A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Pharmacological immunomodulation enhances peripheral nerve regeneration

To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38µm±1.16 and COL/MDP=3.54µm±1.16) compared to NOR (6.19µm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

Retrospective canine skin peripheral nerve sheath tumors data with emphasis on histologic, immunohistochemical and prognostic factors

Abstract: In this retrospective study was determined the frequency of canine skin peripheral nerve sheath tumors (PNST) in cases diagnosed by the Setor de Patologia Veterinária of the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, between the years 2000 and 2012. The canine profiles, as well as histological, immunohistochemical and prognostic aspects of the tumors were based on 70 samples, comprising 40 females, 29 males and one unspecified sample. Between 2000 and 2012, 2,984 skin tumors of dogs were diagnosed in the SPV-UFRGS, totaling 2.34% of skin neoplasms in dogs. Animals that comprised the largest amount of samples (43%) were those with no breed (SRD), followed by German Shepherds (10%). Females were more affected than males (40/70 - 57% and 29/70 - 41% respectively). Skin PNST of this research showed predominant localization on the limbs (40% in the forelimbs and 29% in the hindlimbs); affecting adult dogs, mostly aged between 8 and 11 years (54%). The samples were routinely processed for hematoxylin and eosin, and were also evaluated by toluidine blue and Masson's trichrome staining, and immunohistochemistry (IHC) anti-vimentin, -S-100, -GFAP, -actin, von Willebrand factor and neurofilament. Anisocytosis and anisokaryosis, mitotic index, intratumoral necrosis, invasion of adjacent tissues, tumor location, local recurrence and metastasis were related to the diagnosis of benign (49/70) or malignant tumor (21/70). The Antoni A histological pattern was observed more frequently in benign tumors. The immunohistochemistry helped to diagnose PNST, and anti-vimentin and anti-protein S-100 showed the highest rates of immunostaining. Throughout statistical analysis of animals with tumor recurrence, it was found that the chance of an animal with a malignant peripheral nerve sheath tumor to develop recurrence is 4.61 times higher than in an animal that had a benign tumor.

Comparison between one-dimensional uncoupled and convection-conduction conjugated approaches in finned surface heat transfer

This work studies the forced convection problem in internal flow between concentric annular ducts, with radial fins at the internal tube surface. The finned surface heat transfer is analyzed by two different approaches. In the first one, it is assumed one-dimensional heat conduction along the internal tube wall and fins, with the convection heat transfer coefficient being a known parameter, determined by an uncoupled solution. In the other way, named conjugated approach, the mathematical model (continuity, momentum, energy and K-epsilon equations) applied to tube annuli problem was numerically solved using finite element technique in a coupled formulation. At first time, a comparison was made between results obtained for the conjugated problem and experimental data, showing good agreement. Then, the temperature profiles under these two approaches were compared to each other to analyze the validity of the one-dimensional classical formulation that has been utilized in the heat exchanger design.

Computer programs for analysis of droplets sprayed on water sensitive papers

The use of water-sensitive papers is an important tool for assessing the quality of pesticide application on crops, but manual analysis is laborious and time-consuming. Thus, this study aimed to evaluate and compare the results obtained from four software programs for spray droplet analysis in different scanned images of water-sensitive papers. After spraying, papers with four droplet deposition patterns (varying droplet spectra and densities) were analyzed manually and by means of the following computer programs: CIR, e-Sprinkle, DepositScan and Conta-Gotas. The diameter of the volume and number medians and the number of droplets per target area were studied. There is a strong correlation between the values measured using the different programs and the manual analysis, but there is a great difference between the numerical values measured for the same paper. Thus, it is not advisable to compare results obtained from different programs.

Interactions of ANP and ANG II in tubular nephron acidification

(ANP, 1 &micro;M) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H+-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H+-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF. We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange. ANP (1 &micro;M) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [Ca2+]i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension. The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 &micro;M) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times the basal level. In contrast, the addition of ANP (1 &micro;M) to the cell suspension led to a very rapid 60% decrease in [Ca2+]i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i. These results may indicate a role of [Ca2+]i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II. The data are compatible with stimulation of Na+/H+ exchange by increases of [Ca2+]i in the lower range, and inhibition at high [Ca2+]i levels

Spreading depression is facilitated in adult rats previously submitted to short episodes of malnutrition during the lactation period

