Untreated Sheep Research Articles

Effects of three kinds of Chinese herbs on growth performance, oocysts output and gut microbiota in growing lambs with coccidiosis.

Coccidiosis is a protozoan intestinal disease that reduces the production of the sheep industry and causes large economic losses for sheep. Although chemically synthesised drugs are routinely employed to treat coccidiosis in sheep, the anticoccidial drug resistance and drug residues in edible meat have prompted an urgent search for alternatives. Herein, the anticoccidial properties of diclazuril, a conventional anticoccidial drug, and Allium sativum, Houttuynia cordata and Portulaca oleracea were assessed. Forty 45-day-old lambs naturally infected with Eimeria spp. were selected and randomly divided into five groups. The results showed that the sheep treated for coccidiosis had considerably decreased average daily gain (ADG) during both administration and withdrawal of the drug compared to the control group. Furthermore, at days 14, 21, 28 and 35, respectively, the three herbs and diclazuril had similar anticoccidial effects, with lower oocysts per gram (OPG) than the control group. On day 78, OPG in the three herbal groups was significantly lower than in the diclazuril group. In addition, the abundance and composition of the gut microbiota were changed in sheep treated with the three herbs and diclazuril compared to the untreated sheep. Moreover, some intestinal microorganisms have a correlation with OPG and ADG when using Spearman correlation analysis. In summary, our results suggest that all three herbs produce anticoccidial effects similar to diclazuril and modulate the balance of gut microbiota in growing lambs.

SYNOVIAL FLUID PROTEOMICS AND BIOINFORMATICS HIGHLIGHTS POTENTIAL MECHANISMS OF UMBILICAL CORD-MSC INDUCED RADIOLOGICAL IMPROVEMENT IN AN OVINE MODEL OF OSTEOARTHRITIS

AbstractObjectivesA promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect.MethodsFour weeks after a medial meniscus transection surgery, sheep were injected with 107 hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMinerTM) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses.ResultshUC-MSC treated animals demonstrated significantly less joint space narrowing. Nineteen SF proteins were differentially abundant in treated cf. control sheep (FC±2.0; p<0.05). Biglycan (a small leucine-rich proteoglycan of the cartilage extracellular matrix) abundance was increased by 2.1 fold in treated compared to untreated sheep (p=0.024). IPA indicated that lipid synthesis (z-score=1.772; p=0.00267) and immune cell migration pathways (cell movement of mononuclear leukocytes: z-score=1.761; p=0.00259), amongst others, were likely to be activated in the treated sheep. Conversely, tissue damage (z-score=−2; p=0.00019), senescence (z-score=−1.981; p=0.00007) and necrosis (z-score=−1.728; p=0.00829) associated pathways as well as inflammation (z-score=−1.718; p=0.00057) and vascular permeability (z-score=−1.698; p=0.00002) were likely to be inhibited in treated cf. untreated sheep.ConclusionshUC-MSC treatment prevented/delayed OA progression, demonstrated via a reduction in joint space narrowing. SF proteome bioinformatics revealed potential mechanisms of therapeutic action related to immunomodulation and the inhibition of multiple cell death, and tissue damage associated pathways. Further, a potential predicted upregulation in lipid synthesis in treated sheep represents a novel mechanism warranting further investigation. Additional work is required to validate these discovery phase proteomic findings in studies which specifically target and manipulate the proposed mechanisms highlighted.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

ABC-transporter gene expression in ivermectin-susceptible and resistant Haemonchus contortus isolates

