Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants.

AIMS: To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis-infected dairy herds. METHODS AND RESULTS: Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one-stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two-stage plant. CONCLUSIONS: Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two-stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.

The genetic resistance to gastro-intestinal strongylids in Appenninica sheep: preliminary results.

Heritability parameters of resistance to gastro-intestinal strongylids, measured as FEC (Faecal Egg Count), were evaluated in the Appenninica sheep breed. FEC heritability coefficient was 0.11 +/- 0.061 while FEC repeatability coefficients were 0.58 +/- 0.085 and 0.76 +/- 0.223 in adult females and lambs respectively. Subjects were classified, based on FEC, into three different levels of resistance to strongylids. Ewes belonging to the 'resistant class' should be conveniently exploited in mating schemes, in order to provide a method, alternative to drug administration, for a long-term parasite control; this would result particularly helpful under those production systems, such as organic farming, where the use of drugs is not allowed or limited.

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease.

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.

Age-related changes in the cell proliferation of ATPC+ mouse ascites tumour.

The growth of ATPC+, an ascites tumour derived from a spontaneous mammary carcinoma in BALB/c+ mice, was studied at different ages. It was observed that the number of cells increases rapidly during the first 5 days after implantation. Thereafter, the cell number increases more slowly, reaching a plateau after 8 days. This slowing-down is not due to a reduction in the growth fraction but to a lengthening of the cell cycle. Between 5 and 8 days the duration of all phases increases, including the S phase, which increases from 5.2 h in 5-day tumours to 8.2 h in 8-day tumours. In 12-day tumours both the cell cycle and S phase are only slightly longer than in 8-day tumours whereas the growth fraction is reduced. The slowing-down of cell growth can be attributed to growth fraction reduction rather than cell loss, which is maximal in the 5-day tumour. At this age the time course of the percent labelled cells and of the number of grains/nucleus suggests reutilization of [3H]-thymidine. Incorporation of [3H]-thymidine/cell decreases sharply in 12-day tumours due to a reduced availability of thymidine, which is degraded to thymine in the in vivo ascitic fluid faster than in 8-day tumours. This indicates an age-related change in the ascitic fluid composition.

Changes in oncogene expression in ascite tumour cells during ageing.

The expression of two oncogenes, c-myc and c-fos, was studied in an ascitic tumour (ATPC+) at different times after implantation. The specific mRNA synthesis was analysed by Northern blot analysis. The presence of the oncogene proteins was shown by immunofluorescence using flow cytometry and referred to the distribution of the cells in the different cell phases. The results show that both oncogenes are expressed by ATPC+ tumour cells. c-myc is expressed 5, 8 and 12 days after implantation, although with a different intensity, and the protein is mainly present in S or S+G2 phase cells. The c-fos oncogene is expressed only 12 days after tumour implantation and the cells labelled with the specific antibody are mainly in G1 phase. We conclude that c-myc is principally correlated with proliferative activity, whereas c-fos is expressed by non-cycling cells.

Sex differences on the electrocardiographic pattern of cardiac repolarization: possible role of testosterone.

BACKGROUND: Cardiac repolarization has been shown to be shorter and faster in men compared with women. In this study, we examined the electrocardiographic pattern of repolarization in patients with abnormal plasma levels of testosterone to gain insight into the role that this hormone plays in modulating repolarization. METHODS AND RESULTS: Repolarization was measured in 27 castrated men, 26 women with virilization, and 53 control subjects pair-matched for age and sex. Repolarization in castrated men was slower and longer than that of normal men. Women with virilization exhibited a shorter and faster repolarization than normal women and castrated men. These differences are the opposite of those found in the normal population. Finally, the changes observed in castrated men may be reverted by testosterone. CONCLUSIONS: We conclude that testosterone plays an important role in modulating cardiac repolarization.

Animal model of acute pulmonary thromboembolism treated by local recirculation of streptokinase through the lung.

Massive acute pulmonary thromboembolism has a high mortality within the first few hours. Surgical intervention can remove only larger thrombi. Systemic fibrinolytic administration requires many hours for adequate treatment. We describe an anesthetized dog model of acute, massive, disseminated pulmonary thromboembolism achieved by injection of 1.7 ml/kg of 1-hour-old thrombi directly into the pulmonary artery. The emboli were lysed with 50,000 IU streptokinase recirculated for 20 minutes through the isolated pulmonary vascular bed by use of a roller pump while the systemic bed was supported by conventional cardiopulmonary bypass. On reestablishing natural circulation all hemodynamic parameters returned to preembolism values. Success of lysis was histologically confirmed. Perfusion of the pulmonary vascular bed without inclusion of streptokinase in the perfusate worsened the hemodynamic state of the animals compared with an untreated nonperfused control group.

