Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISA test and its use for detection in rabbit meat cuts

Toxoplasma gondii causes severe disease both to man and livestock and its detection in meat after slaughtering requires PCR or biological tests. Meat packages contain retained exudate that could be used for serology due to its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or meat juice, after blood content correction. These assays were adequate regardless of the storage time up to 120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat juice may be useful for quality control for toxoplasmosis monitoring. (C) 2011 Elsevier Ltd. All rights reserved.

Impact of lung remodelling on respiratory mechanics in a model of severe allergic inflammation

We developed a model of severe allergic inflammation and investigated the impact of airway and lung parenchyma remodelling on in vivo and in vitro respiratory mechanics. BALB/c mice were sensitized and challenged with ovalbumin in severe allergic inflammation (SA) group. The control group (C) received saline using the same protocol. Light and electron microscopy showed eosinophil and neutrophil infiltration and fibrosis in airway and lung parenchyma, mucus gland hyperplasia, and airway smooth muscle hypertrophy and hyperplasia in SA group. These morphological changes led to in vivo (resistive and viscoelastic pressures, and static elastance) and in vitro (tissue elastance and resistance) lung mechanical alterations. Airway responsiveness to methacholine was markedly enhanced in SA as compared with C group. Additionally, IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid were higher in SA group. In conclusion, this model of severe allergic lung inflammation enabled us to directly assess the role of airway and lung parenchyma inflammation and remodelling on respiratory mechanics. (C) 2007 Elsevier B.V. All rights reserved.

Total Pancreatectomy: Porcine Model for Inducing Diabetes - Anatomical Assessment and Surgical Aspects

Background: The swine is an essential model for carrying out preclinical research and for teaching complex surgical procedures. There is a lack of experimental models describing anatomical and surgical aspects of total pancreatectomy in the pig. Materials and Methods: The experiments were performed on 10 white male swine weighing 27-33 kg. The animals were premedicated with midazolam (0.4 mg/kg, i.m.) and ketamine (4 mg/kg, i.m.). Anesthesia was induced with propofol (1-2 mg/kg, i.v.) and was maintained with propofol and fentanyl (0.3 mg and 0.1 mu g/kg/min, respectively, i.v.). The surgical period ranged from 44 to 77 min. The pancreas anatomy, and the main arterial, venous and pancreatic duct anatomy were assessed. Results: The pancreas anatomy was composed of 3 lobes, the `splenic`, `duodenal` and `connecting` lobe which is attached to the anterior portion of the portal vein. The splenic artery and the junction of the splenic vein and portal vein were divided. The left gastric artery was dissected and separated from its origin at the splenic artery. The head of the pancreas is disposed in a C shape. The pancreas was dissected and liberated from the right portion of the portal vein and the infrahepatic vena cava. The pancreas was separated from the duodenum preserving the pancreaticoduodenal artery, then we performed the total pancreatectomy preserving the duodenum, common bile duct and spleen. Conclusion: Total pancreatectomy with duodenum, bile duct and spleen preservation in the pig is feasible and an important instrument for research purposes and teaching surgical technique. Copyright (C) 2010 S. Karger AG, Basel

Inflammation and Remodeling in Infantile, Juvenile, and Adult Allergic Sensitized Mice

Background: Airway structural changes occur early in childhood asthma, but it is unknown whether the development of airway alterations in children is similar to that of adults. We compared inflammation and remodeling parameters in allergic sensitized infantile, juvenile, and adult mice. Methods: Infantile mice (18D) were sensitized with three intraperitoneal injections (i.p.) of ovalbumin (OVA) at days 5 and 7 and challenged with OVA at days 14-16. The 18D1 group received an additional challenge at days 9-11. The juvenile mice (40D) received challenges at days 22-24 and 36-38. Adult mice (100D) were sensitized at days 60-62 and received three inhalations at days 77-79 and 96-98. Animals were submitted to whole body plethysmography. Airway eosinophils, CD3+ T-lymphocytes, IL-5+ cells, mucus content, collagen and reticular fibers density, and smooth muscle thickness were quantified. Results: All sensitized animals presented with airway hyperresponsiveness, without differences in eosinophil cell density The density of CD3+ T-cells was higher in the 100D and 1801 groups than in the 18D and 40D groups. Infantile sensitized groups demonstrated increased interleukin-5 expression in the airways. Infantile mice demonstrated more mucus in the bronchiolar epithelium than the 40D and 100D mice. The 18D animals demonstrated less collagen than the 18D1 group. Juvenile and adult mice had increased airway smooth muscle thickness when compared to age-matched controls, but no differences were observed in the infantile groups. Conclusion: We have shown that infantile mice develop inflammatory and structural alterations in the airways that are partially different from those developed in older animals. Pediatr Pulmonol. 2011;46:650-665. (C) 2011 Wiley-Liss, Inc.

