Human-animal interactions and machine-animal interactions in animals under human care: A summary of stakeholder and researcher perceptions and future directions.

Animals under human care are exposed to a potentially large range of both familiar and unfamiliar humans. Human-animal interactions vary across settings, and individuals, with the nature of the interaction being affected by a suite of different intrinsic and extrinsic factors. These interactions can be described as positive, negative or neutral. Across some industries, there has been a move towards the development of technologies to support or replace human interactions with animals. Whilst this has many benefits, there can also be challenges associated with increased technology use. A day-long Animal Welfare Research Network workshop was hosted at Harper Adams University, UK, with the aim of bringing together stakeholders and researchers (n = 38) from the companion, farm and zoo animal fields, to discuss benefits, challenges and limitations of human-animal interactions and machine-animal interactions for animals under human care and create a list of future research priorities. The workshop consisted of four talks from experts within these areas, followed by break-out room discussions. This work is the outcome of that workshop. The key recommendations are that approaches to advancing the scientific discipline of machine-animal interactions in animals under human care should focus on: (1) interdisciplinary collaboration; (2) development of validated methods; (3) incorporation of an animal-centred perspective; (4) a focus on promotion of positive animal welfare states (not just avoidance of negative states); and (5) an exploration of ways that machines can support a reduction in the exposure of animals to negative human-animal interactions to reduce negative, and increase positive, experiences for animals.

Automated dairy cattle lameness detection utilizing the power of artificial intelligence; current status quo and future research opportunities.

Lameness represents a major welfare and health problem for the dairy industry across all farming systems. Visual mobility scoring, although very useful, is labour-intensive and physically demanding, especially in large dairies, often leading to inconsistencies and inadequate uptake of the practice. Technological and computational advancements of artificial intelligence (AI) have led to the development of numerous automated solutions for livestock monitoring. The objective of this study was to review the automated systems using AI algorithms for lameness detection developed to-date. These systems rely on gait analysis using accelerometers, weighing platforms, acoustic analysis, radar sensors and computer vision technology. The lameness features of interest, the AI techniques used to process the data as well as the ground truth of lameness selected in each case are described. Measures of accuracy regarding correct classification of cows as lame or non-lame varied with most systems being able to classify cows with adequate reliability. Most studies used visual mobility scoring as the ground truth for comparison with only a few studies using the presence of specific foot pathologies. Given the capabilities of AI, and the benefits of early treatment of lameness, longitudinal studies to identify gait abnormalities using automated scores related to the early developmental stages of different foot pathologies are required. Farm-specific optimal thresholds for early intervention should then be identified to ameliorate cow health and welfare but also minimise unnecessary inspections.

Initial validation of an intelligent video surveillance system for automatic detection of dairy cattle lameness.

Introduction: Lameness is a major welfare challenge facing the dairy industry worldwide. Monitoring herd lameness prevalence, and early detection and therapeutic intervention are important aspects of lameness control in dairy herds. The objective of this study was to evaluate the performance of a commercially available video surveillance system for automatic detection of dairy cattle lameness (CattleEye Ltd). Methods: This was achieved by first measuring mobility score agreement between CattleEye and two veterinarians (Assessor 1 and Assessor 2), and second, by investigating the ability of the CattleEye system to detect cows with potentially painful foot lesions. We analysed 6,040 mobility scores collected from three dairy farms. Inter-rate agreement was estimated by calculating percentage agreement (PA), Cohen's kappa (κ) and Gwet's agreement coefficient (AC). Data regarding the presence of foot lesions were also available for a subset of this dataset. The ability of the system to predict the presence of potentially painful foot lesions was tested against that of Assessor 1 by calculating measures of accuracy, using lesion records during the foot trimming sessions as reference. Results: In general, inter-rater agreement between CattleEye and either human assessor was strong and similar to that between the human assessors, with PA and AC being consistently above 80% and 0.80, respectively. Kappa agreement between CattleEye and the human scorers was in line with previous studies (investigating agreement between human assessors) and within the fair to moderate agreement range. The system was more sensitive than Assessor 1 in identifying cows with potentially painful lesions, with 0.52 sensitivity and 0.81 specificity compared to the Assessor's 0.29 and 0.89 respectively. Discussion: This pilot study showed that the CattleEye system achieved scores comparable to that of two experienced veterinarians and was more sensitive than a trained veterinarian in detecting painful foot lesions.

