Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

Treponema denticola in microflora of bovine periodontitis

Periodontitis in cattle is an infectious purulent progressive disease associated with strict anaerobic subgingival biofilm and is epidemiologically related to soil management at several locations of Brazil. This study aimed to detect Treponema species in periodontal pockets of cattle with lesions deeper than 5mm in the gingival sulcus of 6 to 24-month-old animals considered periodontally healthy. We used paper cones to collect the materials, after removal of supragingival plaques, and kept frozen (at -80°C) up to DNA extraction and polymerase chain reaction (PCR) using T. amylovorum, T. denticola, T. maltophilum, T. medium and T. vincentii primers. In periodontal pocket, it was possible to identify by PCR directly, the presence of Treponema amylovorum in 73% of animals (19/26), T. denticola in 42.3% (11/26) and T. maltophilum in 54% (14/26). Among the 25 healthy sites, it was possible to identify T. amylovorum in 18 (72%), T. denticola in two (8%) and T. maltophilum in eight (32%). Treponema medium and T. vincentii were not detected over all 51 evaluated samples. The presence of Treponema amylovorum, T. maltophilum and, in particular, the widely recognized T. denticola in subgingival microflora brings an original and potencially important contribution in studies of the bovine periodontitis.

Monitoring and molecular characterization of group Drotavirus in Brazilian poultry farms

Abstract: Rotaviruses are etiological agents of diarrhea both in humans and in several animal species. Data on avian Group D rotaviruses (RVD) are scarce, especially in Brazil. We detected RVD in 4 pools of intestinal contents of broilers, layer and broiler breeders out of a total of 111 pools from 8 Brazilian states, representing an occurrence of 3.6%, by a specific RVD RT-PCR targeting the VP6 gene. Phylogenetic tree confirmed that the Brazilian strains belong to group D and 3 of the sequences were identical in terms of amino acid whereas one showed 99.5% identity with the others. The sequences described in this study are similar to other sequences previously detected in Brazil, confirming the conserved nature of the VP6 protein.

Ação trófica de aditivos fitogênicos, glutamina e ácido glutâmico sobre a Bursa de Fabrícius e intestino delgado de frangos de corte

Abstract The aim of this study was to evaluate the effect of phytogenic additives and glutamine plus glutamic acid, associated or not, on histomorphometry of bursa of Fabricius and small intestine, oocyst count and lesion scores, and carbon turnover of duodenal mucosa of broiler chickens infected with Eimeria acervulina. A total of 450 male broiler chickens was distributed into a completely randomized design with six treatments and three replications. Treatments consisted of control diet (CD); CD + coccidiosis vaccine; CD + antibiotic performance enhancers and anticoccidial (APE/AC); CD + glutamine and glutamic acid (Gln/Glu); CD + phytogenic additives (PA); CD + Gln/Glu + PA. Birds on treatment CD + vaccine were vaccinated via drinking water at three days of age against coccidiosis. At 16 days of age all birds of all treatments were inoculated orally and individually with 500,000 oocysts of Eimeria acervulina. There was no treatment effect on lesion score in the intestinal epithelium of birds. The smaller number of excreted oocysts was observed in groups of birds fed diets containing APE/AC and PA. Were observed better results of villus height and crypt depth for duodenum and ileum of birds of treatments containing Gln/Glu at 7 days of age, and Gln/Glu and PA at 21 days of age. Higher percentage of cortical area from bursa follicles was observed in birds fed diets supplemented with Gln/Glu and PA at 7, 14 and 21 days of age. Increased turnover of intestinal mucosa was observed in treatments containing Gln/Glu, indicating acceleration in development and regeneration of damaged tissue. Glutamine plus glutamic acid and phytogenic additives can provide improvements to structure, and thus to intestinal function, as well as to better immune response against the infectious challenges. Phytogenic additives can be used for coccidiosis control of broiler chickens where the use of antibiotic performance enhancers and anticoccidials is prohibited.

