Temperature Dependence of the Extrinsic Incubation Period of Orbiviruses in Culicoides Biting Midges

The rate at which viruses replicate and disseminate in competent arthropod vectors is limited by the temperature of their environment, and this can be an important determinant of geographical and seasonal limits to their transmission by arthropods in temperate regions.Here, we present a novel statistical methodology for estimating the relationship between temperature and the extrinsic incubation period (EIP) and apply it to both published and novel data on virus replication for three internationally important orbiviruses (African horse sickness virus (AHSV), bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV)) in their Culicoides vectors. Our analyses show that there can be differences in vector competence for different orbiviruses in the same vector species and for the same orbivirus in different vector species. Both the rate of virus replication (approximately 0.017-0.021 per degree-day) and the minimum temperature required for replication (11-13°C), however, were generally consistent for different orbiviruses and across different Culicoides vector species. The estimates obtained in the present study suggest that previous publications have underestimated the replication rate and threshold temperature because the statistical methods they used included an implicit assumption that all negative vectors were infected.Robust estimates of the temperature dependence of arbovirus replication are essential for building accurate models of transmission and for informing policy decisions about seasonal relaxations to movement restrictions. The methodology developed in this study provides the required robustness and is superior to methods used previously. Importantly, the methods are generic and can readily be applied to other arbovirus-vector systems, as long as the assumptions described in the text are valid

Maze testing of sheep for early detection of Batten disease : A thesis submitted in fulfilment of the requirements for the Degree of Master of Science at Lincoln University

Batten disease is a group fatal inherited neurodegenerative diseases typically manifesting in humans in childhood. The diseases are genetically heterogeneous and characterised by cognitive loss, psychomotor deterioration, retinal degeneration, brain atrophy, seizures and premature death. Treatments for Batten disease are at an early stage, but this study is part of the development of gene therapy for the disease caused by mutations in CLN5 and CLN6. Many of the forms of Batten disease that occur in humans have also occurred spontaneously in large animals, including ovine species. Two naturally occurring sheep models of NCL are maintained at Lincoln University, a CLN6 form in South Hampshire sheep and a CLN5 form in Borderdale sheep. Affected sheep are normal at birth, but develop clinical symptoms at around 10-14 months of age. These include progressive loss of sight, and psychomotor decline, as a result of severe cortical loss and loss of retinal photoreceptors. Premature death generally occurs at around 2 years of age. Cognitive decline is a common symptom of human Batten disease and also occurs in the CLN5 and CLN6 ovine forms of the disease. The purpose of this study was to investigate if a closed-field maze test could be a useful method for determining loss of cognition in sheep with Batten disease. It was hypothesised that cognitive decline may affect an affected sheep’s ability to transit a mazes, hence offering a potential method for early diagnosis of the disease. This would help speed up the optimisation of therapeutic interventions, by providing an early measure of their efficacy. The study consisted of three experiments. The first experiment involved the development of a maze that was navigable by the sheep, and complex enough to discern between normal and affected animals. Time to complete, and path length, determined by high precision GPS, were both used as measures of the sheep’s ability. The second experiment used the final iteration of the maze developed in experiment one, to determine when differences could be seen between normal and affected animals, and measure the effectiveness of the gene therapy treatment. The final experiment used a more cognitively complex maze that used visual cues to indicate the correct path through the maze. This study established that sheep were able to navigate a complex maze. The performance of the normal cohort of both breeds was the same. All animals were slowest and took their longest paths when first exposed to the maze, but were faster and took shorter paths in subsequent transits. The untreated affected cohorts of both genotypes were generally slower and took a longer path length through the maze than their unaffected counterparts. The loss of ability of individual affected (both untreated and treated) animals to negotiate the maze correlated well with other measures of disease progression. Due to the very variable performance of affected and treated animals, the experiment did not achieve the objective of being able to discern the onset of Batten disease at an earlier stage than other measures currently in use

Absence of a causal relationship between environmental and body temperature in dairy cows (Bos taurus) under moderate climatic conditions

(1). Continuous body temperature records from dairy cows for 46 days of summer and contemporary data for climate temperature humidity index (THI) were analysed. (2). A large component of the body temperature data was not explained by changes in THI. (3). Outside a normal range of values (above 72 for THI and 39.05 °C for body temperature), there was evidence of a causal relationship with a time delay of about 120 minutes. (4). Both variables had a prominent circadian component, but these were more likely to be due to common and/or independent causes than to any direct relationship

C-type natriuretic peptide forms in pregnancy: maternal plasma profiles during ovine gestation correlate with placental and fetal maturation