End-of-life decision-making of dairy cattle and calves: A survey of British farmers and veterinary surgeons.

Background: The study aim was to characterise issues faced by farmers and veterinary surgeons when making end-of-life decisions for dairy cattle. Methods: Online surveys were distributed to British dairy farmers and veterinary surgeons for 20 weeks from November 2020. Results: There were 83 responses (37 farmers, 46 veterinary surgeons). Among youngstock, the risk of unassisted/natural death (2.6% ± 0.3%) was almost double the risk of euthanasia (1.4% ± 0.3%; p = 0.003). The opposite, however, was true in the milking herd: the risk of euthanasia (2.3% ± 0.3%) was greater than unassisted/natural death (1.6% ± 0.2%; p = 0.05). A fallen stock collector (62%) typically performed euthanasia and most farms (66%) did not have anyone trained to perform euthanasia. Most deaths within the milking herd were attributed to 'unknown or not recorded' (median 15% of deaths). The factors that farmers most frequently reported as strongly influencing their decision of when to euthanase an animal relative to the onset of disease were 'failure to respond to treatment' (89%), 'veterinary advice' (89%) and 'severity of disease' (88%). On average, veterinarians had moderate or high confidence that 60% of dairy farm clients 'are performing euthanasia in a timely manner so as to prevent unnecessary suffering'. Veterinary surgeons had variable agreement on the time to euthanasia for various conditions. Conclusions: The survey highlighted end-of life decision-making successes and areas for improvement on dairy farms. An evidence-based, decision-support framework may help end-of-life decision-making, particularly for complex diseases.

Evaluation of Horn Bud Wound Healing Following Cautery Disbudding of Dairy Calves With and Without the Use of Oxytetracycline Aerosol Spray.

Oxytetracycline is commonly applied as a topical agent to burn lesions post cautery disbudding of calves. Judicial use of antibiotics dictates that they should only be used where necessary to reduce the development of resistance in target bacteria. The objective of this study, therefore, was to evaluate the efficacy of topical oxytetracycline spray on wound healing post cautery disbudding of dairy calves over a 6-week period. Dairy calves were disbudded by veterinarians, technicians, or veterinary surgeons, using a standard cautery disbudding protocol. Oxytetracycline spray was randomly applied to the right or left horn bud of each animal (OXY), while the other horn bud received no antibiotic spray (NA). The outcomes measured were wound diameter (WD) and lesion score (LS), either normal healing (NH) or abnormal healing (AH). These assessments were conducted every 14 days following disbudding, until 42 days. A total of 360 animals completed the study. There was a difference in wound diameter and lesion score on day 14 post disbudding between the two groups. Cautery lesions sprayed with oxytetracycline (OXY) were 0.5 ± 0.15 mm smaller than NA lesions (P = 0.001), and there were fewer abnormal healing lesions for OXY compared to the NA (2.5 vs. 11%, respectively; P ≤ 0.001). There were no differences at day 28 and day 42 post disbudding, and on day 42, 34% of wounds had healed in both groups. In summary, the authors were unable to demonstrate a difference in healing between the groups using the described methods.

Hypoxia induces an increase in intestinal permeability and pulmonary arterial pressures in neonatal Holstein calves despite feeding the flavonoid rutin.