Molecular epidemiology of classical swine fever in Italy.

To gain an insight into the molecular epidemiology of classical swine fever (CSF) in Italy, virus isolates originating from outbreaks that occurred between 1985 and 2000 in wild boar or in domestic pigs in mainland Italy and in Sardinia were analysed by genetic typing. For this, a fragment (190 nucleotides) of the E2 glycoprotein gene was sequenced and phylogenetic analyses were performed, including older Italian isolates and isolates from recent outbreaks in Europe for comparison. The results show that in mainland Italy, several independent epidemiological events occurred in the last decade. In the north of the country, viruses of genotype 2.2 have persisted in wild boar, causing sporadic outbreaks in domestic pigs. In contrast, viruses of subgroups 2.1 and 2.3 appeared only intermittently in different regions of the mainland. In 1997, classical swine fever virus (CSFV) isolates belonging to the subgroup 2.1 and genetically and epidemiologically related to the Dutch isolate in Venhorst, affected domestic pigs exclusively. The isolates of subgroup 2.3, derived from wild boar as well as from domestic pigs were closely related to isolates from Germany and Poland. In Sardinia, CSF is an endemic in wild boar and affects domestic pigs also. Genetic typing showed that viruses of subgroups 1.1 and 2.3 have been present, the last ones being unrelated to the mainland viruses of the same subgroup. Due to the large quantities of pig and wild boar meat imported in some parts of Italy, it cannot be established if these viruses were always present in either the mainland or Sardinia, or if they represent recent introductions.

Classical swine fever virus: a ring test to evaluate RT-PCR detection methods.

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Two sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 34 samples of random primed cDNA. These had been synthesised from viral RNA representative of seven different genetic subtypes of CSFV. The other set comprised 40 clinical samples containing tonsil, spleen, whole blood or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the clinical samples were also compared with those obtained by virus isolation and antigen ELISA.ELISA. Both RT-PCR and RT-nested PCR appeared to give some false positive results. Several of the PCR tests appear suitable in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratories, and to show that improved control procedures can eliminate problems due to false positive reactions.A limited comparison of extraction and reverse transcription procedures showed similar results in each of three participating laboratories, even though the methods were not standardised.

Effect of electrode impedance in improved buffer amplifier for bioelectric recordings.

We analysed the effects of electrode impedance on the transfer response of a one-stage improved buffer amplifier. The electrode DC resistance (R(d)) modifies the one-stage buffer transfer response. We found a limit electrode resistance (R(d(lim))) which depends on the transfer damping factor (epsilon). If R(d) is lower than 86.5 komega, the transfer response of the buffer fulfils American Heart Association (AHA) recommendations, but when R(d) is greater than R(d(lim)) it must be cautiously weighed up because its influence in the transfer response becomes appreciable. The maximum R(d) that can be driven by the buffer is 1.2 Momega. Higher values do not fulfil AHA recommendations. Therefore, electrodes with higher impedance should not be used with this kind of buffer. In contrast, when this buffer is used to build in an instrumentation amplifier (IA) for bipolar recording, the common-mode rejection ratio (CMRR) is sensitive to the electrode type used.

Statistical analysis of individual assignment tests among four cattle breeds using fifteen STR loci.

Assignment tests based on multilocus genotypes are becoming increasingly important to certify quality and origin of livestock products and assure food safety and authenticity. The purpose of this study was to determine the potential of microsatellites (STR) for determining the breed origin of beef products among cattle breeds present in the market. We typed 19 STR in 269 animals from 4 cattle breeds. Based on Wright's F-statistics, 4 loci were discarded, and the remaining 15 loci (FIT = 0.101, FST = 0.089, and FIS = 0.013) were used to compute the likelihood that each multilocus genotype of the total sample was drawn from its true breed instead of another breed. To avoid occurrence of zero likelihood when one or more alleles were missing from a tested breed, sample allele frequencies were estimated assuming uniform prior distributions. Log-likelihood ratio [log(LR)] distributions of the individual assignments were determined for all possible breed contrasts, and their means and SD were used to infer the true-positive and false-positive rates at several values of the log(LR). The posterior probability that the animals of a presumed breed were actually drawn from that breed instead of any another breed was then calculated. Given an observed value of log(LR) > 0 and assuming equal priors, these probabilities were > 99.5% in 10 of 12 possible breed contrasts. For the 2 most closely related breeds (FST = 0.041), this probability was 96.3%, and the probability of excluding the origin of an animal from an alleged breed when it was actually derived from another breed was similar.