Periprocedural prognostic factors in coronary interventions – retrospective studies

Background: Approximately 11,000 revascularization procedures, either percutaneous coronary interventions (PCI) or coronary artery bypass grafting surgery (CABG), are performed yearly in Finland for coronary artery disease. Periprocedural risk factors for mortality and morbidity as well as long-term outcome have been extensively studied in general populations undergoing revascularization. Treatment choice between PCI and CABG in many high risk groups and risk-stratification, however, needs clarification and there is still room for improvement in periprocedural outcomes. Materials and methods: Cohorts of patients from Finnish hospitals revascularized between 2001 and 2011 were retrospectively analyzed. Patient records were reviewed for baseline variables and postprocedural outcomes (stroke, myocardial infarction, quality of life measured by the EQ-5D –questionnaire, repeat revascularization, bleeding episodes). Data on date and mode of death was acquired from Statistics Finland. Statistical analysis was performed to identify predictors of adverse events and compare procedures. Results: Postoperative administration of blood products (red blood cells, fresh frozen plasma, platelets) after isolated CABG independently and dose-dependently increases the risk of stroke. Patients 80 years or older who underwent CABG had better survival at 5 years compared to those who underwent PCI. After adjusting for baseline differences survival was similar. Patients on oral anticoagulation (OAC) for atrial fibrillation (AF) treated with CABG had better survival and overall outcome at 3 years compared to PCI patients. There was no difference in incidence of stroke or bleeding episodes. Differences in outcome remained significant after adjusting for propensity score. Lower health-related quality of life (HRQOL) scores as measured by the visual analogue scale (VAS) of the EQ-5D questionnaire at 6 months after CABG predicted later major adverse cardiac and cerebrovascular events (MACCE). Deteriorating function and VAS scores between 0 and 6 months on the EQ-5D also independently predicted later MACCE. Conclusions: Administration of blood products can increase the risk of stroke after CABG and liberal use of transfusions should be avoided. In the frail subpopulations of patients on OAC and octogenarians CABG appears to offer superior long-term outcome as compared to PCI. Deteriorating HRQOL scores predict later adverse events after CABG. Keywords: percutaneous coronary intervention, coronary artery bypass grafting, age over 80, transfusion, anticoagulants, coronary artery disease, health-related quality of life, outcome.

Plasma cortisol levels in captive wild felines after chemical restraint

Eight Panthera onca (Po), 13 Felis concolor (Fc), 7 Felis yagouaroundi (Fy), 7 Felis tigrina (Ft) and 5 Felis pardalis (Fp) specimens from São Paulo State zoos were used. All animals were restrained with darts containing 10 mg/kg ketamine and 1 mg/kg xylazine. Venous blood samples were collected as soon as possible (within 15-20 min) and serum was frozen until the time for cortisol quantification. Cortisol was determined using a solid phase radioimmunoassay with an intra-assay coefficient of 8.51%. Data were analyzed statistically by the Kruskal-Wallis test, followed by Dunn's multiple comparisons test, and the one-sample t-test, with the level of significance set at P<0.05. Data are reported as means ± SEM. Cortisol levels differed among the captive felines: Po = 166 ± 33a, Fc = 670 ± 118b, Fy = 480 ± 83b, Ft = 237 ± 42ab, Fp = 97 ± 12a nmol/l (values followed by different superscript letters were significantly different (P<0.001)). Since most of the veterinary procedures on these species involve chemical restraint, these results show the necessity of preventive measures in order to minimize the effect of restraint stress on more susceptible species

Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

Adrenocortical autoantibodies (ACA), present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF) on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA) technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13) and granulomatous (N = 9). ACA-PLPA were detected in 10/22 (45%) patients: 9/13 (69%) with the idiopathic form and 1/9 (11%) with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%): 10/13 (77%) with the idiopathic form and 1/9 (11%) with the granulomatous form. Twelve of the 13 idiopathic addisonians (92%) were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time). However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