The purposes of this study were to determine whether the naturally occurring flavonoid quercetin, as its glucorhamnoside rutin, reduces intestinal permeability and susceptibility to hypoxia-induced pulmonary hypertension in neonatal Holstein calves. A 2 × 2 between-subjects factorial design was conducted using Holstein steers (n = 16). Factors included oxygen level (simulated altitude of 4,572 m vs. 975 m) and quercetin supplementation as its glucorhamnoside rutin (4 g of quercetin per day vs. 0 g per day). Two days after arrival (d 0 of study) the calves were blocked by body mass into treatment groups, and treatments were initiated. Pulmonary arterial pressure, echocardiography, and serum concentrations of orally administered lactulose (0.45 g/kg) and mannitol (0.15 g/kg) were measured on d 12, 13, and 14, respectively. Calves were euthanized on d 15 and pulmonary tissues collected for semiquantitative scoring of histological lesions. Data were analyzed using linear regression, generalized estimating equations, and 2-sample proportion tests. Hypoxia, but not rutin, was found to be associated with intestinal permeability. The lactulose-mannitol ratio was 0.54 ± 0.13 (standard error) in hypoxic calves and 0.02 ± 0.13 in normoxic controls. Hypoxia increased mean pulmonary arterial pressure. Calves fed rutin under hypoxic conditions tended to have a lower mean pulmonary arterial pressure (59 ± 7 mmHg) than control calves (80 ± 7 mmHg) but similar pressures under normoxic conditions. Paradoxically, however, a greater proportion of calves fed rutin had histological evidence of pulmonary arteriolar medial hypertrophy and adventitial hyperplasia than did controls. In conclusion, the findings of this study indicate that hypoxia increased intestinal permeability in neonatal calves. The flavonoid quercetin, as its glucorhamnoside rutin, had no protective effect on intestinal permeability, and, although it tended to reduce the severity of hypoxia-induced pulmonary hypertension, a greater proportion of calves fed rutin had histological lesions consistent with pulmonary arteriolar remodeling.

Maternal influences on oral and faecal microbiota maturation in neonatal calves in beef and dairy production systems.

BACKGROUND: The dam is considered an important source of microbes for the calf; consequently, the development of calf microbiota may vary with farming system due to differences between the contact the calf has with the dam. The objective of this study was to characterise the early changes in the composition of oral and faecal microbiota in beef and dairy calves (N = 10) using high-throughput sequencing of the 16S rRNA gene. The microbiota of calves was compared to selected anatomical niches on their dams which were likely to contribute to the vertical transfer of microbes. RESULTS: A total of 14,125 amplicon sequence variants (ASVs) were identified and taxonomically assigned. The oral microbiota of calves and their dams were composed of more similar microbes after the first 4 weeks of life than immediately after calving. The faecal microbiota of four-week old calves was composed of microbes which were more similar to those found in the oral microbiota of calves and adult cows than the faecal microbiota of adult cows. Specific ASVs were identified in the oral microbiota of four-week old calves that were also present in cow niches at calving, whereas very few ASVs were present in the calf faecal microbiota at four-weeks of age were present in any adult cow niche at calving. These results were observed in both beef and dairy calves. CONCLUSIONS: We did not observe any marked differences in the maturation of the oral and faecal microbiota between beef or dairy calves, despite dairy calves having very limited contact with their dam. This suggests the development of gastrointestinal microbiota in calves may not be affected by continued vertical transmission of microbes from the dam. Although the calf faecal microbiota changed over the first four-weeks of life, it was composed of microbes which were phylogenetically closer to those in the oral microbiota of calves and adult cows than the faeces of adult cows. There was little evidence of persistent microbial seeding of the calf faeces from anatomical niches on the cow at calving in either beef or dairy animals.

Cardiopulmonary remodeling in fattened beef cattle: a naturally occurring large animal model of obesity-associated pulmonary hypertension with left heart disease.

The obesity epidemic in developed societies has led to increased cardiovascular diseases including pulmonary hypertension associated with left heart disease (PH-LHD), the largest and fastest-growing class of PH. Similar to obese humans, PH and heart failure (HF) are increasingly recognized in North American fattened beef cattle. We hypothesized that PH and HF in fattened beef cattle are novel, phenotypically distinct manifestations of bovine PH arising from left ventricular (LV) dysfunction similar to obesity-related PH-LHD in humans. We conducted a semi-quantitative histopathological assessment of cardiopulmonary tissues obtained from fattened beef cattle suffering end-stage HF compared to asymptomatic cattle of equivalent age undergoing the same fattening regimens. In HF animals we observed significant LV fibrosis, abundant cardiac adipose depots, coronary artery injury, and pulmonary venous remodeling recapitulating human obesity-related PH-LHD. Additionally, striking muscularization, medial hypertrophy, adventitial fibrosis, and vasa vasorum hyperplasia in the pulmonary arterial circulation were associated with sequela of pathologic right ventricular (RV) remodeling suggesting combined pulmonary venous and arterial hypertension. The association between obesity, pathologic cardiopulmonary remodeling, and HF in fattened beef cattle appears to recapitulate the complex pathophysiology of obesity-associated PH-LHD in humans. This novel, naturally occurring, and large animal model may provide mechanistic and translational insights into human disease.

