Expression analysis of TLR signaling pathway genes under lipopolysaccharide-induced and E. coli F17-infected sheep intestinal epithelial cells.

Escherichia coli (E. coli) F17 is one of the main pathogens causing diarrhea in young livestock. The specific F17 fimbriae and lipopolysaccharide (LPS) in the surface components of E. coli F17 induces immune activation via interacting with the intestinal epithelial cells (IECs)-expressed innate immune toll-like receptors (TLRs) signaling pathway. In this study, the expression patterns of eight canonical genes from the TLR signaling pathway (IL-6, IL-8, IL-1ß, TLR4, MyD88, CD14, TNF-α and TRAF6) were analyzed in LPS-induced IECs, E. coli F17-infected IECs and ileum tissue of E. coli F17-infected lambs. The results showed that increased expression levels of all the studied genes were observed following post-LPS-induced and E. coli F17-infected treatment, with TLR4 having the highest up-regulated expression multiple (compared to NC, fold change = 17.94 and 20.11, respectively), and CD14 having the lowest up-regulated expression multiple (fold change = 2.68 and 1.59, respectively), and higher expression levels of all the studied TLR signaling pathway genes were observed in ileum tissue of E. coli F17 antagonistic (AN) lambs than in E. coli F17 sensitive (SE) lambs. Furthermore, when compared to LPS-induced IECs, E. coli F17-infected IECs showed a more pronounced increase in the expression of IL6, TLR4 and TNF-α, indicating the different roles of these genes in the IECs resistance to E. coli F17 infection. Our results demonstrate that the TLR signaling pathway likely promotes immune activation and provide the first evidence that TLRs have a significant potential to protect against E. coli F17 infections.

Detection of aluminium hydroxide-induced granulomas in sheep by computed tomography: A feasible approach for small ruminant lentiviruses diagnosis and research.

Aluminium (Al) hydroxide use as adjuvant induces local formation of long-lasting subcutaneous granulomas in sheep. Macrophages within these granulomas have been identified as a new small ruminant lentivirus (SRLV) replication site in naturally infected animals. Diagnosis of Al hydroxide-induced granulomas in sheep is mostly based on postmortem observations but little information is available on in vivo detection. Computed tomography (CT) is used for studying these reactions in other animal species. To determine if CT could be a tool for in vivo diagnosis and research of subcutaneous Al hydroxide-induced granulomas in sheep. A retrospective survey on thoracic CT scans was performed on 46 adult sheep. Analysis included absence or presence, number and location of subcutaneous nodules. Thoracic CT scans and pathological studies were prescribed to two further sheep. Single or multiple subcutaneous nodules were detected in 26 (56.52%) sheep. One or two nodules per animal were most often observed (36.95%). Size ranged between 1.5 and 4.5 cm. Pre-contrast two-dimensional (2D) CT images showed focal or multifocal increases in subcutaneous tissue thickness. Post-contrast 2D CT images revealed hypointense areas in the centre. Histopathology indicated the presence of granulomas composed by a large number of activated macrophages, surrounding a central core of necrosis. Large intracytoplasmic Al-positive aggregates were demonstrated by lumogallion staining. CT is a useful tool to detect subcutaneous Al hydroxide-induced granulomas in vivo in sheep. CT provides a diagnostic and research tool that can be very useful in future works in Al hydroxide-induced pathology, SRLV infection, or both.

Hepatogenous photosensitization in ruminants and horses caused by the ingestion of Chamaecrista serpens in Brazil.