Cannabidiol injected into the bed nucleus of the stria terminalis modulates baroreflex activity through 5-HT(1A) receptors

Cannabidiol (CBD) is a non-psychotomimetic constituent of the Cannabis sativa plant that inhibits behavioral and cardiovascular responses to aversive situations. facilitating 5-HT(1A)-mediated neurotransmission. Previous results from our group suggest that the bed nucleus of the stria terminalis (BNST) may be involved in CBD`s anti-aversive effects. To investigate whether the cardiovascular effects of the CBD could involve a direct drug effect on the BNST, we evaluated the effects of CBD microinjection into this structure on baroreflex activity. We also verified whether these effects were mediated by the activation of 5-HT(1A) receptors. Bilateral microinjection of CBD (60 nmol/100 nL) into the BNST increased the bradycardiac response to arterial pressure increases. However, no changes were observed in tachycardiac responses evoked by arterial pressure decreases. Pretreatment of the BNST with the selective 5-HT(1A) receptor antagonist WAY100635 (0.37 nmol/100 nL) prevented CBD effects on the baroreflex activity. Moreover, microinjection of the 5-HT(1A) receptor agonist 8-OH-DPAT (4 nmol/100 nL) caused effects that were similar to those observed after the microinjection of CBD, which were also blocked by pretreatment with WAY100635. In conclusion, the present studies show that the microinjection of CBD into the BNST has a facilitatory influence on the baroreflex response to blood pressure increases, acting through the activation of 5-HT(1A) receptors. (C) 2010 Elsevier Ltd. All rights reserved.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100 mu mol/L) and indomethacin (100 mu mol/L) greatly impaired ACh responses in vessels from SHRSP. lberiotoxin (0.1 mu mol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1 mu mol/L.), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34; 10 mu mol/L) and charybdotoxin (0.1 mu mol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10 mu mol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats. (Translational Research 2009; 154:183-193)

Role of dorsal raphe nucleus 5-HT(1A) and 5-HT(2) receptors in tonic immobility modulation in guinea pigs

Tonic immobility (TI) is an innate defensive behavior characterized by a state of physical inactivity and diminished responsiveness to environmental stimuli. Behavioral adaptations to changes in the external and internal milieu involve complex neuronal network activity and a large number of chemical neurotransmitters. The TI response is thought to be influenced by serotonin (5-HT) activity in the central nervous system (CNS) of vertebrates, but the neuronal groups involved in the mechanisms underlying this behavior are poorly understood. Owing to its extensive afferents and efferents, the dorsal raphe nucleus (DRN) has been implicated in a great variety of physiological and behavioral functions. in the current study, we investigated the influence of serotonergic 5-HT(1A) and 5-HT(2) receptor activity within the DRN on the modulation of TI behavior in the guinea pig. Microinjection of a 5-HT(1A) receptor agonist (8-OH-DPAT, 0.01 and 0.1 mu g) decreased TI behavior, an effect blocked by pretreatment with WAY-100635 (0.033 mu g), a 5-HT(1A) antagonist. In contrast, activation of 5-HT(2) receptors within the DRN (alpha-methyl-5-HT, 0.5 mu g) increased the TI duration, and this effect could be reversed by pretreatment with an ineffective dose (0.01 mu g) of ketanserine. Since the 5-HT(1A) and 5-HT(2) agonists decreased and increased, respectively, the duration of TI, different serotonin receptor subtypes may play distinct roles in the modulation of TI in the guinea pig. (C) 2009 Elsevier B.V. All rights reserved.