Mammalian efflux transporters of the ATP-binding cassette (ABC) regulate cellular levels of endo- and xenobiotics by transporting molecules across cell membranes and are involved in diverse biological processes. Over-expression of these ABC transporters has been involved in macrocyclic lactone resistance. The main goal of this work was to compare the gene expression of the whole ABC-transporter superfamily in isolates of the sheep nematode Haemonchus contortus with different degrees of susceptibility to ivermectin (IVM). Additionally, the effects of in vivo IVM treatment were evaluated in the resistant isolates. Parasite-free Corriedale lambs were artificially infected with either IVM-susceptible or IVM-resistant H. contortus isolates. The differential expression of ABC transcripts in H. contortus female worms with differential susceptibility to IVM were assessed by RNA-seq. Additionally, the transcription levels of ABC-transporter genes in IVM-resistant adult worms recovered from treated sheep at 12 and 24 h after IVM administration were compared to those of IVM-R worms collected from untreated sheep. The comparative analysis of the ABC-transcripts revealed some minor differences in the expression levels of HCON_00042800 (pgp-3), HCON_00020200.mod (ced-7c), HCON_00085890 (abt-4), HCON_00063000 (pmp-5) and HCON_00116670 (wht-8), indicating that, at transcriptional level, these ABC-genes alone cannot explain resistance in H. contortus. HCON_00130060 (pgp-9.2) was highly differentially expressed in resistant isolates compared to susceptible ones, which agrees with previous reports suggesting that pgp-9 may be one of the most relevant candidates contributing to the multi-genic nature of the IVM resistance trait.

DXA reference values of the humanoid sheep model in preclinical studies.

BackgroundMerino land sheep are a popular pre-clinical large animal model in research on systemic skeletal diseases such as osteoporosis. Interpretation of studies is difficult because many reference parameters are missing or not established. This study aims to determine the reference parameters of the skeletal system (peak bone mass = PBM, T-Score). A defined standard allows an easier comparison of the study data of the animal model with human studies (T-Score).Materials and methodsA total of 116 Dual Energy X-ray Absorptiometry DXA measurements were performed on 74 untreated sheep. The average age of the animals was 57 months. The BMD, BMC, and fat content of the sheep were determined by the relevant human region of interest (ROI). From this, the PBM and from this the T-score for each of the animals were calculated.ResultsUsing 682 DXA measurements BMD and BMC were determined to provide an indication to PBM. For BMD a significant correlation to the age of the animals was observed (p = 0.043). A significant correlation was also seen for BMC (B) (p ≤ 0.001). In the age-dependent analysis, a widespread of values above the linear regression line was measured for both BMD and BMC between the 50th and 90th months of life. From an age of about 90 months, a wider spread of values below the linear regression line was found, although the average values continued to rise.DiscussionThe evaluation of the 116 DXA measurements allowed the determination of the PBM for merino land sheep. With the help of the PBM, a T-score was calculated for each animal. The statistical analysis shows significant differences in BMD values between the different animal groups in each of the four ROIs investigated. Individual control or sham groups per study are therefore not sufficient. To improve comparability, an independent reference group should be established.ConclusionAn independent reference group for PBM and a T-score was established from four to six-year-old animals. The bone density increases with the age of the animals. Around the fourth year of life, a first peak could be observed. Also, after the seventh year of life, a further peak with the beginning plateau phase was observed. When compiling a group of animals for an osteoporosis model, animals from the age of seven years should, therefore, be used.

Bone Morphogenetic Protein 7 Expression in Alveolar Bone Addition With Autologous Blood, Lyophilized Bone and Atelocollagen.

The biomaterials used in guided bone regeneration have undergone significant diversification in recent years. This study aimed to evaluate alveolar bone addition and bone morphogenetic protein 7 (BMP7) expression using an improved autologous and xenogeneic biomaterial. Chronic marginal periodontitis was induced in sheep; the intervention group received bone addition as periodontal therapy, using a composite system with lyophilized bovine bone enriched with atelocollagen type 1, platelet-rich plasma and advanced platelet-rich fibrin (A-PRF). Six weeks after the intervention, the dentoalveolar structures were evaluated using hematoxylin-eosin and immunohistochemical staining, to evaluate bone addition and BMP7 expression. The untreated sheep showed inflammation, periodontal ligament destruction, remnants of calculus and bacterial plaque as well as foreign bodies in the desmodontal space, without sings of repair. In the treated sheep, fibroblasts/fibrosis, cartilage and/or new bone, cellular cementum and desmodontium, along with remnants of biomaterial with various degrees of cellularity were observed. In the untreated group, the presence of BMP7 was found in osteoblasts and osteocytes while in the treated group, it was mainly found in the biomaterial remnants, while immunohistochemical staining was less intense in the newly formed osteo-periodontal tissues. Quantitative analysis using the Mann-Whitney U-test showed highly statistically significant differences between the two groups, demonstrating the efficiency of this composite system. The current composite system meets all the necessary conditions for promising guided alveolar bone regeneration.