This study aimed to describe the first reports of outbreaks of hepatogenous photosensitization in cattle, sheep, and horses caused by spontaneous ingestion of Chamaecrista serpens, as well as to reproduce poisoning in sheep experimentally. Eleven photodermatitis outbreaks of unknown cause occurred in cattle, sheep and horses on nine farms in the semiarid region of Bahia, northeastern Brazil, between July 2017 and July 2020. Cutaneous lesions of photosensitization initiated until one week after the animals were introduced in paddocks invaded by the plant at the beginning of the rainy season. The photosensitive skin lesions were progressive and consisted of hyperemia, edema, ulcerative-crusted lesions with necrosis, especially in non-pigmented skin areas. The lesions in young animals were more severe. The animals avoided the sun and exhibited hyporexia, weight loss, restlessness, irritability, and severe itching. An experimental study was made using seven sheep, and resulted in photodermatitis, similar to that observed in the natural poisoning, seven days after the beginning of plant ingestion. Two sheep were reserved for the control group. Serum biochemistry changes indicated liver injury caused by the plant. Skin biopsies and liver biopsy guided by ultrasound were performed. The one sheep that had more pronounced skin lesions was euthanized and necropsied. At the necropsy, the liver was enlarged, diffusely pale, and firm, with an evident lobular pattern and an empty gallbladder. Histopathology revealed similar skin and liver lesions in samples from biopsies and the necropsy. There was a marked disorganization of the cords of hepatocytes associated with degenerative necrotic changes on the liver. The cutaneous injuries included orthokeratotic hyperkeratosis, hypergranulosis, acanthosis, and extensive areas of epidermic necrosis and ulceration. Three sheep were protected from sunlight and the lesions regressed within 45 days after the plant's consumption ceased. In conclusion, C. serpens causes hepatogenous photosensitization in ruminants and horses, and should be included in the list of differential diagnoses in cases of photosensitive dermatitis.

Replication of Small Ruminant Lentiviruses in Aluminum Hydroxide-Induced Granulomas in Sheep: a Potential New Factor for Viral Dissemination.

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue.IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection.

Developmental Programming: Contribution of Epigenetic Enzymes to Antral Follicular Defects in the Sheep Model of PCOS.

Prenatal testosterone (T)-treated sheep, similar to women with polycystic ovary syndrome (PCOS), manifest oligo-/anovulation, hyperandrogenism, and polyfollicular ovary. The polyfollicular ovarian morphology, a result of persistence of antral follicles, arises, in part, by transcriptional changes in key mediators of follicular development that, in turn, are driven by epigenetic mechanisms. We hypothesized that prenatal T excess induces, in a cell-specific manner, transcriptional changes in key mediators of follicular development associated with relevant changes in epigenetic machinery. Expression levels of key mediators of follicular development, DNA methyltransferases (DNMTs), and histone de-/methylases and de-/acetylases were determined in laser-capture microdissection-isolated antral follicular granulosa and theca and ovarian stromal cells from 21 months of age control and prenatal T-treated sheep (100 mg IM twice weekly from gestational day 30 to 90; term: 147 days). Changes in histone methylation were determined by immunofluorescence. Prenatal T treatment induced the following: (i) cell-specific changes in gene expression of key mediators of follicular development and steroidogenesis; (ii) granulosa, theca, and stromal cell-specific changes in DNMTs and histone de-/methylases and deacetylases, and (iii) increases in histone 3 trimethylation at lysine 9 in granulosa and histone 3 dimethylation at lysine 4 in theca cells. The pattern of histone methylation was consistent with the expression profile of histone de-/methylases in the respective cells. These findings suggest that changes in expression of key genes involved in the development of the polyfollicular phenotype in prenatal T-treated sheep are mediated, at least in part, by cell-specific changes in epigenetic-modifying enzymes.

Granulomas Following Subcutaneous Injection With Aluminum Adjuvant-Containing Products in Sheep.

The use of vaccines including aluminum (Al)-based adjuvants is widespread among small ruminants and other animals. They are associated with the appearance of transient injection site nodules corresponding to granulomas. This study aims to characterize the morphology of these granulomas, to understand the role of the Al adjuvant in their genesis, and to establish the presence of the metal in regional lymph nodes. A total of 84 male neutered lambs were selected and divided into 3 treatment groups of 28 animals each: (1) vaccine (containing Al-based adjuvant), (2) adjuvant-only, and (3) control. A total of 19 subcutaneous injections were performed in a time frame of 15 months. Granulomas and regional lymph nodes were evaluated by clinicopathological means. All of the vaccine and 92.3% of the adjuvant-only lambs presented injection-site granulomas; the granulomas were more numerous in the group administered the vaccine. Bacterial culture in granulomas was always negative. Histologically, granulomas in the vaccine group presented a higher degree of severity. Al was specifically identified by lumogallion staining in granulomas and lymph nodes. Al median content was significantly higher ( P < .001) in the lymph nodes of the vaccine group (82.65 µg/g) compared with both adjuvant-only (2.53 µg/g) and control groups (0.96 µg/g). Scanning transmission electron microscopy demonstrated aggregates of Al within macrophages in vaccine and adjuvant-only groups. In these two groups, Al-based adjuvants induce persistent, sterile, subcutaneous granulomas with macrophage-driven translocation of Al to regional lymph nodes. Local translocation of Al may induce further accumulation in distant tissues and be related to the appearance of systemic signs.

