tissue concentrations of alkylphenols were relatively low (< 10–400 μg/kg) in all animals and tissues. Phthalates were detected in tissues of both control and treated animals at relatively high concentrations (> 20,000 μg/kg in many tissue samples). The use of sludge as a fertilizer was not associated with consistently increased concentrations of either alkylphenols or phthalates in the tissues of animals grazing treated pastures relative to levels in control animal tissues. Concentrations of the two classes of chemicals differed but were of a similar order of magnitude in liver and muscle as well as in fat. Concentrations of each class of compound were broadly similar in tissues derived from ewes and lambs. Although there were significant differences (p < 0.01 or p < 0.001) between years (cohorts) in mean tissue concentrations of both nonylphenol (NP) and phthalate in each of the tissues from both ewes and lambs, the differences were not attributable to either the age (6 months or 5 years) of the animal or the duration of exposure to treatments. Octylphenol concentrations were generally undetectable. There was no consistent cumulative outcome of prolonged exposure on the tissue concentrations of either class of pollutant in any ewe tissue. Mean tissue concentrations of phthalate were higher (p < 0.001) in the liver and kidney fat of male compared with female lambs. We suggest that the addition of sewage sludge to pasture is unlikely to cause large increases in tissue concentrations of NP and phthalates in sheep and other animals with broadly similar diets and digestive systems (i.e., domestic ruminants) grazing such pasture.

Phthalate and alkyl phenol concentrations in soil following applications of inorganic fertiliser or sewage sludge to pasture and potential rates of ingestion by grazing ruminants

Soil concentrations of dioctyl phthalate (DOP) and the alkyl phenols, octyl phenol (OP) and nonyl phenol (NP), after repeated surface applications of sewage sludge to pastures, were investigated. Liquid sludge was applied at a rate of 2.25 tonnes dry matter (DM) per hectare to each of three treated (T) plots on three occasions during the summer and two occasions in the early spring over a period of 2.5 years. Control (C) plots were treated with inorganic fertiliser containing amounts of nitrogen equivalent to those applied to the treated plots. At between 69 and 81 days after the application of sludge, 15 separate soil samples were collected from one half of each of the plots (Experiment 1). Concentrations (microg g(-1)) of DOP were higher (P < 0.001) than those of NP, while those of OP were generally below detectable levels. Mean soil concentrations of DOP were not significantly different in T and C plots [0.233 vs. 0.155 microg g(-1); standard error of the difference (SED) = 0.046; not significant (NS)], partly because there was already a relatively large amount of DOP present. NP concentrations were, however, significantly higher in T than in C plots (0.021 vs. 0.013 microg g(-1) SED = 0.002; P < 0.05). There was no consistent change over time in the mean soil concentrations of these compounds when sampled at intervals of 3-6 months. Concentrations in soil samples collected at monthly intervals following sludge application indicated that the variation in concentrations of these endocrine-disrupting compounds (EDC) was unrelated to time since sludge application. Rates of soil ingestion, expressed as the percentage of DM intake represented by soil, were higher during the winter than the summer (5.40 vs. 1.17; SED = 0.360; P < 0.001) and estimated daily intakes of DOP and NP were up to 150 microg and 8 microg, respectively. It is concluded that the application of sewage sludge to pasture does not increase soil concentrations of phthalate (as DOP) or alkyl phenols. Thus, the risk of increased exposure to these EDC as a result of sludge application is small. However, the small effect of sludge application on soil concentrations may be largely a reflection of the relatively high concentrations of DOP already present in the soil, which may be biologically significant.

Selective pressurized liquid extraction of estrogenic compounds in soil and analysis by gas chromatography-mass spectrometry.

A selective pressurized liquid extraction (SPLE) method, followed by gas chromatography-mass spectrometry (GC-MS), for the simultaneous extraction and clean-up of estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), estriol (E3) and bisphenol A (BPA) from soil samples is described. The on-line clean-up of soil by SPLE was achieved using different organic matter retainers, including silica, alumina and Florisil, the most effective being silica. Thus, different amounts of silica, in conjunction with different extraction solvents (acetone, ethyl acetate, isohexane and dichloromethane), either alone or in combination, were used to extract the target chemicals from spiked soil samples. It was shown that 3g silica resulted in satisfactory rates of recovery of target compounds and acetone:dichloromethane (1:3, v/v) was efficient in extracting and eluting estrogenic compounds for SPLE. Variables affecting the SPLE efficiency, including temperature and pressure were studied; the optimum parameters were 60°C and 1500 psi, respectively. The limits of detection (LODs) of the proposed method were 0.02-0.37 ng g(-1) for the different estrogenic chemicals studied. The outputs using the proposed method were linear over the range from 0.1 to 120 ng g(-1) for E1, E2, EE2, 0.2-120 ng g(-1) for E3, and 0.5-120 ng g(-1) for BPA. The optimized method was further verified by performing spiking experiments in natural soil matrices; good rates of recovery and reproducibility were achieved for all selected compounds and the method was successfully applied to soil samples from Northeast Scotland, for the determination of the target compounds.

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception

This paper reports patterns of accumulation of selected EDCs in adult and fetal livers of sheep exposed to sewage sludge-treated pastures, at different times and at two stages of development.