Lactating rat dams were submitted to short episodes (1, 2 or 3 weeks) of nutritional restriction by receiving the "regional basic diet" (RBD, with 8% protein) of low-income human populations of Northeast Brazil. Their pups were then studied regarding the developmental effects on body and brain weights. When the rats reached adulthood, cortical susceptibility to the phenomenon of spreading depression (SD) was evaluated by performing electrophysiological recordings on the surface of the cerebral cortex. SD was elicited at 20-min intervals by applying 2% KCl for 1 min to a site on the frontal cortex and its occurrence was monitored at 2 sites in the parietal region by recording the electrocorticogram and the slow potential change of SD. When compared to control rats fed a commercial diet with 23% protein, early malnourished rats showed deficits in body and brain weights (10% to 60% and 3% to 15%, respectively), as well as increases in velocity of SD propagation (10% to 20%). These effects were directly related to the duration of maternal dietary restriction, with pups malnourished for 2 or 3 weeks presenting more intense weight and SD changes than those malnourished for 1 week. The effects of 1-week restrictions on SD were less evident in the pups malnourished during the second week of lactation and were more evident in pups receiving the RBD during the third week. The results indicate that short episodes of early malnutrition during the suckling period can affect body and brain development, as well as the cortical susceptibility to SD during adulthood. The data also suggest that the third week of lactation is the period during which the brain is most sensitive to malnutrition, concerning the effects on SD

Characterization of nerve-induced relaxation of gastrointestinal sphincteric smooth muscle in a South American opossum (Didelphis albiventris)

The presence of inhibitory nonadrenergic noncholinergic (NANC) intrinsic innervation of the circular muscle of the gastrointestinal sphincters of the South American (SA) opossum was investigated in vitro. Isolated circular muscle strips from the esophagogastric and ileocolonic junctions but not from the gastroduodenal (pylorus) region developed spontaneous tension. Tetrodotoxin (TTX, 1 µM) augmented the spontaneous tension only in the ileocolonic junction strips. Electrical field stimulation of esophagogastric and ileocolonic junction strips caused frequency-dependent responses consisting of a relaxation at lower frequencies (<1 Hz) and a biphasic response or contraction at higher frequencies. In the strips from the pyloric region electrical field stimulation abolished the spontaneous activity at lower frequencies and induced contractions at higher frequencies. The responses elicited by electrical field stimulation in the three sphincters were abolished by TTX (1 µM). Electrical field-induced contractions were reduced while relaxations were enhanced by atropine (1 µM). In the presence of atropine (1 µM) and guanethidine (3 µM), electrical field stimulation, nicotine and ATP induced frequency- or concentration-dependent relaxations of the three sphincters that were abolished by TTX (1 µM). Isoproterenol and sodium nitroprusside caused concentration-dependent relaxations which were TTX-resistant. These findings indicate that the sphincteric circular muscle of the SA opossum gastrointestinal tract is relaxed by the activation of intrinsic NANC nerves and therefore can be used as a model for the study of the mechanisms involved in these responses

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

Sera from chronic chagasic patients depress cardiac electrogenesis and conduction

We report results obtained with sera from 58 chronic chagasic patients that were evaluated for effects on heart rate and atrioventricular (AV) conduction in isolated rabbit hearts and screened for the presence of muscarinic and beta-adrenergic activity. We show that sera from 26 patients decreased heart rate, while 10 increased it and 22 had no effect. Additionally, sera from 20 of the 58 patients blocked AV conduction. Muscarinic activation seems to be involved in both effects, but is not the only mechanism, since atropine did not antagonize the decrease in heart rate in 23% of sera or AV block in 40%. Sera from patients with complex arrhythmias were significantly more effective in depressing both heart rate and AV conduction. Sera that induce increases in heart rate seem to operate exclusively through beta-adrenergic activation. Two of these sera, evaluated with respect to intercellular communication in primary cultures of embryonic cardiomyocytes were able to block gap junction conductance evaluated by a dye injection technique after 24-h exposure. The mechanisms underlying this uncoupling effect are currently being investigated.