Intraruminal infusion of oligofructose alters ruminal microbiota and induces acute laminitis in sheep.

The objectives of this study were to examine the clinical response, changes in ruminal bacterial microbiota, and inflammatory response in lamellar tissues during oligofructose-induced laminitis. Ten fistulated sheep were randomly assigned into a control group ( = 5) and a treatment group ( = 5). The treatment group was infused with oligofructose (21 g/kg BW) by rumen cannula, and the control group was sham-treated with saline. Results showed that all 5 sheep treated with oligofructose developed anorexia and diarrhea 8 to 12 h after the administration of oligofructose. By 12 to 24 h after treatment, the treatment group developed lameness and roach back. Compared with the control group, oligofructose administration decreased ( < 0.001) the rumen pH and concentrations of total VFA and increased ( < 0.001) the level of lactic acid in the rumen. Microbial data analysis revealed that oligofructose infusion increased the abundance of ( = 0.009) and ( = 0.008) and decreased the percentage of unclassified Christensenellaceae ( = 0.028), unclassified Ruminococcaceae ( = 0.009), ( = 0.016), unclassified Lachnospiraceae ( = 0.009), and ( = 0.009) compared with the control group. Oligofructose infusion decreased the ACE ( = 0.047) and Shannon ( = 0.009) indices compared with the control group. The histomorphology analysis revealed that oligofructose overload resulted in damage to the dermoepidermal junction in the lamellar tissue of sheep. Quantitative real-time PCR results showed that compared with the control group, the mRNA expression of membrane-type metalloproteinase-1 ( = 0.049) was downregulated whereas the expression of proinflammatory IL-6 ( = 0.004) and matrix metalloprotease-9 ( = 0.037) was upregulated in the lamellar tissues of the oligofructose treatment group. In general, the present study provides the foundation for a sheep model of oligofructose-overload-induced acute laminitis that could be used in later experiments. Our findings suggest that intraruminal infusion of oligofructose altered ruminal microbiota and resulted in acute laminitis and that the inflammatory damage to the lamellae tissue may be related to the upregulation of matrix metalloprotease-9. The information generated will provide more insight into the systemic effects of lameness caused by oligofructose overload in sheep.

Sheep lymph-nodes as a biological indicator of environmental exposure to fluoro-edenite.

A significantly increased incidence of pleural mesothelioma in Biancavilla (Sicily, Italy) has been attributed to exposure to fluoro-edenite (FE), a fibrous amphibole extracted from a local stone quarry. The lymph-nodes draining the pulmonary lobes of sheep grazing around the town were examined, to gain insights into fibre diffusion. The pasture areas of six sheep flocks lying about 3km from Biancavilla were located using the global positioning system. The cranial tracheobronchial and one middle mediastinal lymph-node as well as four lung tissue samples were collected from 10 animals from each flock and from 10 control sheep for light and scanning electron microscopy (SEM) examination. The lymph-nodes from exposed sheep were enlarged and exhibited signs of anthracosis. Histologically, especially at the paracortical level, they showed lymph-follicle hyperplasia with large reactive cores and several macrophages (coniophages) containing grey-brownish particulate interspersed with elements with a fibril structure, forming aggregates of varying dimensions (coniophage nodules). Similar findings were detected in some peribronchiolar areas of the lung parenchyma. SEM examination showed that FE fibres measured 8-41µm in length and 0.4-1.39µm in diameter in both lymph-nodes and lung tissue. Monitoring of FE fibres in sheep lymph-nodes using appropriate techniques can help set up environmental pollution surveillance.

Biliary atresia: From Australia to the zebrafish.