Chronic versus acute effects of amiodarone on the Vmax-conduction velocity relationship and on the space constant in canine myocardium.

Isolated tissue experiments (canine ventricular epicardium) and computer simulations were used to characterize the relationship between changes in maximum rate of depolarization during upstroke (Vmax) and the conduction velocity (theta) induced after long-term therapy with amiodarone and to compare these data with those obtained after acute superfusion with either desethylamiodarone or the parent compound. After chronic amiodarone, the changes in Vmax were linearly related to the square of the changes in theta during longitudinal propagation (LP) (slope = 0.93, r = 0.93, p = NS with respect to slope = 1), whereas during transverse propagation (TP), the slope of the relationship between both variables was slightly decreased (slope = 0.88, r = 0.96, p < 0.05 with respect to slope = 1). Similar results were observed after acute superfusion with desethylamiodarone (LP), (slope = 1, r = 0.05, p = NS with respect to slope = 1). In contrast, a significant increase in slope (p < 0.05 with respect to slope = 1) was observed after acute superfusion (slope = 1.45, r = 0.85 and slope = 1.48, r = 0.75 during LP and TP, respectively). In addition, the space constant (lambda) after chronic amiodarone (1.05 +/- 0.06 mm) was not significantly different from control (0.98 +/- 0.04 mm), but was slightly though significantly increased after acute amiodarone administration (1.07 +/- 0.03 mm, p < 0.03). Data are mean +/- SEM. Experimental data from chronic amiodarone were well fitted in a one-dimensional Beeler-Reuter-based discrete cable by reducing sodium conductance (GNa) exclusively. In contrast, data from acute superfusion were fitted only when junctional resistance (rj) and GNa were simultaneously reduced. These data suggest that acute amiodarone may modify both active and passive membrane properties whereas chronic amiodarone appears to alter only the active properties; the data further indicate that desethylamiodarone may play an important role in the mechanism of action of chronic amiodarone treatment.

The use of (3H) thymidine-adsorbed on activated charcoal for the study of non-premitotic DNA synthesis in newborn rat hepatocytes.

DNA synthesis in newborn rat hepatocytes was studied in the first three days of life by means of repeated injections of (3H) thymidine. One group of animals was treated with the label adsorbed on activated charcoal (experimental group) and another group (controls) was given the label diluted in saline. The specific activity of DNA was higher in control group, but its increase was not linear with time; in the experimental group, the radioactivity was lower, but its increase with time was linear. The percentage of labeled nuclei was higher in the experimental animals than in the controls and increased linearly with time. The average number of grains/nucleus was considerably smaller in the experimental group than in the controls, in which also the percentage of labeled cells showed considerable variations during the first three days of life. It is concluded that activated charcoal adsorption increases label availability with time and, by keeping a lower label concentration in the pool, reduces the risk of radiation damage.

A comparative in vitro study of the closing characteristics of Björk-Shiley and Bicer-Val tilting disc mitral valve prostheses.

The replacement of mitral valves has become a relatively common procedure in cardiac surgery. Information concerning losses due to backflow through the valve may be used to assess and compare the physical characteristics of commercially available prostheses. A simple apparatus based on an artificial ventricle was used to measure closing volume, leakage volume, total volume, leakage rate and closing time on a beat by beat basis for Björk-Shiley and Bicer Val prosthetic mitral valves. The Björk-Shiley valve opened to 60 degrees and displayed a smaller closing volume than the Bicer Val, which opened to 75 degrees; and the Björk-Shiley displayed higher leakage than the Bicer Val, thus reducing its advantage in terms of closing volume. Overall, the Bicer-Val showed about 1 ml greater reflux per beat than the Björk-Shiley. Study of a Bicer-Val, modified to have an opening angle of 80 degrees, confirmed that valve closing volume was a function of opening angle.