Who were the male founders of rural Brazilian Afro-derived communities? A proposal based on three populations

Background: Brazilian Quilombos are Afro-derived communities founded mainly by fugitive slaves between the 16(th) and 19(th) centuries; they can be recognized today by ancestral and cultural characteristics. Each of these remnant communities, however, has its own particular history, which includes the migration of non-African derived people. Methods: The present work presents a proposal for the origin of the male founder in Brazilian quilombos based on Y-haplogroup distribution. Y haplogroups, based on 16 binary markers (92R7, SRY2627, SRY4064, SRY10831.1 and .2, M2, M3, M09, M34, M60, M89, M213, M216, P2, P3 and YAP), were analysed for 98 DNA samples from genetically unrelated men from three rural Brazilian Afro-derived communities-Mocambo, Rio das Ras and Kalunga-in order to estimate male geographic origin. Results: Data indicated significant differences among these communities. A high frequency of non-African haplogroups was observed in all communities. Conclusions: This observation suggested an admixture process that has occurred over generations and directional mating between European males and African female slaves that must have occurred on farms before the slaves escaped. This means that the admixture occurred before the slaves escaped and the foundation of the quilombo.

Spontaneous reversibility of damage to outer hair cells after sodium salicylate induced ototoxicity

Background: High sodium salicylate doses can cause reversible hearing loss and tinnitus, possibly due to reduced outer hair cell electromotility. Sodium salicylate is known to alter outer hair cell structure and function. This study determined the reversibility and cochlear recovery time after administration of an ototoxic sodium salicylate dose to guinea pigs with normal cochlear function. Study design: Prospective experimental investigation. Methods: All animals received a single 500 mg sodium salicylate dose, but with different durations of action. Function was evaluated before drug administration and immediately before sacrifice. Cochleae were processed and viewed using scanning electron microscopy. Results: Changes in outer hair cell function were observed to be present 2 hours after drug administration, with recovery of normal anatomy beginning after 24 hours. Subsequently, derangement and distortion of cilia reduced, with effects predominantly in row three. At 168 hours, cilia were near-normal but with mild distortions which interfered with normal cochlear physiology. Conclusions: Ciliary changes persisted for up to 168 hours after ototoxic sodium salicylate administration.

Gentamicin attenuates gentamicin-induced ototoxicity - Self-protection

Aminoglycoside antibiotics cause considerable toxicity to the inner ear. A progressive hearing loss at high frequencies resulted from the loss of hair cells in the base of the cochlea and a constant preoccupation with finding a treatment that protects against their toxic effects. A self-protection phenomenon to high ototoxic doses of gentamicin is proposed in this paper. Thirty-eight adult guinea pigs with normal hearing were tested using Preyer`s reflex and the distortion product otoacoustic emission (DPOAE) test, and their cochleae were analyzed by scanning electron microscopy. To the four groups investigated, group I (control) and group II (low dose, 10 mg/kg/day for 30 days) showed a normal DPOEA and normal outer hair cells; group III (high dose, 160 mg/kg/day for 10 days) showed the absence of DPOEA and damage to the outer hair cells; and group IV (low dose, 10 mg/kg/day for 30 days followed by a high dose of 160 mg/kg/day for 10 days) showed a normal DPOEA and normal outer hair cells. These results demonstrate that there was a considerable self-protection phenomenon by gentamicin.

Cigarette Smoking Exacerbates Nonalcoholic Fatty Liver Disease in Obese Rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal-regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase-1 and procollagen-alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010;51:1567-1576.)

Genetic associations between carcass traits measured by real-time ultrasound and scrotal circumference and growth traits in Nelore cattle

The aim of the present study was to evaluate the genetic correlations among real-time ultrasound carcass, BW, and scrotal circumference (SC) traits in Nelore cattle. Carcass traits, measured by real-time ultrasound of the live animal, were recorded from 2002 to 2004 on 10 farms across 6 Brazilian states on 2,590 males and females ranging in age from 450 to 599 d. Ultrasound records of LM area (LMA) and backfat thickness (BF) were obtained from cross-sectional images between the 12th and 13th ribs, and rump fat thickness (RF) was measured between the hook and pin bones over the junction between gluteus medius and biceps femoris muscles. Also, BW (n = 22,778) and SC ( n = 5,695) were recorded on animals born between 1998 and 2003. The BW traits were 120, 210, 365, 450, and 550-d standardized BW (W120, W210, W365, W450, and W550), plus BW (WS) and hip height (HH) on the ultrasound scanning date. The SC traits were 365-, 450-, and 550-d standardized SC (SC365, SC450, and SC550). For the BW and SC traits, the database used was from the Nelore Breeding Program-Nelore Brazil. The genetic parameters were estimated with multivariate animal models and REML. Estimated genetic correlations between LMA and other traits were 0.06 (BF), -0.04 ( RF), 0.05 (HH), 0.58 (WS), 0.53 (W120), 0.62 (W210), 0.67 (W365), 0.64 ( W450 and W550), 0.28 (SC365), 0.24 (SC450), and 0.00 ( SC550). Estimated genetic correlations between BF and with other traits were 0.74 ( RF), -0.32 (HH), 0.19 (WS), -0.03 (W120), -0.10 (W210), 0.04 (W365), 0.01 (W450), 0.06 ( W550), 0.17 (SC365 and SC450), and -0.19 (SC550). Estimated genetic correlations between RF and other traits were -0.41 (HH), -0.09 (WS), -0.13 ( W120), -0.09 ( W210), -0.01 ( W365), 0.02 (W450), 0.03 (W550), 0.05 ( SC365), 0.11 ( SC450), and -0.18 (SC550). These estimates indicate that selection for carcass traits measured by real-time ultrasound should not cause antagonism in the genetic improvement of SC and BW traits. Also, selection to increase HH might decrease subcutaneous fat as correlated response. Therefore, to obtain animals suited to specific tropical production systems, carcass, BW, and SC traits should be considered in selection programs.