Mineral elements in sheep cheese

The indigenous Travnik (Vlasic) cheese is produced in central Bosnia in the area of Mt. Vlasic. This cheese belongs to the group of white soft cheeses, ripened in brine under anaerobic conditions, and made from raw, thermally untreated sheep’s milk. The production technology is very simple and is adapted to mountainous conditions. Cheese is an important source of essential nutrients, in particular proteins, fat, vitamins and minerals. The aim of this study was to determine the concentration of macroelements (Ca, Na, K, Mg, P) and microelements (Zn, Fe, Cu) in 15 samples of Travnik sheep cheese using the atomic absorption spectrometry (AAS) and ultraviolet–visible spectrometry (UV-VIS) methods. A tendency of increasing Mg and K, Fe and Ca, Fe and Mg, and Fe and P was determined in the examined samples, but without statistical significance. The correlation coefficient values showed statistical validity at the level of high significance of differences (P<0.01).

Delivery of Fviii-mcoET3 to Previously Untreated Sheep Using Human Placental Cells Enables Durable Elevation of Plasma FVIII Levels and Avoids Inhibitor Formation

Hemophilia A (HA) is characterized by a decrease in the functional clotting protein factor VIII (FVIII). Current hemophilia treatments consist of either prophylactic or on-demand administration of FVIII protein. Despite the vast improvements and availability of these treatments, which have increased the life expectancy and quality of life of HA patients, up to 30% of patients who receive FVIII infusions develop inhibitors to FVIII, rendering subsequent treatments ineffective and placing the patient at risk of a life-threatening bleeding event. The current method used to eradicate inhibitors is immune tolerance induction (ITI), in which the patient is repeatedly infused with high doses of FVIII protein to induce tolerance. While effective in some patients, ITI is extremely expensive, the mechanism whereby tolerance is induced is not well understood, and as many as 35% of patients fail to become tolerized. In this study, we created a Master Cell Bank of human placental cells (PLC) transduced with a lentiviral vector (0.5 vector copies/diploid genome equivalent) to produce high levels (4.9IU/10^6 cells/24 hr) of mcoET3, an expression/secretion-optimized FVIII protein (PLC-mco). We hypothesized that the immunomodulatory properties of PLC could be exploited to deliver FVIII while preventing inhibitor formation, and to determine whether the route of administration impacted the efficacy and safety of this cell-based treatment. To accomplish these objectives, we administered one of the following to normal healthy juvenile sheep recipients: a) 3 weekly IV infusions of 4x10^6 PLC-mco/kg (calculated to provide ~20IU/kg mcoET3 each 24 hr); or b) a single intraperitoneal (IP) infusion of 10^7 PLC-mco/kg or c) to enable comparison of the relative immunogenicity of mcoET3 when delivered as a bolus protein injection versus when constitutively secreted from transplanted PLC-mco, we also included a control/reference group in which we administered 5 weekly IV injections of 20 IU/kg recombinant mcoET3 protein. Prior to the first injection, and once per week for 5 weeks after the first injection, we collected blood from each animal and isolated plasma and peripheral blood mononuclear cells (PBMC). The plasma was used to determine FVIII activity by aPTT and to assess the presence of mcoET3-specific IgM and IgG by ELISA. PBMC were used to perform ELISpot assays to determine whether mcoET3-specific Th1 and Th2 cells developed following each infusion paradigm. After IV PLC injection, the sheep occasionally exhibited transient labored breathing; this was never observed in animals that received IV protein infusions nor those that received PLC via the IP route. One week after the IP injection of PLC-mcoET3, plasma FVIII levels had increased by 6.3% over baseline and then further increased to remain in a range between 63-108% for weeks 2-5. Following IV PLC infusions, sheep showed a 12.9% increase in plasma FVIII levels at 1-week post-infusion which persisted in weeks 2-4 and rose to a maximum of 30% five weeks after the first injection. As expected, given FVIII's short half-life, the sheep that received mcoET3 protein exhibited baseline plasma FVIII levels when evaluated 1 week after each IV protein infusion. To evaluate humoral immunity in these animals, ELISA analysis demonstrated that all sheep in this study were devoid of mcoET3-specific IgM antibodies at all time points. Similarly, no mcoET3-specific IgG antibodies were ever detected in sheep transplanted IV or IP with PLC-mco. In contrast, the IV infusion of recombinant mcoET3 protein resulted in the generation of a specific IgG response by 2 weeks after the first infusion. With respect to cell-mediated anti-mcoET3 immunity, ELISpot assays performed on PBMC demonstrated that the only cohort that developed Th1 or Th2 cells specific to mcoET3 was the one that received mcoET3 protein. In this group, Th2 cells became detectable by 3 weeks after the first protein infusion, while Th1 cells only became evident by week 4. Taken together, our results show that human PLC can be transduced to produce high levels of FVIII protein, and that these cells can then be transplanted into previously untreated animals to yield a prolonged increase in plasma FVIII levels. Our findings also support our hypothesis that delivering a fVIII transgene via PLC enables avoidance of an immune response to a FVIII protein (mcoET3) that is immunogenic to sheep when delivered as an IV bolus. Disclosures Doering: Expression Therapeutics, LLC: Current equity holder in private company, Patents & Royalties, Research Funding; Kilpatrick, Townsend & Stockton: Consultancy.