Preliminary evidence that hydrostatic edema may contribute to the formation of diffuse alveolar damage in a Holstein calf model.

Background: Two notable findings of clinically healthy feedlot cattle suggest they may have pulmonary hydrostatic edema during the finishing phase of production: increased pulmonary arterial wedge pressures and pulmonary venous hypertrophy. The goal of this study was to determine if increased pulmonary arterial wedge pressure (PAWP) in a Holstein calf could lead to diffuse alveolar damage consistent with the early, exudative phase of acute interstitial pneumonia of feedlot cattle. Methods: Six male Holstein dairy calves were given daily subcutaneous injections of the nonspecific ß-adrenergic agonist isoprenaline (10 mg/kg/d), to induce left ventricular diastolic dysfunction, or sterile water for 14 days. On Day 14, pulmonary arterial pressures and wedge pressures were measured, echocardiography performed, and the ratio of mitral valve flow velocity (E) to septal lengthening velocity (e') calculated. Calves were euthanized on Day 15 and lung lesions semi-quantitatively scored. Results: Mean PAWP was 12 ± 1 mm Hg in calves that received isoprenaline and 7 ± 1 mm Hg in controls ( P = 0.01). Calves that received isoprenaline tended to have greater relative wall thickness than control calves ( P = 0.15) and greater E/e' ratios ( P = 0.16), suggestive of concentric hypertrophy and diastolic dysfunction, respectively. Calves that received isoprenaline also tended to have a left ventricle and interventricular septum that was 29 ± 10 g heavier than control calves ( P = 0.10) when controlling for body mass. Hyaline membranes, the hallmark feature of diffuse alveolar damage, were evident in lung sections from all calves that received isoprenaline but none of the controls. Conclusions: Consistent with prior pathological and physiological studies of feedlot cattle, this study provides preliminary evidence that cattle presenting with clinical signs and pathology consistent with early stage acute interstitial pneumonia could be attributable to hydrostatic edema associated with left ventricular failure.

Successful treatment of suckling Red Angus calves for bovine respiratory disease is not associated with increased mean pulmonary arterial pressures at weaning.

The purposes of this study were to determine if the successful treatment of bovine respiratory disease (BRD) in suckling calves was associated with a long-term increase in mean pulmonary arterial pressure (mPAP) and, to screen for associations between blood leukogram variables and mPAP. A cohort of Red Angus calves (n = 74) were followed from birth to weaning at an altitude of 975 m. Calves were weaned at 172 ± 14 d when their mPAP was measured and whole blood collected. Thirty calves that had been treated for BRD (34 to 45 d prior) and 30 calves that had not required treatment for BRD were sampled. Treatment for BRD had no effect on mPAP (P = 0.37). Mean mPAP was 48 ± 8 mm Hg (± SD) with a minimum of 34 mm Hg and a maximum at 69 mm Hg. Weaning weight and sex tended to be associated with mPAP, but they explained just 5% of the variation in mPAP (P = 0.08; Adj. r2 = 0.05). Fibrinogen (P = 0.008) and absolute lymphocyte count (P = 0.06) were negatively associated with mPAP, whereas absolute monocyte count was positively associated with mPAP (P = 0.01). The findings of this study suggest that pre-weaning treatment for BRD does not increase a calves' post-weaning risk of congestive right heart failure. Further, components of the immune and acute phase response system may play a role in the development and progression of pulmonary hypertension.

Surgical placement of a wireless telemetry device for cardiovascular studies of bovine calves.

