Low abundance of mitofusin 2 in dairy cows with moderate fatty liver is associated with alterations in hepatic lipid metabolism.

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells.

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H2O2 (600 µM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H2O2, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H2O2. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H2O2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H2O2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H2O2-induced cytotoxicity. The relevance of this kinase in vivo merits further study.

Associations of periparturient plasma biochemical parameters, endometrial leukocyte esterase and myeloperoxidase, and bacterial detection with clinical and subclinical endometritis in postpartum dairy cows.

This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P<0.05). Albumin and BUN levels had weak negative associations with endometrial PMN% at W3. Pathogenic bacterial detection rates were higher in the cows with endometritis at W3 and W5. Conception rate at first artificial insemination tended to be lower (P=0.057) in the cows diagnosed with endometritis at W3 than in the healthy cows. In conclusion, associations were found between endometrial LE and endometritis, but not for MPO and endometritis. Diagnosing endometritis in W3 may be the best moment to predict subsequent fertility.

Ectonucleotidase and adenosine deaminase as inflammatory marker in dairy cows naturally infected by Dictyocaulus viviparus.

The aim of this study was to evaluate the influence of dictyocaulosis (mild or severe) on enzymes of NTPDase, 5'-nucleotidase, and adenosine deaminase (ADA) of dairy cows naturally infected by Dictyocaulus viviparus. Blood and faeces were collected from 22 dairy cows of the same farm to evaluate NTPDase (ATP and ADP substrate), 5'-nucleotidase, and ADA activities on days 0 (pre-treatment) and 10 (post-treatment). Seric activities of NTPDase (ATP substrate), 5'-nucleotidase, and ADA were lower (P<0.05) in D. viviparus infected animals compared to uninfected cows. The number of D. viviparus larvae per gram of faeces varied among the animals, and they showed different degrees of severity according to respiratory clinical signs of the disease (cough and nasal discharge). Later, these cows were divided into two groups: those with mild (n=10) and severe (n=12) disease. Cows with severe disease showed higher NTPDase activity (ATP substrate) than those with mild disease (P≤0.05). The opposite occurred with NTPDase (ADP substrate), 5'-nucleotidase, and ADA in cows with severe disease, that is, the enzymatic activity of these seric enzymes significantly decreased (P≤0.05) compared to animals with mild disease. Infected animals showed reduced NTPDase activity (ATP and ADP substrate) after treatment. No enzymatic changes were observed for 5'-nucleotidase, and ADA pre- and post-treatment (P>0.05). Based on these results, we conclude that dictyocaulosis alters NTPDase, 5'-nucleotidase, and ADA activities of cow naturally infected by the parasite, in consequence the enzymes act as inflammatory markers.

Activities of ectonucleotidases and adenosine deaminase in platelets of cattle experimentally infected by Fasciola hepatica.

The enzymatic activities of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) are important in regulating the concentration of adenine nucleotides, molecules known to be involved on platelet aggregation. Fasciolosis causes coagulation disorders that have not been completely elucidated. Taking into consideration the association between the purinergic system and hemostasis, this study aimed to evaluate the enzymatic activities of NTPDase (hydrolyze ATP and ADP), 5'-nucleotidase (hydrolyze AMP) and ADA (deamination of adenosine) in platelets from cattle experimentally infected by Fasciola hepatica on days 20, 40, 60 and 80 post-infection (PI). For this study, 10 healthy Friesian steers were separated into two groups: the group A (n = 5) was used as uninfected control, and the group B was composed of steers experimentally infected by F. hepatica (n = 5). The number of platelets did not differ between groups in the periods evaluated. Reduction of NTPDase (p < 0.05) hydrolysing ATP (days 20, 40 and 60 PI), and ADP (days 40, 60 and 80 PI), and on 5'-nucleotidase hydrolyzing AMP (days 40 and 60 PI) was observed. A reduction (p < 0.05) in ADA activity on day 20 PI, as well as an increase (p < 0.05) in ADA activity on days 40 and 60 PI was observed when compared to the control. Based on these results, we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of cattle infected by F. hepatica. Considering the importance of the purinergic system in hemostasis, it is believed that those changes may contribute to the coagulation impairment observed in acute fasciolosis described in the literature.

Cattle naturally infected by Eurytrema coelomaticum: Relation between adenosine deaminase activity and zinc levels.