Genetic associations between age at first calving and heifer body weight and scrotal circumference in Nelore cattle

P>Age at first calving (AFC) measures the entry of heifers into the beef cattle production system. This trait can be used as a selection criterion for earlier reproductive performance. Using data from Nelore cattle participating in the `Program for Genetic Improvement of the Nelore Breed` (PMGRN-Nelore Brazil), bi-trait analyses were performed using the restricted maximum likelihood method, based on an AFC animal model and the following traits: female body weight adjusted to 365 (BW365) and 450 (BW450) days of age, and male scrotal circumference adjusted to 365 (SC365), 450 (SC450), 550 (SC550) and 730 (SC730) days of age. The heritability estimates for AFC ranged from 0.02 +/- 0.02 to 0.04 +/- 0.02. The estimates of additive direct heritabilities (with standard error) for BW365, BW450, SC365, SC450, SC550 and SC730 were 0.36 +/- 0.07, 0.38 +/- 0.07, 0.48 +/- 0.07, 0.65 +/- 0.07, 0.64 +/- 0.07 and 0.42 +/- 0.07, respectively, and the genetic correlations with AFC were -0.38, -0.33, 0.10, -0.13, -0.13 and 0.06, respectively. In the herds studied, selection for SC365, SC450, SC550 or SC730 should not cause genetic changes in AFC. Selection based on BW365 or BW450 would favor smaller AFC breeding values. However, the low magnitude of direct heritability estimates for AFC in these farms indicates that changes in phenotypical expression depend mostly on non-genetic factors.

Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B

Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-alpha levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK(1) receptor antagonist) and SR48968 (tachykinin NK(2) receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT(3) receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly hi.-her than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-alpha. Increased production of TNF-alpha and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products. (C) 2009 Elsevier B.V. All rights reserved.

Genetic and environmental factors affecting ultrasound measures of longissimus muscle area and backfat thickness in Nelore cattle

The aim of the present study was to evaluate the genetic and environmental factors affecting records of longissimus muscle area (LMA) and back fat thickness (BF) obtained between the 12th and 13th ribs, and rump fat thickness (RF) between the hook and pin bones, measured by real-time ultrasound in Nelore cattle. Also, weight records of 22,778 animals born from 1998 to 2003, in ten farms across six Brazilian states were used. Carcass traits as measured by ultrasound of the live animal were recorded from 2002 to 2004 in 2590 males and females with ages varying from 450 to 599 days. Fixed models including farm, year and season of birth, sex and type of feed effects, and the covariates age of dam (AOD) and age of animal at measurement were used to study the effect of environmental factors on these traits. The genetic parameters for LMA, BF and RF were estimated with two and three-trait animal models with 120-day weights using a restricted maximum likelihood method. All environmental effects significantly affected carcass traits, with the exception of year of birth for BF and RF and AOD for LMA. The heritability estimates for LMA, BF and RF were 0.35, 0.51 and 0.39, respectively. Standard errors obtained in one-trait analyses were from 0.07 to 0.09. Genetic correlation estimates between LMA and the two traits of subcutaneous fat were low (close to zero) and 0.74 between BF and RF, indicating that the selection for LMA should not cause antagonism in the genetic improvement of subcutaneous fat measured by real-time ultrasound. (C) 2007 Elsevier B.V. All fights reserved.