Circulating concentrations of C-type natriuretic peptide (CNP) and a related amino terminal fragment (NTproCNP) were measured at weekly intervals from preconception to 3 wk postpartum in ewes with twins (n = 8) and nonpregnant ewes (n = 8). In contrast to low and stable values in nonpregnant ewes (CNP, 0.75 ± 0.08; NTproCNP, 22 ± 2 pmol/liter), CNP forms increased abruptly at 40–50 d of gestation and rose to peak values (CNP, 31 ± 5, NTproCNP, 270 ± 16 pmol/liter) at about d 120. Approximately 7 d prepartum, the concentration of both CNP forms fell precipitously to preconception values immediately postpartum. In separate studies, circulating maternal CNP forms were positively related to fetal number at d 120. Consistent with a major contribution from the placenta to circulating levels, the concentrations of CNP forms were elevated in the placentome (cotyledon: CNP, 18 ± 4, NTproCNP, 52 ± 10 pmol/g; caruncle: CNP, 13 ± 3, NTproCNP, 31 ± 6 pmol/g) and much higher than those of intercaruncular uterine tissue (CNP, 0.19 ± 0.05, NTproCNP, 0.98 ± 0.2 pmol/g) in late-gestation ewes (P < 0.001, n = 4). These distinctive patterns of maternal plasma CNP forms, positive relation with fetal number, and greatly elevated protein concentrations in the placentome demonstrate the hormone's strong relation to placental and fetal maturation. The findings provide a firm basis for future studies of the functional role of CNP in fetal-maternal welfare

Pre-weaning growth of red deer calves is not determined by ability of hinds to produce milk

Three experiments were carried out to determine whether growth of suckling red deer calves is determined by the potential of their mothers to produce milk. In the first experiment red deer hinds (n = 10, calves 6 weeks old) were treated either with bovine somatotrophin (bST, 54 mg s.c. injected every 2 weeks for 8 weeks then 108 mg every 2 weeks for a further 8 weeks) or saline. There was no effect of bST treatment on calf or hind liveweight, calf liveweight gain or body condition score of hinds. The second experiment used red and red-wapiti crossbred deer calves (n = 8–11) suckled by red deer dams that had been treated with bST or had received excipient only for 12 weeks from when the calves were 5 weeks old. Calf liveweight was affected by genotype (wapiti-red crossbreds were heavier than their red counterparts) but there was no effect of bST treatment of the hinds on calf growth in either of the genotypes. Although bST treatment of the suckled hinds elevated their plasma insulin-like growth factor-1 concentration it had no effect on milk yield. A third experiment ruled out the possibility that bST ingested by calves in milk from treated hinds would have had any influence on growth of calves in the other experiments. From these results it is concluded that the inherent demand from suckling calves, rather than the ability of adequately nourished hinds to produce milk, determines growth rate of red deer calves from birth to weaning

Differential response of C-type natriuretic peptide to estrogen and dexamethasone in adult bone

C-type natriuretic peptide (CNP) is crucial in promoting endochondral bone growth in mammals including humans but whether this paracrine hormone participates in maintaining bone integrity in the mature skeleton is unknown. Accordingly we studied changes in plasma and bone tissue CNP in anoestrus adult ewes receiving short term anabolic (estrogen) or catabolic (dexamethasone) treatment for 7 days. CNP and the aminoterminal fragment of the CNP prohormone (NTproCNP) were measured in plasma and extracts of cancellous bone excised from vertebral, iliac, tibial and marrow tissues. Concentrations of CNP peptides were much higher in vertebral and iliac extracts than those of tibial or marrow. Both plasma CNP and NTproCNP increased rapidly after estrogen followed by a later rise in bone alkaline phosphatase. Vertebral and iliac (but not tibial or marrow) CNP peptide content were significantly increased by estrogen. Consistent with a skeletal source, plasma NTproCNP was significantly associated with vertebral tissue CNP. In contrast, bone tissue CNP peptide content was unaffected by dexamethasone despite suppression of plasma CNP peptides and bone alkaline phosphatase. We postulate that increases in trabecular bone CNP reflect new endosteal bone formation in these estrogen responsive tissues whereas reduced plasma CNP peptides after dexamethasone, without change in cancellous bone content, reflects reductions in cortical bone turnover. © 2014 Elsevier Inc. All rights reserved