The enzyme adenosine deaminase (ADA) is critical for modulating the immune system, and in the presence of zinc, its activity is catalyzed. The aim of this study was to evaluate the ADA activity in pancreas of cattle naturally infected by Eurytrema coelomaticum in relation to the results of zinc levels, pathological findings and parasite load. For this study 51 slaughtered cattle were used. The animals were divided into two groups: Group A consisting of animals naturally infected by E. coelomaticum (n=33) and Group B of uninfected animals (n=18). Blood and pancreas were collected of each animal for analysis of zinc and ADA, respectively. Infected cattle showed a reduction on seric levels of zinc, and decreased ADA activity in the pancreas (P>0.05). A positive correlation between zinc levels and ADA activity was observed. Thus, high parasite load and severity of histopathologic lesions affect the ADA activity in pancreas, as well as the zinc levels in serum of infected animals (negative correlation between these variables). Therefore, we can conclude that cattle infected by E. coelomaticum have low ADA activity in pancreas, which can be directly related to zinc reduction, responsible for ADA activation and catalyzes.

Oxidative stress and changes in adenosine deaminase activity of cattle experimentally infected by Fasciola hepatica.

The aim of this study was to evaluate the oxidative stress in serum and liver and adenosine deaminase (ADA) activity of cattle experimentally infected by Fasciola hepatica. The group A consisted of five healthy animals (uninfected), and the group B was composed of five animals orally infected with 200 metacercariae of F. hepatica. On days 20, 40, 60 and 80 post-infection (PI) serum was collected to measure oxidative stress variables. On day 100 PI, animals were humanely euthanized and liver samples were collected. Infected animals showed lower (P < 0·05) seric ADA activities on days 40 and 60 PI but higher (P < 0·05) in the liver tissue compared with uninfected animals. Seric and hepatic reactive oxygen species (ROS) were higher (P < 0·05) in infected compared with uninfected animals. Hepatic thiobarbituric acid reactive substances were higher (P < 0·05) in infected animals. Catalase and glutathione S-transferase activities were lower in liver tissue of infected animals, while glutathione peroxidase was higher compared with uninfected (P < 0·05). In summary, we conclude that oxidative stress occurs in cattle experimentally infected by F. hepatica, mainly due to excessive ROS production in the course of fasciolosis, contributing to hepatic damage, and that increased in hepatic ADA activity may contribute to the inflammatory process.

Involvement of Matrix Metalloproteinases and their Inhibitors in Bovine Cystic Ovarian Disease.

The most important regulators of tissue remodelling during ovarian follicular growth, development, ovulation and atresia are gonadotropins, steroid hormones, growth factors and different proteolytic enzymes. Matrix metalloproteinases (MMPs) such as collagenase or gelatinase (i.e. MMP-1, -8, -2 and -9) and associated tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4) control connective tissue remodelling during follicular rupture. In this study, we hypothesized that an imbalance in the MMP-TIMP system may be an intra-ovarian component that contributes to the pathogenesis of cystic ovarian disease (COD) in cows. Taking into account that the control of MMP activity by TIMPs could determine their effects in both physiological and pathological conditions, MMP and TIMP mRNA and protein expression was examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively, in ovaries from control cows and cows with COD. Expression of mRNA encoding MMP-2, TIMP-1 and TIMP-2 was lower in follicular cysts than in control pre-ovulatory follicles, while the results by IHC showed this imbalance only for TIMP-2 protein expression. Additional analysis by zymography to evaluate the gelatinase activity of MMP-2 and MMP-9 demonstrated higher MMP-2 activity in follicular fluid (FF) of cysts than in FF of pre-ovulatory follicles. On the other hand, MMP-9 activity was increased in follicular cysts and absent in the FF of pre-ovulatory follicles. These findings suggest that the altered mRNA expression and activities of the MMP-TIMP system may be related to the failure in ovulation and follicular development observed in COD.

Association between liver failure and hepatic UDP-glucuronosyltransferase activity in dairy cows with follicular cysts.

Uridine 5'-diphospho-glucuronosyltransferase (UGT) liver activity was measured using estradiol-17ß as a substrate in dairy cows with follicular cysts. The activity was significantly lower than that in dairy cows with normal estrous cycles (P<0.01). Liver disorders, such as fatty liver and hepatitis, were observed in half cows with follicular cysts, and liver UGT activity was lower than that in cows with normal estrus cycles. In addition, the liver UGT activity was significantly lower in dairy cows with follicular cysts without liver disorders than in dairy cows with normal estrous cycles. Therefore, the cows were divided into those with low, middle and high liver UGT activities, and liver disorder complication rates were investigated. The complication rate was significantly higher in the low- (78.1%) than in the middle- (22.2%) and high-level (8.3%) groups, suggesting that liver disorders are closely associated with the development of follicular cysts in dairy cows and that steroid hormone metabolism is delayed because of reduced liver UGT activity, resulting in follicular cyst formation. We conclude that reduced estradiol-17ß glucuronidation in the liver and liver disorders are associated with follicular cyst occurrence in dairy cows.