This review is based upon an invited lecture for the 52nd Annual Meeting of the British Association of Paediatric Surgeons, July 2015. The aetiology of biliary atresia (BA) is at best obscure, but it is probable that a number of causes or pathophysiological mechanisms may be involved leading to the final common phenotype we recognise clinically. By way of illustration, similar conditions to human BA are described, including biliary agenesis, which is the normal state and peculiar final pattern of bile duct development in the jawless fish, the lamprey. Furthermore, there have been remarkable outbreaks in the Australian outback of BA in newborn lambs whose mothers were exposed to and grazed upon a particular plant species (Dysphania glomulifera) during gestation. More recent work using a zebrafish model has isolated a toxic isoflavonoid, now named Biliatresone, thought to be responsible for these outbreaks. Normal development of the bile ducts is reviewed and parallels drawn with two clinical variants thought to definitively have their origins in intrauterine life: Biliary Atresia Splenic Malformation syndrome (BASM) and Cystic Biliary Atresia (CBA). For both variants there is sufficient clinical evidence, including associated anomalies and antenatal detection, respectively, to warrant their aetiological attribution as developmental BA. CMV IgM +ve associated BA is a further variant that appears separate with distinct clinical, histological, and immunohistochemical features. In these it seems possible that this involves perinatal obliteration of a normally formed duct system. Although still circumstantial, this evidence appears convincing enough to perhaps warrant a different treatment strategy. This then still leaves the most common (more than 60% in Western series) variant, now termed Isolated BA, whereby origins can only be alluded to.

Experimental hyperlipidemia induces insulin resistance in sheep.

This study aimed to evaluate the effects of intravenous infusion of a soybean-based lipid emulsion on some blood energy-related metabolites and insulin sensitivity indexes in sheep. Four clinically healthy ewes were assigned into a 2-treatment, 2-period cross-over design. Either normal saline (NS) or lipid emulsion (LE) was intravenously introduced at a rate of 0.025 mL·kg(-1) min(-1) for 6 h. The concentrations of blood nonesterified fatty acid (NEFA), beta-hydroxybutyrate, triglyceride, cholesterol, urea, creatinine, cortisol, glucose, and insulin were measured at different time points. After 6 h, intravenous glucose tolerance test was performed. Lipid infusion elicited an increase (P < 0.05) in the NEFA, beta-hydroxybutyrate, and triglyceride concentrations compared with the baseline value and NS infusion. Infusion of NS did not influence blood glucose concentration; however, LE infusion increased plasma glucose concentration (P < 0.05). At time point 12 h, serum insulin concentrations were increased (P < 0.05) in NS treatment; however, such an increase was not observed in the LE treatment. Insulin sensitivity index for the LE infusion was lower (P < 0.05) than that for the NS treatment. The glucose effectiveness was not (P > 0.05) different among treatments. In the LE treatment, acute-phase insulin responses increased (P < 0.05) and disposition index decreased (P < 0.001) compared with NS treatment. The results showed that experimentally induced NEFA in blood could cause insulin resistance in sheep. The current model could be used to evaluate the pathogenesis of conditions associated with increased lipid mobilization and insulin resistance.

Pharmacokinetics of florfenicol after intravenous administration in Escherichia coli lipopolysaccharide-induced endotoxaemic sheep.