Effect of chemical treatment of jute fiber on thermo-mechanical properties of jute and sheep wool fiber reinforced hybrid polypropylene composites

Use of combination of natural fiber in hybrid composite ensures environment-friendly product and a balanced pursuit of required properties. Present research focuses on preparing a completely new hybrid composite reinforcing jute and sheep wool fiber into polypropylene (PP) matrix using a hot press machine. Jute fiber was chemically treated with 5% NaOH and diazonium salt at alkali, acidic, and neutral media to increase compatibility with sheep wool fiber and PP matrix. Total jute and sheep wool fiber loading was fixed at 15 wt%, with a ratio of 3:1. Both treated and untreated jute fibers were utilized during composite preparation. Mechanical, structural, thermal, and water absorption tests of prepared composites were subsequently conducted. Five percentage of NaOH treatment reduced the hydroxyl group in jute fiber, while diazonium salt treatment converted the hydroxyl group into the diazo group. Hybrid PP composite containing neutral media diazonium salt-treated jute fiber and untreated sheep wool fiber had the best of mechanical properties, thermal stability, and lowest water absorption among all prepared composites.

Effects of intrauterine insulin-like growth factor-1 therapy for fetal growth restriction on adult metabolism and body composition are sex specific.

Fetal growth restriction (FGR) is associated with compromised growth and metabolic function throughout life. Intrauterine therapy of FGR with intra-amniotic insulin-like growth factor-1 (IGF1) enhances fetal growth and alters perinatal metabolism and growth in a sex-specific manner, but the adult effects are unknown. We investigated the effects of intra-amniotic IGF1 treatment of FGR on adult growth and body composition, adrenergic sensitivity, and glucose-insulin axis regulation. Placental embolization-induced FGR was treated with four weekly doses of 360 µg intra-amniotic IGF1 (FGRI) or saline (FGRS). Offspring were raised to adulthood (18 mo: FGRI, n = 12 females, 12 males; FGRS, n = 13 females, 10 males) alongside offspring from unembolized and untreated sheep (CON; n = 12 females, 21 males). FGRI females had increased relative lean mass compared with CON but not FGRS (P < 0.05; 70.6 ± 8.2% vs. 61.4 ± 8.2% vs. 67.6 ± 8.2%), decreased abdominal adipose compared with CON and FGRS (P < 0.05; 43.7 ± 1.2% vs. 49.3 ± 0.9% vs. 48.5 ± 1.0%), increased glucose utilization compared with FGRS but not CON (P < 0.05; 9.6 ± 1.0 vs. 6.0 ± 0.9 vs. 7.6 ± 0.9 mg·kg-1·min-1), and increased β-hydroxybutyric acid:nonesterified fatty acid ratio in response to adrenaline compared with CON and FGRS (P < 0.05; 3.9 ± 1.4 vs. 1.1 ± 1.4 vs. 1.8 ± 1.4). FGRS males were smaller and lighter compared with CON but not FGRI (P < 0.05; 86.8 ± 6.3 vs. 93.5 ± 6.1 vs. 90.7 ± 6.3 kg), with increased peak glucose concentration (10%) in response to a glucose load but few other differences. These effects of intra-amniotic IGF1 therapy on adult body composition, glucose-insulin axis function, and adrenergic sensitivity could indicate improved metabolic regulation during young adulthood in female FGR sheep.