Background: Domestic cattle ( Bos taurus) are naturally susceptible to hypoxia-induced pulmonary arterial hypertension; consequently, the bovine calf has been used with considerable success as an animal model of the analogous human condition. Studies to date, however, have relied on instantaneous measurements of pressure and cardiac output. Here, we describe the surgical technique for placement of a fully implantable wireless biotelemetry device in a bovine calf for measurement of pulmonary arterial and left ventricular pressures, right ventricular output, and electrocardiogram. Methods: Three, 2-month old bovine calves underwent left-sided thoracotomies. A transit-time flow probe was placed around the pulmonary artery and solid-state pressure catheters inserted into the pulmonary artery and left ventricle. Biopotential leads were secured to the epicardium. The implant body was secured subcutaneously, dorso-caudal to the incision. Results: The implant and sensors were successfully placed in two of the three calves. One calf died from ventricular fibrillation following left ventricular puncture prior to pressure sensor insertion. Anatomical discrepancies meant that either 4 th or 5 th rib was removed. The calves recovered quickly with minimal complications that included moderate dyspnea and subcutaneous edema. Conclusions: Left thoracotomy is a viable surgical approach for wireless biotelemetry studies of bovine calf cardiovascular function. The real-time, contemporaneous collection of cardiovascular pressures and output, permits pathophysiological studies in a naturally susceptible, large animal model of pulmonary arterial hypertension.

Right Ventricular Longitudinal Strain Is Depressed in a Bovine Model of Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension and resulting right ventricular (RV) dysfunction are associated with significant perioperative morbidity and mortality. Although echocardiography permits real-time, noninvasive assessment of RV function, objective and comparative measures are underdeveloped, and appropriate animal models to study their utility are lacking. Longitudinal strain analysis is a novel echocardiographic method to quantify RV performance. Herein, we hypothesized that peak RV longitudinal strain would worsen in a bovine model of pulmonary hypertension compared with control animals. METHODS: Newborn Holstein calves were randomly chosen for induction of pulmonary hypertension versus control conditions. Pulmonary hypertension was induced by exposing animals to 14 days of hypoxia (equivalent to 4570 m above sea level or 430 mm Hg barometric pressure). Control animals were kept at ambient pressure/normoxia. At the end of the intervention, transthoracic echocardiography was performed in awake calves. Longitudinal wall strain was analyzed from modified apical 4-chamber views focused on the RV. Comparisons between measurements in hypoxic versus nonhypoxic conditions were performed using Student t test for independent samples and unequal variances. RESULTS: After 14 days at normoxic versus hypoxic conditions, 15 calves were examined with echocardiography. Pulmonary hypertension was confirmed by right heart catheterization and associated with reduced RV systolic function. Mean systolic strain measurements were compared in normoxia-exposed animals (n = 8) and hypoxia-exposed animals (n = 7). Peak global systolic longitudinal RV strain after hypoxia worsened compared to normoxia (-10.5% vs -16.1%, P = 0.0031). Peak RV free wall strain also worsened after hypoxia compared to normoxia (-9.6% vs -17.3%, P = 0.0031). Findings from strain analysis were confirmed by measurement of tricuspid annular peak systolic excursion. CONCLUSIONS: Peak longitudinal RV strain detected worsened RV function in animals with hypoxia-induced pulmonary hypertension compared with control animals. This relationship was demonstrated in the transthoracic echocardiographic 4-chamber view independently for the RV free wall and for the combination of the free and septal walls. This innovative model of bovine pulmonary hypertension may prove useful to compare different monitoring technologies for the assessment of early events of RV dysfunction. Further studies linking novel RV imaging applications with mechanistic and therapeutic approaches are needed.

Impaired alveolar-arterial oxygen transfer is associated with reduced milk yield in primiparous post-partum dairy heifers at moderate altitude.

Domestic cattle have limited cardiopulmonary reserve for their body size and oxygen requirements. Therefore, it is plausible that impaired alveolar-arterial gas exchange may be detrimental to energetically expensive traits such as milk production which, like all aerobic processes, requires oxygen. The degree of alveolar-arterial oxygen transfer impairment can be determined by estimating the alveolar-arterial oxygen (A-a O2) pressure gradient from arterial blood-gas tensions. The degree of oxygen transfer impairment is proportional to the A-a O2 pressure gradient: the higher the A-a O2 pressure gradient the less oxygen is transferred to the blood for a given ventilation rate. In this study two cohorts of Holstein-Friesian heifers were followed on one northern Colorado dairy farm. Arterial blood-gas analyses were performed up to 9 d post-calving. Heifers were grouped into quartiles based on A-a O2 pressure gradient so that relative comparisons could be made. Heifers in the lowest (Q1) and highest (Q4) quartile had the least and greatest impairment of alveolar-arterial oxygen transfer, respectively. We hypothesised that milk yield over 60 d would be greatest for heifers in Q1 and would decrease with quartile increments. Hyperventilation, as indicated by hypocapnia, was notable. Despite hypoxia, haematocrit was low. Alveolar-arterial O2 pressure gradient was associated with milk production (P=0·03) when controlling for cohort, treatment for disease and calving difficulty score. Heifers in Q1 produced 1992 kg (95% CI=1858, 2127 kg) of milk when controlling for all other variables. Relative to heifers in Q1, heifers in Q2, Q3 and Q4 produced 130 kg (95% CI=313, -52 kg; P=0·45), 285 kg (95% CI=474, 96 kg; P=0·004) and 169 kg (95% CI=395, -57 kg; P=0.14) less milk, respectively. In conclusion, efficacy of alveolar-arterial oxygen transfer was associated with milk yield in dairy heifers on one farm at moderate altitude.

Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure.

Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension.

An investigation into beef calf mortality on five high-altitude ranches that selected sires with low pulmonary arterial pressures for over 20 years.

Producer reports from ranches over 2,438 meters in southwest Colorado suggest that the mortality of preweaned beef calves may be substantially higher than the national average despite the selection of low pulmonary pressure herd sires for over 20 years. Diagnostic investigations of this death loss problem have been limited due to the extensive mountainous terrain over which these calves are grazed with their dams. The objective of the current study was to determine the causes of calf mortality on 5 high-altitude ranches in Colorado that have been selectively breeding sires with low pulmonary pressure (<45 mmHg) for over 20 years. Calves were followed from branding (6 weeks of age) in the spring to weaning in the fall (7 months of age). Clinical signs were recorded, and blood samples were taken from sick calves. Postmortem examinations were performed, and select tissue samples were submitted for aerobic culture and/or histopathology. On the principal study ranch, 9.6% (59/612) of the calves that were branded in the spring either died or were presumed dead by weaning in the fall. In total, 28 necropsies were performed: 14 calves (50%) had lesions consistent with pulmonary hypertension and right-sided heart failure, and 14 calves (50%) died from bronchopneumonia. Remodeling of the pulmonary arterial system, indicative of pulmonary hypertension, was evident in the former and to varying degrees in the latter. There is a need to better characterize the additional risk factors that complicate pulmonary arterial pressure testing of herd sires as a strategy to control pulmonary hypertension.

Onchocerca armillata contains the endosymbiotic bacterium Wolbachia and elicits a limited inflammatory response.

Human onchocerciasis, also known as River Blindness, is a debilitating disease caused by the filarial nematode Onchocerca volvulus. Many, but not all, filarial nematodes carry within their tissues endosymbiotic, Rickettsia-like bacteria of the genus Wolbachia. Onchocerca spp. infections in cattle offer the most relevant, analogous host-parasite model system. West African cattle are commonly co-infected with four Onchocerca spp.; two of these are Wolbachia-positive (Onchocerca gutturosa and Onchocerca ochengi), and the remainder are of unknown Wolbachia status (Onchocerca dukei and Onchocerca armillata). Previous studies have suggested that worm survival is dependent on this bacterium. O. armillata, an abundant parasite of African cattle that has received little attention, is a primitive species that may lack Wolbachia. The objectives of this study were to determine if O. armillata carries Wolbachia and to provide preliminary descriptions of the host inflammatory cell environment around the adult worms. The findings may support or refute the hypothesis that a prime contribution of Wolbachia is to permit long-term survival and reproduction of certain Onchocerca spp. (including O. volvulus in humans). O. armillata adult worms were found in the aorta of 90.7% of cattle (n=54) slaughtered at an abattoir in Ngaoundéré, Adamawa Region, Cameroon. The presence of Wolbachia in O. armillata was confirmed by a specific anti-Wolbachia surface protein antibody detected using a peroxidase conjugate (immunohistochemistry) and PCR for detection of Wolbachia-specific sequences within DNA extracts from frozen worms. Tissue sections stained with haematoxylin and eosin showed the host cell response to be dominated by macrophages and fibroblasts. This is unusual compared with nodule-dwelling Wolbachia-positive Onchocerca spp., where the host response is typically characterised by granulocytes, and suggests that the mechanisms for worm survival employed by this species (which is probably motile) may differ.

Trophic functions of nucleotides in the central nervous system.