Experiments in different animal species have shown that febrile conditions, induced by Escherichia coli lipopolysaccharide (LPS), may alter the pharmacokinetic properties of drugs. The objective was to study the effects of a LPS-induced acute-phase response (APR) model on plasma pharmacokinetics of florfenicol (FFC) after its intravenous administration in sheep. Six adult clinically healthy Suffolk Down sheep, 8 months old and 35.5 ± 2.2 kg in body weight (bw), were distributed through a crossover factorial 2 × 2 design, with 4 weeks of washout. Pairs of sheep similar in body weight were assigned to experimental groups: Group 1 (LPS) was treated with three intravenous doses of 1 µg/kg bw of E. coli LPS before FFC treatment. Group 2 (control) was treated with an equivalent volume of saline solution (SS) at similar intervals as LPS. At 24 h after the first injection of LPS or SS, an intravenous bolus of 20 mg/kg bw of FFC was administered. Blood samples (5 mL) were collected before drug administration and at different times between 0.05 and 48.0 h after treatment. FFC plasma concentrations were determined by liquid chromatography. A noncompartmental pharmacokinetic model was used for data analysis, and data were compared using a Mann-Whitney U-test. The mean values of AUC0-∞ in the endotoxaemic sheep (105.9 ± 14.3 µg·h/mL) were significantly higher (P < 0.05) than values observed in healthy sheep (78.4 ± 5.2 µg·h/mL). The total mean plasma clearance (CLT ) decreased from 257.7 ± 16.9 mL·h/kg in the control group to 198.2 ± 24.1 mL·h/kg in LPS-treated sheep. A significant increase (P < 0.05) in the terminal half-life was observed in the endotoxaemic sheep (16.9 ± 3.8 h) compared to the values observed in healthy sheep (10.4 ± 3.2 h). In conclusion, the APR induced by the intravenous administration of E. coli LPS in sheep produces higher plasma concentrations of FFC due to a decrease in the total body clearance of the drug.

Survival of bone marrow mesenchymal stem cells labelled with red fluorescent protein in an ovine model of collagenase-induced tendinitis.

OBJECTIVE: The aim of this study was to track the survival and efficacy of allogeneic bone marrow mesenchymal stem cells (BM-MSC) marked with red fluorescent protein (BM-MSCRFP) in an ovine model of collagenase-induced tendinopathy. METHODS: Bone marrow was harvested from one donor sheep and BM-MSC were isolated, cultivated and transfected with red fluorescent protein (BM-MSCRFP). Collagenase was injected into both Achilles tendons in the remaining nine sheep. After two weeks the left tendon was injected with a solution of 6 x 106 BM-MSCRFP and fibrin glue, while only fibrin glue was administered to the contra-lateral tendon in each sheep. After three, four and six weeks the tendons were harvested and evaluated for morphology, collagen I deposition, presence of CD34+ cells, and fluorescent labelled BM-MSC. RESULTS: We demonstrated that delivery of BM-MSC into tendon lesions had positive effects on the injured tendons. The BM-MSCRFP survived at three, four and six weeks after treatment, leading to better quality healing of tendons as compared to the controls, where no labelled cells were detected. Interestingly, we demonstrated high expression of CD34+ cells in tendons that had been treated with BM-MSCRFP. CLINICAL RELEVANCE: Mesenchymal stem cell allografts have a positive effect on tendon healing and local injection of BM-MSC directly into the tendon allows the homing of BM-MSC for good efficiency of engraftment.

Lung cancer in mice induced by the jaagsiekte sheep retrovirus envelope protein is not maintained by rare cancer stem cells, but tumorigenicity does correlate with Wnt pathway activation.

JSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non-small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope-induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling.

An outbreak of granulomatous peritonitis caused by injectable selenium in a flock of Merino sheep.

During meat inspection, unusual pigmented lesions were found in the abdomens of 411 sheep from a flock raised in the Northern Tablelands of New South Wales. In each affected sheep there were multiple discrete, soft, yellow homogeneous plaques beneath the parietal peritoneum and extending into marginating facial planes of the diaphragm and body wall. Microscopically, the lesions consisted of focal granulomatous peritonitis with intracellular acicular refractile golden-brown crystals. Energy dispersive X-ray spectroscopy revealed intralesional barium and selenium, two components of an injectable selenium compound administered to the sheep 6-8 months prior, which contains the yellow pigment, iron oxide. The mechanism of subperitoneal deposition of the compound could not be confirmed, but is presumed to have involved intraperitoneal injection of barium selenate. Meat inspectors and diagnosticians should consider barium selenate injection-site granulomas as a possible explanation for yellow pigmented lesions, especially in livestock from selenium-deficient areas. Animal care providers should be aware that incorrect administration of barium selenate can result in losses from condemnation or downgrading of meat product.

Influence of glutamine infusion on ubiquitin, caspase-3, cathepsins L and B, and m-calpain expression in sheep with nutritionally induced metabolic acidosis.