Histologic safety of transcranial focused ultrasound neuromodulation and magnetic resonance acoustic radiation force imaging in rhesus macaques and sheep

BackgroundNeuromodulation by transcranial focused ultrasound (FUS) offers the potential to non-invasively treat specific brain regions, with treatment location verified by magnetic resonance acoustic radiation force imaging (MR-ARFI). ObjectiveTo investigate the safety of these methods prior to widespread clinical use, we report histologic findings in two large animal models following FUS neuromodulation and MR-ARFI. MethodsTwo rhesus macaques and thirteen Dorset sheep were studied. FUS neuromodulation was targeted to the primary visual cortex in rhesus macaques and to subcortical locations, verified by MR-ARFI, in eleven sheep. Both rhesus macaques and five sheep received a single FUS session, whereas six sheep received repeated sessions three to six days apart. The remaining two control sheep did not receive ultrasound but otherwise underwent the same anesthetic and MRI procedures as the eleven experimental sheep. Hematoxylin and eosin-stained sections of brain tissue (harvested zero to eleven days following FUS) were evaluated for tissue damage at FUS and control locations as well as tissue within the path of the FUS beam. TUNEL staining was used to evaluate for the presence of apoptosis in sheep receiving high dose FUS. ResultsNo FUS-related pre-mortem histologic findings were observed in the rhesus macaques or in any of the examined sheep. Extravascular red blood cells (RBCs) were present within the meninges of all sheep, regardless of treatment group. Similarly, small aggregates of perivascular RBCs were rarely noted in non-target regions of neural parenchyma of FUS-treated (8/11) and untreated (2/2) sheep. However, no concurrent histologic abnormalities were observed, consistent with RBC extravasation occurring as post-mortem artifact following brain extraction. Sheep within the high dose FUS group were TUNEL-negative at the targeted site of FUS. ConclusionsThe absence of FUS-related histologic findings suggests that the neuromodulation and MR-ARFI protocols evaluated do not cause tissue damage.

The role of lateral pterygoid muscle in the traumatic temporomandibular joint ankylosis: A gene chip based analysis.

Traumatic temporomandibular joint ankylosis (TMJA) is a common disease and disorder of the temporomandibular joint (TMJ); however, its pathogenesis has yet to be completely elucidated. In the authors' previous studies, the lateral pterygoid muscle (LPM) was confirmed to exert a function in distraction osteogenesis (DO) during the healing of a condylar fracture, which resulted in the formation of excess bone. The aim of the present study was to investigate alterations in the expression of any associated genes via an Affymetrix GeneChip method. The traumatic TMJA model was fabricated by a condylar fracture in the TMJ area of sheep with either a dissected LPM (LPD) or normal (LPN). The untreated sheep served as a control. At 4- and 12 weeks post-surgery, the condylar zone was isolated to perform the gene chip analysis, which was performed according to a standard Affymetrix protocol. The validated genes were further evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The gene chip analysis indicated that the LPN gene expression pattern was similar compared with the DO process, while LPD was similar to that of normal bone fracture healing. The validated genes were collagen type II α1 chain, C-type lectin domain family 3 member A, interleukin 1A, cartilage oligomeric matrix protein, chondromodulin (LECT1), calcitonin receptor (CALCR), transforming growth factor (TGF)-β1, Fos proto-oncogene (FOS), bone γ-carboxyglutamate protein and bone morphogenic protein (BMP)7, among which, BMP7, LECT1, CALCR and FOS were confirmed by RT-qPCR. In conclusion, the present study demonstrated that LPM exerts a DO effect during the pathogenesis of traumatic TMJA, which may provide a novel target for preventing TMJA.