NTPDase and 5'-nucleotidase as inflammatory markers in cattle naturally infected by Eurytrema coelomaticum.

The aim of this study was to evaluate seric NTPDase and 5'nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P<0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P=0.37), as well as 5'-nucleotidase activity (P=0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho=0.66, P<0.001) and the parasitic load on the pancreas (rho=0.65, P<0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho=0.29, P=0.03), and 5'nucleotidase activity (rho=0.35, P=0.01). Based on the results of NTPDase and 5'nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response.

Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells.

The bovine papillomavirus E1 helicase is essential for viral replication. In dividing cells, DNA replication maintains, but does not increase, the viral genome copy number. Replication is limited by low E1 expression and an E1 nucleocytoplasmic shuttling mechanism. Shuttling is controlled in part by phosphorylation of E1 by cellular kinases. Here we investigate conserved sites for phosphorylation by kinase CK2 within the E1 nuclear localization signal. When these CK2 sites are mutated to either alanine or aspartic acid, no change in replication phenotype is observed, and there is no effect on the subcellular distribution of E1, which remains primarily nuclear. This demonstrates that phosphorylation of E1 by CK2 at these sites is not a factor in regulating viral DNA replication in dividing cells.

Expression of Cyclooxygenase-2 in naturally occurring bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are benign skin tumours characterized by epithelial and dermal proliferation and induced by Bovine papillomaviruses (BPVs). Cyclooxygenase (COX) 1 and 2 are enzymes involved in pathological conditions, such as inflammation and epithelial carcinogenesis. Here we investigated biochemically and immunohistochemically COX-2 expression in bovine cutaneous fibropapillomas. Eight of twelve fibropapillomas (67%) showed COX-2 positive immunosignal mostly in the cytoplasm of the basal cell layer, while the normal skin did not stain. Biochemical analysis confirmed the expression of COX-2 in tumour samples. This study shows COX-2 expression in cutaneous fibropapillomas, suggesting a contribution in epithelial tumour development.

Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia.

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.

Prevalence of paunch calf syndrome carriers in Italian Romagnola cattle.

The term 'paunch calf syndrome' encompasses the multi-organic lethal developmental dysplasia reported in the Romagnola breed of cattle and is characterised by facial deformities, an enlarged and floating abdomen containing considerable abdominal effusion, and hepatic fibrosis. Paunch calf syndrome is caused by a missense mutation in the KDM2B gene (c.2503G>A) that is thought to lead to an amino acid exchange (p.D835N). In this study, the prevalence of carriers of the mutant KDM2B allele (and thus the frequency of the allele) was assessed in selected subpopulations of Romagnola cattle. The prevalence of carriers within top-ranked Romagnola sires over the years 2007-2012 was 29.3% (allele frequency 14.6%). In young bull calves, 30.9% were carriers with an allele frequency of 15.4%.

Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell.

UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Ovarian localization of 11ß-hydroxysteroid dehydrogenase (11ßHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and 11ßHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11ßHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11ßHSD1 in tertiary follicles and follicular cysts and a decrease in 11ßHSD2 in follicular cysts. Moderate expression of 11ßHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11ßHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11ßHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.

Comparative potential therapeutic effect of sesame oil and peanut oil against acute monocrotaline (Crotalaria) poisoning in a rat model.

BACKGROUND: Many Crotalaria plant species contain hepatotoxic pyrrolizidine alkaloids (such as monocrotaline) that can cause acute and chronic poisoning in cattle and other animals. HYPOTHESIS: Peanut oil, atropine sulfate, and antidiarrheal agents are used to treat acute monocrotaline poisoning. The effect of sesame on acute monocrotaline poisoning has never been investigated. ANIMALS: Fifty male Sprague-Dawley rats were used for toxicity studies. METHODS: Experiment 1: Group I, control. Groups II-IV were given monocrotaline (205.2 mg/kg) and euthanized 6, 12, and 24 hours later. Experiment 2: Group I, control. Group II monocrotaline alone (205.2 mg/kg). Groups III-VI were given monocrotaline (205.2 mg/kg) and 1 hour later, Groups III and IV were given sesame oil (1 and 2 mL/kg) and Groups V and VI were given peanut oil (1 and 2 mL/kg). RESULTS: Monocrotaline significantly decreased (P < .05) serum amylase activity, but, over time, increased (P < .05) pancreatic and lung injury. AST and ALT activity and liver injury peaked at 24 hours. Sesame oil and peanut oil (P < .05) inhibited the changes in all tested parameters in acute monocrotaline poisoning. Although peanut oil inhibited acute monocrotaline poisoning, it induced steatosis, but sesame oil did not. CONCLUSION AND CLINICAL IMPORTANCE: We hypothesize that early pancreatic and lung injury and late liver injury contribute to acute monocrotaline poisoning and that sesame oil is more efficacious than peanut oil against acute monocrotaline poisoning in rats. However, additional studies are needed to confirm that these oils have the same effects in cattle and other animals.