Differential effects of amiodarone on Vmax and conduction velocity in anisotropic myocardium.

Despite its widespread clinical use, the precise mechanism of action of amiodarone (AMI) has not been completely defined. We examined the effects of AMI (20 micrograms/ml) on Vmax and on conduction velocity (theta) during longitudinal (LP) and transverse (TP) propagation with respect to fiber orientation, in 10 strips of uniform anisotropic epicardial muscle obtained from the left ventricle of adult canine hearts. Mean values +/- SEM (standard error of the mean) were calculated as normalized values (beat 50/beat 1) after 4 h of AMI superfusion at five different basic cycle lengths (BCL). Vmax decreased from 0.99 +/- 0.01 at a BCL of 5,000 ms to 0.43 +/- 0.03 at a BCL of 300 ms during LP. During TP, Vmax decreased from 0.99 +/- 0.01 at a BCL of 5,000 ms to 0.54 +/- 0.05 at a BCL of 300 ms. The differences in the relative changes between both directions at a BCL of 300 ms, as well as at intermediate values of 1,000, 500, and 400, were significant (p less than 0.01). theta during LP (theta L) was depressed from 0.99 +/- 0.01 at a BCL of 5,000 ms to 0.80 +/- 0.04 at a BCL of 300 ms. In contrast, theta during TP (theta T) did not change as the BCL was decreased. In consequence, theta L was significantly more depressed than theta T at BCLs shorter than 1,000 ms (p less than 0.05). Moreover, theta T after AMI was not statistically different from control at any BCL studied. The lack of depression of theta T associated with a marked depression of Vmax during either LP or TP suggests that in addition to its sodium channel blocking properties, AMI could produce a decrease in the effective axial resistivity.

Relationship between Vmax and conduction velocity in uniform anisotropic canine ventricular muscle: differences between the effects of lidocaine and amiodarone.

The purpose of these experiments was to determine if Vmax in anisotropic myocardium varies approximately as the square of the conduction velocity (theta) after the addition of lidocaine (6.5 micrograms/ml) and amiodarone (20 micrograms/ml). We measured Vmax and theta in 16 epicardial strips of uniform anisotropic ventricular muscles, over a wide range of stimulation frequencies. The relationship of Vmax to theta 2 was evaluated by linear regression analysis. We found that the decrease in Vmax was proportional to the square of the decrease in theta in the presence of lidocaine both during longitudinal (LP) and transverse (TP) propagation (mean slope +/- SEM: 0.961 +/- 0.047 and 0.918 +/- 0.068, respectively). The changes in Vmax, in the presence of amiodarone, were not predicted by the quadratic changes in theta during TP. However, during LP, the changes in Vmax and theta were well fitted by the predicted relationship. The slope was significantly different from that of lidocaine (2.399 +/- 0.673 vs. 0.961 +/- 0.047, p less than 0.05). On the other hand, the predicted values of theta, assuming theta = square root of Vmax x k, were significantly more depressed than the measured values. We conclude that in uniform anisotropic ventricular muscle, when the changes in Vmax and theta are solely due to a decrease in sodium conductance, a quadratic relationship between the changes in both variables is seen and the slope of the regression line should be 1, such as we have shown for lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lidocaine on Vmax and conduction velocity in uniform anisotropic canine ventricular muscle: possible role of its binding-rate constants.

We evaluated the effect of lidocaine (5 micrograms/ml) on Vmax and conduction velocity during longitudinal and transverse propagation to fiber orientation in strips of uniform anisotropic ventricular muscle from adult canine hearts. Tissues were markedly anisotropic, with conduction velocities 3.2 times faster during longitudinal propagation than during transverse propagation to the long axis of the fibers, and with the greatest values of Vmax associated with the slowest conduction velocities. After addition of lidocaine, Vmax (normalized values with respect to control for each cycle length, expressed as mean +/- SEM) decreased from 0.99 +/- 0.02 at a cycle length of 1,000 ms to 0.86 +/- 0.02 at a cycle length of 300 ms during longitudinal propagation. During transverse propagation, Vmax decreased from 0.99 +/- 0.03 at a cycle length of 1,000 ms to 0.87 +/- 0.03 at a cycle length of 300 ms. The differences in the relative changes between both directions at these cycle lengths, as well as with intermediate values of 500, 400, and 350 ms, were not significant. Similar results were obtained for conduction velocity. We conclude from these findings that under these experimental conditions the effects of lidocaine are characterized by a relative change both in Vmax and conduction velocity that is almost the same during longitudinal and transverse propagation at all cycle lengths explored. Moreover, the rapid binding-rate constants reported for lidocaine may play a significant role in determining the characteristics of Vmax and conduction velocity depressions in both directions.