Exposure to pastures fertilised with sewage sludge disrupts bone tissue homeostasis in sheep

The femurs of male and female sheep (Ovis aries), aged 18 months, bred on pastures fertilized twice annually with sewage sludge (2.25 tonnes dry matter/ha; Treated; T)) or on pastures treated with inorganic fertilizer (Control; C) were studied, using peripheral Quantitative Computed Tomography (pQCT) and the three-point bending test. Males were maintained on the respective treatments from conception to weaning and then maintained on control pastures while the females were maintained on the respective treatments until slaughter. T rams exhibited increased total bone mineral density (BMD) at the metaphyseal part of femur (+10.5%, p<0.01) compared with C rams but had a reduced total cross sectional area (CSA, -11.5%, p<0.001), trabecular CSA (-17.1%, p<0.01) and periosteal circumference (-5.7%, p<0.001). In the mid-diaphyseal part, T rams had an increased total BMD (+13.8%, p<0.0001) and stiffness (+6.4%, p<0.01) but reduced total CSA (-12.1%, p<0.0001) and marrow cavity (-25.8%, p<0.0001), relative to C rams. In ewes although pQCT analysis of neither the metaphyseal nor the mid-diaphyseal part of the female femur bones showed any significant differences with treatment, the biomechanical method revealed a reduction in load at failure (-17.3%, p<0.01) and stiffness (-10.7%, p<0.05) amongst T ewes. It is concluded that exposure to pollutants present in sewage sludge can perturb bone tissue homeostasis in sheep, but particularly in males.

Accumulation of endocrine disrupting compounds in sheep fetal and maternal liver tissue following exposure to pastures treated with sewage sludge

Fetal tissue concentrations of endocrine disrupting compounds (EDCs), and their relationship to maternal tissue concentrations, are largely unknown, in any species. In particular, the patterns of accumulation in the respective tissues following increased rates of environmental exposure are little known. This study was designed to determine fetal and maternal tissue concentrations of selected EDCs in sheep exposed to background, environmental concentrations of EDCs (pastures treated with inorganic fertiliser; Control; C) or to elevated, environmental concentrations (sludge-treated pastures; Treated; T). Mean log concentrations of diethylhexyl phthalate (DEHP) were similar in adult and fetal livers but there was a significant interaction between stage of development (maternal or fetal) and treatment reflecting the fact that mean concentrations were lower (P < 0.05) in C than T fetuses but not adults. Relative concentrations of polychlorinated biphenyls (PCB) in maternal and fetal tissue differed with congener; concentrations of congener 101 were higher (P < 0.05) in fetal tissue. Neither maternal nor fetal liver concentrations of any of the PCB congeners differed significantly with treatment. Polybrominated diphenyl ethers (PBDE) in the tissue were represented primarily by congeners 47 and 99. PBDE 99 concentrations were higher in maternal than fetal tissue (P = 0.01). None differed with treatment in either maternal or fetal tissues. Concentrations of many polycyclic aromatic hydrocarbons (PAH) were higher in maternal tissue but none differed with treatment in either adult or fetuses. It is concluded that sheep fetal liver EDC concentrations are variably related to those of their dams and in some cases appear to be selectively accumulated in fetuses. Differential accumulation of individual pollutants may have important implications for the assessment of risk from exposure.

Concentrations and geographic distribution of selected organic pollutants in Scottish surface soils.

Concentrations of selected persistent organic pollutants (POPs) representing three chemical classes (polycyclic aromatic hydrocarbons (PAH), polybrominated diphenyl ethers (PBDE) and polychlorinated biphenyls (PCB) and the organic pollutant diethylhexyl phthalate (DEHP), were determined in surface soil samples (0-5 cm) collected at 20 km grid intersects throughout Scotland over a three-year period. Detectable amounts of all chemical classes and most individual congeners were present in all samples. There were no consistent effects of soil or vegetation type, soil carbon content, pH, altitude or distance from centres of population on concentrations which exhibited extreme variation, even in adjacent samples. It is concluded that soil POPs and DEHP concentrations and associated rates of animal and human exposure were highly variable, influenced by multiple, interacting factors, and not clearly related to local sources but possibly related to wet atmospheric deposition and the organic carbon content of the soil.

Effect of long-term feed restriction on seasonal endocrine changes in Soay sheep

Groups of 15 adult, castrated, male Soay sheep were housed under natural daylength conditions at 57 degrees N and fed a complete diet ad libitum (AL) or at a restricted rate (R) of 35 g dry matter (DM)/kg(0.75) initial liveweight per day. The diet was based on barley and dried grass pellets and contained an estimated 11.6 MJ of metabolisable energy, 83% DM and 140 g crude protein/kg DM. In the AL animals, higher levels of feed intake during the periods of long daylength were associated with shorter inter-meal intervals (p<0. 001), a greater meal frequency (p<0.001), and a greater proportion of time spent eating (p<0.001) together with a greater rate of feed ingestion (p<0.001) and an increased meal size (p<0.001). Mean plasma concentrations of insulin, prolactin, insulin-like growth factor 1 (IGF-1), triiodothyronine (T(3)), and thyroxine (T(4)) were higher (p<0.001) in the spring or summer than in the autumn. Mean plasma GH concentrations did not differ with month. Compared with R animals, AL animals had higher mean plasma concentrations of insulin (p<0.001), prolactin (p<0.01), T(3) (p<0.01), and T(4) (p<0.01). Plasma GH and IGF-1 concentrations did not differ significantly with treatment. There was a greater increase in plasma insulin concentrations following feeding in R than AL animals (p<0.001) owing to higher pre-feeding concentrations in AL animals and the ingestion of larger amounts of feed by R than AL animals in the period after fresh feed was introduced. There were significant differences between months in this response, in R animals (p<0.01). Mean CSF insulin concentrations were significantly higher in AL than R animals (p<0.05) but were not affected by month. Neither was there a difference between pre-feeding concentrations and concentrations at approximately 12 h after feeding. It is concluded that the differences in the response of plasma insulin concentrations to feeding at different times of year, which were detected in R animals, were attributable, primarily, to differences in the vagally-induced insulin response to feeding and that these differences may provide important feedback signals to the appetite centre.