Effect of bST administration on plasma concentrations of IGF-I and follicular dynamics and ovulation during the interovulatory cycle of sheep and goats

This study used a comparative approach to gather clinical information to assess the effect of bovine somatotropin (bST) on follicular dynamics and ovulation in sheep and goats during an interovulatory cycle. The performance of general markers of ovarian function and specific features of follicular dynamics obtained by daily ultrasonography (US) were used to assess the hypothesis that bST, associated with supraphysiological levels of IGF-I, was able to disrupt the follicular dynamics and ovulation in Highlander ewes and Saanen goats. In Exp 1, 15 ewes and 14 goats were estrous-synchronized (P4-6 days + PGFα d-6) and then allocated to a bST-treated group (50 and 100 mg, Lactotropin®; n = 5 females each) and to an untreated control group (5 ewes and 4 goats) to assess the activity of bST through plasma IGF-I (RIA). In Exp 2, 12 animals from each species were synchronized. At day 6, they were divided into a bST-group (100 mg in sheep and 50 mg in goats, n = 6 each) and an untreated control group (n = 6 each). Starting at day 6 and up to 22 days after ovulation in sheep and 25 days in goats, each female was subjected to daily US (10 mHz probe) to assess follicular and luteal (CL) dynamics and ovulation. This included assessments of both general ovarian features and specific follicular wave features. Our results showed that bST increased plasma IGF-I by day 3 (p < 0.01) when compared to the control group. Moreover, these concentrations were maintained for at least 10 days in sheep and 10 days in goats before returning to pre-treatment concentrations. Increases in IGF-I after bST doses were similar in terms of a daily and total amount (P > 0.10). Results from Exp.2 indicate that in sheep, bST administration had a subtle inhibitory effect on follicular function. However, bST in goats had a stronger influence, extending the interovulatory cycle (P = 0,034), increasing the number of follicular waves during the period (P = 0.003), and reducing the functional potential of large follicles as measured by their lower follicular diameter (P = 0.02), duration of the follicle waves (P = 0.02), and persistence of follicles after reaching their maximum diameters (P = 0.04). In addition, untreated sheep and goats shared common patterns of terminal follicular development and ovulations characterized by overlapping between follicular waves and ovulations of follicles from different waves, features not seen in cattle.

Comparison of footbathing and vaccination to control ovine footrot in an experimentally infected flock

Compare footbathing and vaccination for control of footrot during a transmission period in a sheep flock deliberately infected with multiple strains of Dichelobacter nodosus. The strains included a known virulent strain, a benign strain and several intermediate strains. The resulting footrot was clinically intermediate. A total of 1450 Polwarth sheep aged 1-3 years were allocated to one of five treatment groups: untreated, weekly walkthrough zinc sulfate footbathing, 1-hour stand-in Footrite® footbathing every 3 weeks, vaccination with a commercial multivalent whole-cell vaccine and vaccination with a novel recombinant DNA fimbrial vaccine. There were four replicates, in four paddocks. Of the untreated animals, 76% had footrot. Footbathing, either weekly or every 3 weeks, restricted the prevalence to 6/283 (2%; 97% effective) and 18/275 (6.5%; 91% effective), respectively. This was significantly lower than the prevalence in either the untreated or vaccinated group (P < 0.001). Weekly footbathing resulted in significantly fewer affected sheep than footbathing for 1 h every 3 weeks (P < 0.05). Vaccination with either whole-cell or recombinant vaccines significantly (P < 0.001) reduced the prevalence ((142/280 (51%; 33% effective), 114/278 (41%; 46% effective) respectively), with the recombinant vaccine superior (P < 0.05) to whole-cell vaccination. Significantly (P < 0.05) fewer 1-year-old sheep had footrot than older sheep. A single Footrite treatment reduced the prevalence to 12% (53/445) compared with a prevalence of 57% (27/47) for untreated sheep (79% effective). In this study footbathing was more effective than vaccination at controlling and treating multistrain footrot.