Real-time pressure-volume diagrams for the evaluation of ventricular function.

Using three intraventricular diameter signals obtained from ultrasonic distance gauges and applying the general ellipsoid model to the left ventricle, it was possible to obtain the left ventricular volume signal. Implanting a miniature transducer in the left ventricle the pressure signal was attained. With these two signals the pressure-volume diagrams were constructed on line, and ventricular function during load manoeuvres could be studied from them. Because the whole process was done on line, using a microcomputer, the performance of the left ventricle to load manoeuvres in different conditions could be seen instantly.

Effects of amiodarone and desethylamiodarone on the inward rectifying potassium current (IK1) in rabbit ventricular myocytes.

We examined the effects of amiodarone (AMI) and desethylamiodarone (DAM) on whole-cell inward rectifying potassium current (IK1) in freshly isolated adult rabbit ventricular myocytes by using the whole-cell voltage-clamp technique, as an index of their effects on resting membrane resistance (Rm). Under control conditions, the current showed a strong inward rectification with a maximal inward current measured at -130 mV of -26.4 +/- 1.3 pA/pF and a maximal outward current measured at -50 mV of 3.5 +/- 0.3 pA/pF The current also exhibit a time-dependent activation, with a time constant of activation (tau(a)) that increased with depolarization. The maximal slope conductance normalized to cell capacitance was 0.509 +/- 0.019 nS/pE After exposure to both DAM (50 microM; n = 8) and AMI (50 microM; n = 7), rapid decrease in inward IK1 was observed. Block was restricted almost exclusively to the inward component. DAM caused a significant reduction of the maximal inward current (-20.0 +/- 2.0 pA/pF; p < 0.05), whereas AMI induced an even greater reduction of the same component (-14.1 +/- 1.2 pA/pF; p < 0.05 with respect to control and to DAM). The outward component of IK1 was not changed by either AMI or DAM (4.0 +/- 0.3 pA/pF and 3.4 +/- 0.4 pA/pF, respectively). AMI and DAM also decreased the maximal slope conductance significantly (0.297 +/- 0.019 nS/pF and 0.421 +/- 0.038 nS/pF, respectively). In addition, AMI but not DAM significantly increased the tau(a). However, the voltage dependence of the acceleration of tau(a) remained unchanged after both AMI and DAM exposure. These results allow us to conclude that AMI may induce a greater increase in the resting Rm than its main metabolite. This effect may counterbalance, at least in part, the conduction slowing due to its sodium channel-blocking properties.

Influence of filtering techniques on the time-domain analysis of signal-averaged P wave electrocardiogram.

INTRODUCTION: The advent of signal-averaged ECG (SAECG) systems for P wave analysis has made it important to determine if the use of different filtering techniques in these systems is diagnostically equivalent. METHODS AND RESULTS: Three different high-pass filtering techniques and two cutoff frequency values were used: 29- and 40-Hz Butterworth bidirectional filter (BB29, BB40), 29- and 40-Hz Butterworth unidirectional filter (UB29, UB40), and 29- and 40-Hz least mean square filter (LMS29, LMS40). Normal healthy volunteers (n = 36) and patients with documented paroxysmal atrial fibrillation (n = 23) were analyzed. A custom-built SAECG system and standard bipolar orthogonal leads were used. Noise was reduced to < 0.3 microV. P wave total duration, root mean square voltage of the terminal 20, 30, and 40 msec of the filtered vector magnitude, and the area under the curve between the onset and offset of averaged unfiltered and filtered P wave vector magnitude were analyzed. Only the duration of the P wave showed statistically significant differences between groups, being longer in the PAF group for all filters and cutoff frequencies studied. A bias increment of approximately 20 msec was detected in unidirectional and least mean square filters as compared to the bidirectional filter. Sensitivity, specificity, and predictive accuracy were > 70% for all filters; the BB40 filter yielded the best performance. CONCLUSION: The normality limits derived from one filter cannot be applied directly to recordings obtained from the other filters. Critical limits must be established individually for different software settings.