Provision of AA has shown success in attenuating proteolytic activity in monogastrics suffering from metabolic acidosis. However, it is unknown whether AA supplementation can provide any beneficial effects to ruminants with nutritionally induced metabolic acidosis. The objective of the current study was to examine the effects of glutamine infusion on various protein degradation components across several tissues in sheep with induced metabolic acidosis. Sheep were assigned to a randomized complete block design with 2 x 2 factorial arrangement of treatments (n = 6 sheep/treatment) consisting of a control or acidosis diet, and receiving a saline or L-glutamine infusion. Sheep were fed diets for 10 d and slaughtered on d 11. Liver, kidney, and muscle samples were collected at slaughter and examined for relative messenger RNA (mRNA) expression of ubiquitin, C8, E2, cathepsin L, cathepsin B, caspase-3, and m-calpain, as well as protein expression of ubiquitin. Relative mRNA expression of C8 (P = 0.02), E2 (P = 0.06), and ubiquitin (P = 0.07) was less in kidney in acidotic vs. control sheep. Additionally, mRNA expression of m-calpain in kidney was greater (P = 0.01) as a result of glutamine infusion. There were no significant alterations (P > 0.10) in mRNA of any component as a result of acidosis in the liver or muscle. This study demonstrates the inability of metabolic acidosis to increase expression of the ubiquitin-mediated proteolytic pathway in skeletal muscle; however, downregulation of renal mRNA expression of these components is apparent during the induction of metabolic acidosis.

Prolonged recovery and respiratory depression after fentanyl infusion in a sheep undergoing mitral valve reconstruction.

A sheep was anaesthetized for implantation of a novel device (MitroFast) to replace the posterior leaflet of the mitral valve. Anaesthetic management included a balanced anaesthetic protocol and consisted of propofol or isoflurane combined with fentanyl infusion (0.15-0.4 microg/kg/min). Deliberate hypothermia during cardiopulmonary bypass was set at 34.5-35.5 degrees C. Surgery proceeded uneventfully. Total time of aortic cross-clamping was 35 min and total time on extracorporeal circulation was 60 min. Visual inspection, intracardiac pressure testing and transesophageal echocardiography indicated proper functioning of the device. The anaesthetic period was uneventful, but recovery was prolonged with central nervous and respiratory depression and marked hypoxaemia. Administration of naloxone (1.5 microg/kg, repeated twice at 15-20 min intervals) reversed the central nervous and attenuated the respiratory depressions. An initially low rate of urine production normalized after rewarming and a single intravenous administration of furosemide.

Assessment of proliferative activity by AgNOR and PCNA in prostatic tissues of ram lambs implanted with zeranol.

The aim of this study was to assess cellular proliferation using silver-stained nucleolar organizer regions (AgNOR) and the proliferating cell nuclear antigen (PCNA) in various tissues in the prostate of ram lambs implanted with increasing zeranol doses and to compare the sensitivity of different tissues of lamb prostate to zeranol. Twenty-four Akkaraman lambs were implanted with increasing zeranol doses, including 12 mg (n = 8), 24 mg (n = 8) and 96 mg (n = 8), with eight lambs serving as controls. After 33 days, the prostate tissues of the lambs were stained using AgNOR and PCNA techniques. The prostate tissues were divided into two compartments--the epithelial tissues, including glandular acinus, collecting duct and penile urethra, and the non-epithelial tissues, including interstitial tissue and striated muscle. AgNOR dots and PCNA index on each prostatic tissue were counted under a light microscope and were evaluated statistically. AgNOR staining in the treatment groups showed a higher score in the non-epithelial tissues than the epithelial components, whereas the PCNA index was significant in the epithelial tissues and non-epithelial tissues had very low PCNA immunostaining. According to the PCNA index, collecting duct epithelium showed more sensitivity to increasing zeranol doses and according to AgNOR counts, there was no difference of sensitivity to zeranol among tissues of the same origin. Both AgNOR counts and PCNA indexes seem to be valuable proliferating markers for the epithelial components of ram prostate, but PCNA index had no significance in relation to the non-epithelial components in contrast to AgNOR counts. Therefore, the controversial results arising from the combined use of both techniques as proliferating markers for the ram prostate should be considered in further studies.