Longitudinal InVivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal invivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

Application of polymer-mesh device to remodel left ventricular–mitral valve apparatus in ischemic mitral regurgitation

ObjectivesIschemic mitral regurgitation (IMR) results from ischemic left ventricular (LV) distortion and remodeling, which displaces the papillary muscles and tethers the mitral valve leaflets apically. The aim of this experimental study was to examine efficacy of an adjustable novel polymer filled mesh (poly-mesh) device to reverse LV remodeling and reduce IMR. MethodsAcute (N = 8) and chronic (8 weeks; N = 5) sheep models of IMR were studied. IMR was produced by ligation of circumflex branches to create myocardial infarction. An adjustable poly-mesh device was attached to infarcted myocardium in acute and chronic IMR models and compared with untreated sham sheep. Two- and 3-dimensional echocardiography and hemodynamic measurements were performed at baseline, post IMR, and post poly-mesh (humanely killed). ResultsIn acute models, moderate IMR developed in all sheep and decreased to trace/mild (vena contracta: 0.50 ± 0.09 cm to 0.26 ± 0.12 cm; P < .01) after poly-mesh. In chronic models, IMR decreased in all sheep after poly-mesh, and this reduction persisted over 8 weeks (vena contracta: 0.42 ± 0.09 cm to 0.08 ± 0.12 cm; P < .01) with significant increase in the slope of end-systolic pressure–volume relationship (1.1 ± 0.5 mm Hg/mL to 2.9 ± 0.7 mm Hg/mL; P < .05). There was a significant reduction in LV volumes from chronic IMR to euthanasia stage with poly-mesh compared with sham group (%end-diastolic volume change −20 ± 11 vs 15% ± 16%, P < .01; %end-systolic volume change −14% ± 19% vs 22% ± 22%, P < .05; poly-mesh vs sham group) consistent with reverse remodeling. ConclusionsAn adjustable polymer filled mesh device reduces IMR and prevents continued LV remodeling during chronic follow-up.

Effect of Losartan on Mitral Valve Changes After Myocardial Infarction

BackgroundAfter myocardial infarction (MI), mitral valve (MV) tethering stimulates adaptive leaflet growth, but counterproductive leaflet thickening and fibrosis augment mitral regurgitation (MR), doubling heart failure and mortality. MV fibrosis post-MI is associated with excessive endothelial-to-mesenchymal transition (EMT), driven by transforming growth factor (TGF)-β overexpression. In vitro, losartan-mediated TGF-β inhibition reduces EMT of MV endothelial cells. ObjectivesThis study tested the hypothesis that profibrotic MV changes post-MI are therapeutically accessible, specifically by losartan-mediated TGF-β inhibition. MethodsThe study assessed 17 sheep, including 6 sham-operated control animals and 11 with apical MI and papillary muscle retraction short of producing MR; 6 of the 11 were treated with daily losartan, and 5 were untreated, with flexible epicardial mesh comparably limiting left ventricular (LV) remodeling. LV volumes, tethering, and MV area were quantified by using three-dimensional echocardiography at baseline and at 60 ± 6 days, and excised leaflets were analyzed by histopathology and flow cytometry. ResultsPost-MI LV dilation and tethering were comparable in the losartan-treated and untreated LV constraint sheep. Telemetered sensors (n = 6) showed no significant losartan-induced changes in arterial pressure. Losartan strongly reduced leaflet thickness (0.9 ± 0.2 mm vs. 1.6 ± 0.2 mm; p < 0.05; 0.4 ± 0.1 mm sham animals), TGF-β, and downstream phosphorylated extracellular-signal–regulated kinase and EMT (27.2 ± 12.0% vs. 51.6 ± 11.7% α-smooth muscle actin–positive endothelial cells, p < 0.05; 7.2 ± 3.5% sham animals), cellular proliferation, collagen deposition, endothelial cell activation (vascular cell adhesion molecule-1 expression), neovascularization, and cells positive for cluster of differentiation (CD) 45, a hematopoietic marker associated with post-MI valve fibrosis. Leaflet area increased comparably (17%) in constrained and losartan-treated sheep. ConclusionsProfibrotic changes of tethered MV leaflets post-MI can be modulated by losartan without eliminating adaptive growth. Understanding the cellular and molecular mechanisms could provide new opportunities to reduce ischemic MR.