In vivo monitoring of viral gene injection therapy in ovine batten disease

Recent progress in gene therapy for CLN5 and CLN6 ovine Batten disease (neuronal ceroid-lipofuscinosis, NCL) has highlighted the need for in vivo monitoring to provide compelling data for translation to human therapies while surveying the long term impacts of these therapies. Sheep have brains similar to those of non-human primates, have similar cognitive ability and are social domesticated animals. Neurological examinations are carried out monthly. Simple maze tests have proved to be informative and data are now being collected via a precision GPS system. Affected sheep can be distinguished from normal sheep from 6 months of age. Routine computer tomography (CT) scanning is simple and economic. This provides 3-D images of the intracranial space, which is decreased in affected animals by 9 months following cranial thickening into the space left by neurodegeneration. It also allows modelling of the increased volume of the lateral and third ventricles in affected sheep compared to unaffected controls and the identification of cranial markers, ideal for developing co-ordinates for stereotactic injections. CT scans are benchmarked against more sophisticated magnetic resonance structural imaging (MRI) at specific times in disease development. While observations of the sheep in the field and in maze studies indicate a relatively early visual impairment that correlates with the atrophy of the visual cortex, electroretinography (ERG) shows a later onset and slower development of retinal degeneration. Differences are seen in dark-adapted electroretinography between controls and CLN5 affected sheep at 7 months and later in CLN6 affected sheep. All these changes are progressive, differences becoming stark with time. The success of our gene therapies beyond the natural endpoint of disease highlights the desirability of being able to gather in vivo data over the long term, to determine the long term efficacy of treatment and to monitor for any other symptoms that may develop, particularly more mild ones that may be amendable to secondary treatments

Nerve conduction velocity as a non-destructive biomarker in the earthworm Aporrectodea caliginosa exposed to insecticides

International audienceEarthworms are important and useful soil organisms, but in agricultural soils, they are potentially exposed to a wide variety of pesticides. Insecticides represent the highest threat to earthworms and many are neurotoxic. There is a need for a reliable, relevant, simple biomarker to assess the sub-lethal effects of neurotoxic insecticides on earthworms under laboratory or field conditions. The Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.5×, 1× (normal field application rate), and 5× concentrations of a carbamate (Pirimor®) and an organophosphate (Lorsban®) insecticides. The nerve conduction velocity (NCV) of the medial giant fibers of A. caliginosa earthworm was recorded on days 0, 1, 2, 3, 4, and 7 to quantify sub-lethal neurotoxic effects. Acetylcholinesterase (AChE) enzyme activity of A. caliginosa homogenates was measured at the conclusion of the experiment. Pirimor® but not Lorsban® induced a significant decrease in NCV on days 3, 4, and 7 at 1× and 5× doses. A significant dose-dependent decrease was observed on AChE activity to Pirimor® at the doses used but not Lorsban®. A clear relationship is observed between AChE activity and NCV in the case of Pirimor®. This study showed that NCV is a sensitive biomarker that correlates well with classical biomarker measurements such as AChE enzyme activity. This technique could be used to study the impact of insecticides on earthworms and also their recovery

Expression of regulatory neuropeptides in the hypothalamus of red deer (Cervus elaphus) reveals anomalous relationships in the seasonal control of appetite and reproduction

© 2016 Elsevier Inc. Red deer are seasonal with respect to reproduction and food intake, so we tested the hypothesis that their brains would show seasonal changes in numbers of cells containing hypothalamic neuropeptides that regulate these functions. We examined the brains of male and female deer in non-breeding and breeding seasons to quantify the production of kisspeptin, gonadotropin inhibitory hormone (GnIH), neuropeptide Y (NPY) and γ-melanocyte stimulating hormone (γ-MSH - an index of pro-opiomelanocortin production), using immunohistochemistry. These neuropeptides are likely to be involved in the regulation of reproductive function and appetite. During the annual breeding season there were more cells producing kisspeptin in the arcuate nucleus of the hypothalamus than during the non-breeding season in males and females whereas there was no seasonal difference in the expression of GnIH. There were more cells producing the appetite stimulating peptide, NPY, in the arcuate/median eminence regions of the hypothalamus of females during the non-breeding season whereas the levels of an appetite suppressing peptide, γ-MSH, were highest in the breeding season. Male deer brains exhibited the converse, with NPY cell numbers highest in the breeding season and γ-MSH levels highest in the non-breeding season. These results support a role for kisspeptin as an important stimulatory regulator of seasonal breeding in deer, as in other species, but suggest a lack of involvement of GnIH in the seasonality of reproduction in deer. In the case of appetite regulation, the pattern exhibited by females for NPY and γ-MSH was as expected for the breeding and non-breeding seasons, based on previous studies of these peptides in sheep and the seasonal cycle of appetite reported for various species of deer. An inverse result in male deer most probably reflects the response of appetite regulating cells to negative energy balance during the mating season. Differences between the sexes in the seasonal changes in appetite regulating peptide cells of the hypothalamus present an interesting model for future studies