In addition to short-term effects, one of the fundamental roles of extracellular nucleotides in the central nervous system involves long-term trophic effects. Physiological outcomes include neurogenesis, neuronal differentiation, glial proliferation, migration, growth arrest and apoptosis. Nucleotides exert these functions via P2-receptor-mediated mechanisms that can also interact with polypeptide-growth-factor-mediated or integrin-mediated signaling pathways. In addition, pathogenic roles for extracellular nucleotides in response to central nervous system injury including trauma and ischemia have been observed after the release of nucleotides by damaged and dying cells and in the development of neuropathic and inflammatory pain. Here, we illuminate the contribution of extracellular nucleotides to the development, growth, cellular plasticity and death of neural cells and the mechanisms regulating these trophic effects.

Opposing effects of P2X(7) and P2Y purine/pyrimidine-preferring receptors on proliferation of astrocytes induced by fibroblast growth factor-2: implications for CNS development, injury, and repair.

Extracellular nucleotides play important trophic roles in development and central nervous system (CNS) injury, but the functions of distinct purinergic receptors and related signaling pathways have not been fully elucidated. In the present study we identified opposing effects of P2X and P2Y receptors on the ability of FGF2 to induce proliferation in primary cultures of rat cortical astrocytes. Low concentrations of ATP enhanced DNA synthesis induced by FGF2, whereas high concentrations inhibited FGF2-induced proliferation. Comparison of concentration-response experiments with ATP and 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) indicated that the inhibitory effect was mediated by P2X(7) receptors. Interestingly, activation of P2X(7) receptors led to a state of reversible growth arrest rather than cell death. Selectivity studies showed that proliferation evoked by epidermal growth factor and platelet-derived growth factor was also inhibited by P2X(7) receptors, but P2X(1) or P2X(3) receptors did not inhibit proliferation induced by FGF2. A marker of mitosis, phosphohistone-3, was reduced by BzATP and increased by UTP, suggesting that the enhancing effect of ATP on FGF2-induced proliferation was mediated by P2 purine/pyrimidine receptors. Phosphorylation of the growth arrest-related protein kinases p38/MAPK and SAPK/JNK was strongly increased by BzATP but only weakly affected by UTP. We conclude that P2Y purine/pyrimidine receptors enhance proliferation induced by FGF2 in astrocytes, whereas stimulation of P2X(7) receptors inhibits proliferation by shifting cells to a state of reversible growth arrest that may be mediated by protein kinase signaling. These trophic actions of P2X(7) and P2Y purine/pyrimidine receptors may contribute to the regulation of CNS development, adult neurogenesis, and the response of astrocytes to injury.

A new in vitro model of the glial scar inhibits axon growth.

Astrocytes respond to central nervous system (CNS) injury with reactive astrogliosis and participate in the formation of the glial scar, an inhibitory barrier for axonal regeneration. Little is known about the injury-induced mechanisms underlying astrocyte reactivity and subsequent development of an axon-inhibitory scar. We combined two key aspects of CNS injury, mechanical trauma and co-culture with meningeal cells, to produce an in vitro model of the scar from cultures of highly differentiated astrocytes. Our model displayed widespread morphological signs of astrocyte reactivity, increases in expression of glial fibrillary acidic protein (GFAP), and accumulation of GFAP in astrocytic processes. Expression levels of scar-associated markers, phosphacan, neurocan, and tenascins, were also increased. Importantly, neurite growth from various CNS neuronal populations was significantly reduced when neurons were seeded on the scar-like cultures, compared with growth on cultures of mature astrocytes. Quantification of neurite growth parameters on the scar model demonstrated significant reductions in neuronal adhesion and neurite lengths. Interestingly, neurite outgrowth of postnatal neurons was reduced to a greater extent than that of embryonic neurons, and outgrowth inhibition varied among neuronal populations. Scar-like reactive sites and neurite-inhibitory patches were found throughout these cultures, creating a patchwork of growth-inhibitory areas mimicking a CNS injury site. Thus, our model showed relevant aspects of scar formation and produced widespread inhibition of axonal regeneration; it should be useful both for examining mechanisms underlying scar formation and to assess various treatments for their potential to improve regeneration after CNS injury. (c) 2008 Wiley-Liss, Inc.

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures.

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.