The greater susceptibility of North Ronaldsay sheep compared with Cambridge sheep to copper-induced oxidative stress, mitochondrial damage and hepatic stellate cell activation.

Sheep of the semi-feral North Ronaldsay (copper-sensitive) and domesticated Cambridge (copper-tolerant) breeds were compared in respect of pathological changes and protein expression in the liver as a result of excessive dietary copper. Acute mitochondrial damage and hepatic stellate cell (HSC) activation with collagen synthesis occurred in response to moderate copper overload in North Ronaldsay but not in Cambridge sheep. Mitochondrial degradative changes occurred either as ballooning degeneration and rupture with subsequent autophagic degradation or as mitochondrial matrical condensation (pyknosis). In North Ronaldsay sheep prolonged exposure to copper produced mitochondrial hyperplasia and hypertrophy, and nuclear damage with necrosis. Cytosolic isocitrate dehydrogenase (IDH), an enzyme responsive to oxidative stress, was induced in the liver of Cambridge sheep receiving a Cu-supplemented diet but was undetectable in the non-supplemented control sheep. Conversely, IDH was detected at similar levels in both control and copper-supplemented North Ronaldsay sheep, indicating a lower threshold response, and an enhanced susceptibility, to oxidative stress. "Upregulation" of mitochondrial thioredoxin-dependent peroxidase reductase (antioxidant protein-1) in the hepatic cytosol of the North Ronaldsay (but not Cambridge) sheep affirmed the increased susceptibility of the mitochondria to Cu-induced oxidative stress in this breed. Likewise the upregulation of cathepsin-D indicated increased lysosomal activity and HSC activation. The findings may be relevant to copper toxicosis in human infants.

Histopathological changes and inflammatory response induced by Tityus discrepans scorpion venom in rams.

Anesthetized rams envenomed s.c. with 40 microg/kg Tityus discrepans scorpion venom developed fasciculation, hypothermia, polyuria, pulmonary wet rales, tachypnea, respiratory distress and arrhythmia. Rams developed a cascade of inflammation reactions, characterized by activation of macrophages, fibroblasts and neutrophils, neutrophil infiltration and aggregation, vasculitis, arteritis and abundant fibrin deposition. At the inoculation site, venom was detected by immunohistochemistry in the extra cellular matrix, lymphatic vessels' and venules' lumen, inside macrophages and surrounding nerves. Extra cellular matrix was degraded at the inoculation site perhaps by activated neutrophils. Envenoming produced hepatocytes with Mallory body-like vacuoles which may be due to the increased plasmatic levels of TNF-alpha and IL6. Venom produced degranulation and vacuolization of acinary cells as well as interstitial swelling and necrosis. Necrosis of the Langerhan's islets occurred occasionally. Lungs showed the most deleterious effects developing wall collapse and necrosis, diffuse injury of the alveolar capillary barrier, interstitial and alveolar fibrin deposits with strong neutrophil infiltration. Massive infiltration of lymphocytes and macrophage occurred in the intestinal submucose, to the point that it modified villi and intestinal folding morphology. Envenomation developed a marked leukocyte aggregation surrounding nerves at the inoculation site. This study reveals that beyond its neurotoxicity, Tityus venom produces a severe and widespread inflammatory syndrome, expressed as histopathological changes at the site of inoculation, as well as in remote organs such as pancreas, lungs, intestine and liver. Our results suggest that not all remote targets are directly affected by the venom but that, as proposed earlier, are modified by inflammation by products produced elsewhere.

Biomedical applications of poisonous plant research.

Research designed to isolate and identify the bioactive compounds responsible for the toxicity of plants to livestock that graze them has been extremely successful. The knowledge gained has been used to design management techniques to prevent economic losses, predict potential outbreaks of poisoning, and treat affected animals. The availability of these compounds in pure form has now provided scientists with tools to develop animal models for human diseases, study modes of action at the molecular level, and apply such knowledge to the development of potential drug candidates for the treatment of a number of genetic and infectious conditions. These advances are illustrated by specific examples of biomedical applications of the toxins of Veratrum californicum (western false hellebore), Lupinus species (lupines), and Astragalus and Oxytropis species (locoweeds).