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

Sheep model for osteoporosis: The effects of peripheral hormone therapy on centrally induced systemic bone loss in an osteoporotic sheep model

Hypothalamic-pituitary disconnection (HPD) leads to low bone turnover followed by bone loss and reduced biomechanical properties in sheep. To investigate the role of peripheral hormones in this centrally induced systemic bone loss model, we planned a hormone replacement experiment. Therefore, estrogen (OHE), thyroxin (OHT) or a combination of both (OHTE) was substituted in ovariectomized HPD sheep, as both hormones are decreased in HPD sheep and are known to have a significant but yet not fully understood impact on bone metabolism. Bone turnover and structural parameters were analyzed in comparison to different control groups – untreated sheep (C), ovariectomized (O) and ovariectomized+HPD sheep (OH). We performed histomorphometric and HR-pQCT analyses nine months after the HPD procedure, as well as biomechanical testing of all ewes studied. In HPD sheep (OH) the low bone turnover led to a significant bone loss. Treatment with thyroxin alone (OHT) mainly increased bone resorption, leading to a further reduction in bone volume. In contrast, the treatment with estrogen alone (OHE) and the combined treatment with estrogen and thyroxin (OHTE) prevented HPD-induced bone loss completely. In conclusion, peripheral hormone substitution was able to prevent HPD-induced low-turnover osteoporosis in sheep. But only the treatment with estrogen alone or in combination with thyroxin was able to completely preserve bone mass and structure. These findings demonstrate the importance of peripheral hormones for a balanced bone remodeling and a physiological bone turnover.

Anthelmintic evaluation of Cucurbita pepo againstgastrointestinal nematodes of sheep

The study was aimed at to investigate the anthelmintic efficacy of aqueous and ethanolic extracts of Cucurbita pepo against gastrointestinal nematodes of sheep by scientifically validated in vitro and in vivo tests. In vitro anthelmintic activity was determined by egg hatch assay, larval migration inhibition assay and larval development assay against eggs and larvae of mixed gastrointestinal nematodes of sheep. The extracts which were found to be effective in in vitro assays were further evaluated by in vivo faecal egg count reduction test against mixed species of sheep gastrointestinal nematodes with 3 different doses (125, 250 and 500 mg kg-1). Haematological and serum parameters were estimated before and after treatment to determine the impact of plant extracts on health of the animal. Aqueous extract of C. pepo demonstrated significant inhibition of egg hatch at 40 and 80 mg/ml. The ED50 value of egg hatch inhibition was 3.51mg/ml. In larval migration inhibition assay, both aqueous and ethanolic extracts of C. pepo demonstrated inhibition of larval migration and the LM50 was 1.75 and 0.32 mg/ml respectively. However, in larval development assay, both aqueous and ethanolic extracts showed only marginal inhibition of larval development at all concentrations tested. In faecal egg count reduction test (FECRT), aqueous extracts of C. pepo at 500 mg/kg caused significant reduction in eggs per gram (96.61 per cent) the reduction being higher than the result obtained with albendazole, the positive control (93.25 per cent). There was a significant difference in EPG reduction between sheep treated with albendazole and C. pepo. Haematological and serum parameters were not affected in both the treated and untreated sheep indicating that the extracts did not cause toxic effects. Based on the results obtained in this study, the aqueous extract of C. pepo possesses anthelmintic activity and could offer an alternative source for the control of gastrointestinal nematodes of sheep.

Response of an ovine laryngeal injury model to a novel fibrosis inhibitor.

Vocal fold injury results in severe voice alteration that limits occupational function and social interaction. An ovine model of laryngeal injury has been developed, validated and utilized to examine laryngeal wound healing and the effect of a novel collagen inhibitor (halofuginone) on surgical wound healing. The study design includes basic research and animal model. An ovine laryngeal model was utilized to study controlled vocal fold injury and healing. Twenty-five sheep were divided into five groups. Sheep underwent right vocal fold injury preceded or followed by administration of halofuginone orally, topically or intralesionally. Biopsies were taken at commencement, 1 month and larynges explanted at 3 months. Specimens were examined for elastin and collagen density and epithelial changes. Pearson correlation statistics and Student's t-tests were used to assess inter-relationships. All sheep tolerated halofuginone. One sheep death occurred in an untreated sheep. Vocal fold tissue demonstrated a predictable histological response to injury. Elastin was significantly reduced post-injury in the glottis. Halofuginone administered orally for 10 weeks prevented elastin loss and demonstrated a trend of reducing collagen density post-injury. In an ovine laryngeal injury model, administration of a fibrosis inhibitor resulted in altered elastin and collagen deposition after injury in the glottis. Further investigation is warranted to examine whether these tissue changes affect vocal fold dynamics.