The relationship between serum biotin and oxidant/antioxidant activities in bovine lameness.

Serum biotin concentrations, erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and plasma thiobarbituric acid reactive substances (TBARS) were measured in 36 dairy cows, 18 of them were healthy and served as control. In the 18 cows with lameness problems, there were 5 cows with interdigital necrobacillosis, 5 cows with subsolar abscessation, 2 cows with solar ulcers, 2 cows with white line disease, 2 cows with chronic laminitis and 2 cows with septic arthritis. The degree of lameness was estimated to be slight in 3 cows, moderate in 11 cows and severe in 4 cows. Plasma fibrinogen levels and TBARS concentrations were increased significantly (P≤0.05) in lame cows compared to control group. The antioxidant enzymes GSH-Px, and CAT concentrations were increased significantly (P≤0.05) in lame cows. The level of reduced glutathione and the activity of SOD were significantly decreased in affected cows compared to healthy ones. Serum biotin levels in healthy cows ranged from 2.25 to 3.5ng/ml while in lame cows, biotin levels ranged from 1.17 to 2.3ng/ml. Biotin levels correlated positively with blood GSH (r=0.870, P≤0.05), (r=0.735, P≤0.05) and with GSH-Px (r=0.539, P≤0.05), (r=0.637, P≤0.05) and with SOD (r=0.637, P≤0.05), (r=0.449, P≤0.05) and with catalase (r=0.533, P≤0.05), (r=0.585, P≤0.05) in both healthy and lameness affected subjects, respectively.

The interplay between Araçatuba virus and host signaling pathways: role of PI3K/Akt in viral replication.

In this study, we describe the interaction between Araçatuba virus (ARAV), a naturally occurring Brazilian vaccinia virus isolated from an outbreak at a dairy farm, and the host cell's signal transduction pathways. Even though ARAV infection led to phosphorylation of MAPKs MEK/ERK, JNK, and p38MAPK, genetic or pharmacological inhibition of these pathways had no impact on viral replication. We also provide evidence that ARAV stimulated the phosphorylation of Akt (PKB) at serine 473 (S473-P), a signaling event that is required for full activation of Akt during the infectious cycle. Furthermore, pharmacological inhibition of PI3K (LY294002) abrogated ARAV-induced Akt activation (S473-P) and affected early and late viral gene expression, which was followed by a decrease in virus yield (~1 log). Taken together, our data shed some light onto the biological differences between ARAV and vaccinia virus strain WR (VACV-WR), which could contribute, at least in part, to the low-virulence phenotype displayed by ARAV. Thus, while the requirement for the PI3K/Akt pathway for successful ARAV replication is also shared with VACV-WR and cowpox virus strain BR (CPXV-BR), ARAV showed a lower replicative capacity, as well as a smaller plaque-size phenotype after infection of A31 cells when compared to VACV-WR.

Differential immunohistochemical expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in cow uteri with adenomyosis during follicular phase.

The present investigation was intended to show a different immunohistochemical profile of matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2 in bovine uteri with adenomyosis during follicular phase. Uterine samples of 32 cows in reproductive age were taken from the medial third of one of the uterine horns and grouped according to the adenomyosis degree (superficial and deep). Tissue sections (4 µm) were incubated overnight at 4°C with monoclonal antibody for matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2. Staining intensities were evaluated in the luminal epithelium, ectopic and dystopic endometrial tissue (stroma, capillaries and glands), endometrial-myometrial border, myometrium, myometrial vessels (middle tunic and endothelium). The matrix metalloproteinase-2 expression was higher for deep adenomyosis samples, showing a differential mean reactivity in superficial endometrium, myometrial vessels, myometrium adjacent to adenomyotic focus and endometrial-myometrial border (P < 0.05). Moreover, matrix metalloproteinase-2 expression was higher in deep adenomyosis samples than that of Tissue inhibitor metalloproteinase-2 in almost all uterine structures analyzed (except for the endometrial and myometrial vessels and endometrial-myometrial border). The opposite was observed in the follicular phase, for both normal specimens and with superficial adenomyosis, where Tissue inhibitor metalloproteinase-2 expression was higher than that of matrix metalloproteinase-2. In conclusion, a differential pattern of matrix metalloproteinase-2 and Tissue inhibitor metalloproteinase-2 was observed in cow uteri with adenomyosis.