The cardiopulmonary reflexes of spontaneously hypertensive rats are normalized after regression of left ventricular hypertrophy and hypertension

Cardiopulmonary reflexes are activated via changes in cardiac filling pressure (volume-sensitive reflex) and chemical stimulation (chemosensitive reflex). The sensitivity of the cardiopulmonary reflexes to these stimuli is impaired in the spontaneously hypertensive rat (SHR) and other models of hypertension and is thought to be associated with cardiac hypertrophy. The present study investigated whether the sensitivity of the cardiopulmonary reflexes in SHR is restored when cardiac hypertrophy and hypertension are reduced by enalapril treatment. Untreated SHR and WKY rats were fed a normal diet. Another groups of rats were treated with enalapril (10 mg kg-1 day-1, mixed in the diet; SHRE or WKYE) for one month. After treatment, the volume-sensitive reflex was evaluated in each group by determining the decrease in magnitude of the efferent renal sympathetic nerve activity (RSNA) produced by acute isotonic saline volume expansion. Chemoreflex sensitivity was evaluated by examining the bradycardia response elicited by phenyldiguanide administration. Cardiac hypertrophy was determined from the left ventricular/body weight (LV/BW) ratio. Volume expansion produced an attenuated renal sympathoinhibitory response in SHR as compared to WKY rats. As compared to the levels observed in normotensive WKY rats, however, enalapril treatment restored the volume expansion-induced decrease in RSNA in SHRE. SHR with established hypertension had a higher LV/BW ratio (45%) as compared to normotensive WKY rats. With enalapril treatment, the LV/BW ratio was reduced to 19% in SHRE. Finally, the reflex-induced bradycardia response produced by phenyldiguanide was significantly attenuated in SHR compared to WKY rats. Unlike the effects on the volume reflex, the sensitivity of the cardiac chemosensitive reflex to phenyldiguanide was not restored by enalapril treatment in SHRE. Taken together, these results indicate that the impairment of the volume-sensitive, but not the chemosensitive, reflex can be restored by treatment of SHR with enalapril. It is possible that by augmenting the gain of the volume-sensitive reflex control of RSNA, enalapril contributed to the reversal of cardiac hypertrophy and normalization of arterial blood pressure in SHR.

Early myelin breakdown following sural nerve crush: a freeze-fracture study

In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.

Catecholamine-induced vasoconstriction is sensitive to carbonic anhydrase I activation

We studied the relationship between alpha- and beta-adrenergic agonists and the activity of carbonic anhydrase I and II in erythrocyte, clinical and vessel studies. Kinetic studies were performed. Adrenergic agonists increased erythrocyte carbonic anhydrase as follows: adrenaline by 75%, noradrenaline by 68%, isoprenaline by 55%, and orciprenaline by 62%. The kinetic data indicated a non-competitive mechanism of action. In clinical studies carbonic anhydrase I from erythrocytes increased by 87% after noradrenaline administration, by 71% after orciprenaline and by 82% after isoprenaline. The increase in carbonic anhydrase I paralleled the increase in blood pressure. Similar results were obtained in vessel studies on piglet vascular smooth muscle. We believe that adrenergic agonists may have a dual mechanism of action: the first one consists of a catecholamine action on its receptor with the formation of a stimulus-receptor complex. The second mechanism proposed completes the first one. By this second component of the mechanism, the same stimulus directly acts on the carbonic anhydrase I isozyme (that might be functionally coupled with adrenergic receptors), so that its activation ensures an adequate pH for stimulus-receptor coupling for signal transduction into the cell, resulting in vasoconstriction.

Apoptosis of sensory neurons and satellite cells after sciatic nerve transection in C57BL/6J mice

The rate of axonal regeneration, after sciatic nerve lesion in adult C57BL/6J mice, is reduced when compared to other isogenic strains. It was observed that such low regeneration was not due just to a delay, since neuronal death was observed. Two general mechanisms of cell death, apoptosis and necrosis, may be involved. By using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, we demonstrated that a large number of sensory neurons, as well as satellite cells found in the dorsal root ganglia, were intensely labeled, thus indicating that apoptotic mechanisms were involved in the death process. Although almost no labeled neurons or satellite cells were observed one week after transection, a more than ten-fold increase in TUNEL labeling was detected after two weeks. The results obtained with the C57BL/6J strain were compared with those of the A/J strain, which has a much higher peripheral nerve regeneration potential. In A/J mice, almost no labeling of sensory neurons or satellite cells was observed after one or two weeks, indicating the absence of neuronal loss. Our data confirm previous observations that approximately 40% of C57BL/6J sensory neurons die after sciatic nerve transection, and indicate that apoptotic events are involved. Also, our observations reinforce the hypothesis that the low rate of axonal regeneration occurring in C57BL/6J mice may be the result of a mismatch in the timing of the neurons need for neurotrophic substances, and production of the latter by non-neuronal cells in the distal stump.