Infrequent p53 gene alterations in ulcerative colitis

The purpose of this study was to determine whether point mutations and loss of the p53 gene take place in ulcerative colitis which is histologically negative for dysplasia. DNA was extracted from 13 frozen rectal or colon biopsies and blood samples. Ulcerative colitis was classified histologically as active (10 cases) and inactive (3 cases). Exons 5-8 were amplified by PCR, treated with exonuclease and shrimp alkaline phosphatase and sequenced by the dideoxy chain termination method with the Sequenase Version 2.0 DNA sequencing kit. PCR products of intron 6 and exon 4 were digested with MspI and AccII, respectively, for RFLP analysis. No p53 gene mutation was detected in these cases. The number of informative patients for loss of heterozygosity (LOH) at the p53 intron 6 was high, 11 out of 12 (92%), whereas no LOH was observed. LOH affecting p53 exon 4 was not detected in lesions from 5 of 12 patients (42%). In ulcerative colitis, tumor progression is similar to that in sporadic colon cancer, and other oncogenes and tumor suppressor genes are likely to be mutated before the p53 gene.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Usefulness of a rapid immunometric assay for intraoperative parathyroid hormone measurements

Intraoperative parathyroid hormone (IO-PTH) measurements have been proposed to improve operative success rates in primary, secondary and tertiary hyperparathyroidism (PHP, SHP and THP). Thirty-one patients requiring parathyroidectomy were evaluated retrospectively from June 2000 to January 2002. Sixteen had PHP, 7 SHP and 8 THP. Serum samples were taken at times 0 (before resection), 10, 20 and 30 min after resection of each abnormal parathyroid gland. Samples from 28 patients were frozen at -70ºC for subsequent tests, whereas samples from three patients were tested while surgery was being performed. IO-PTH was measured using the Elecsys immunochemiluminometric assay (Roche, Mannheim, Germany). The time necessary to perform the assay was 9 min. All samples had a second measurement taken by a conventional immunofluorimetric method. We considered as cured patients who presented normocalcemia in PHP and THP, and normal levels of PTH in SHP one month after surgery and who remained in this condition throughout the follow-up of 1 to 20 months. When rapid PTH assay was compared with a routine immunofluorimetric assay, excellent correlation was observed (r = 0.959, P < 0.0001). IO-PTH measurement showed a rapid average decline of 78.8% in PTH 10 min after adenoma resection in PHP and all patients were cured. SHP patients had an average IO-PTH decrease of 89% 30 min after total parathyroidectomy and cure was observed in 85.7%. THP showed an average IO-PTH decrease of 91.9%, and cure was obtained in 87.5% of patients. IO-PTH can be a useful tool that might improve the rate of successful treatment of PHP, SHP and THP.

Effect of one stretch a week applied to the immobilized soleus muscle on rat muscle fiber morphology

We determined the effect of stretching applied once a week to the soleus muscle immobilized in the shortened position on muscle fiber morphology. Twenty-six male Wistar rats weighing 269 ± 26 g were divided into three groups. Group I, the left soleus was immobilized in the shortened position for 3 weeks; group II, the soleus was immobilized in the shortened position and stretched once a week for 3 weeks; group III, the soleus was submitted only to stretching once a week for 3 weeks. The medial part of the soleus muscle was frozen for histology and muscle fiber area evaluation and the lateral part was used for the determination of number and length of serial sarcomeres. Soleus muscle submitted only to immobilization showed a reduction in weight (44 ± 6%, P = 0.002), in serial sarcomere number (23 ± 15%) and in cross-sectional area of the fibers (37 ± 31%, P < 0.001) compared to the contralateral muscles. The muscle that was immobilized and stretched showed less muscle fiber atrophy than the muscles only immobilized (P < 0.05). Surprisingly, in the muscles submitted only to stretching, fiber area was decreased compared to the contralateral muscle (2548 ± 659 vs 2961 ± 806 µm², respectively, P < 0.05). In conclusion, stretching applied once a week for 40 min to the soleus muscle immobilized in the shortened position was not sufficient to prevent the reduction of muscle weight and of serial sarcomere number, but provided significant protection against muscle fiber atrophy. In contrast, stretching normal muscles once a week caused a reduction in muscle fiber area.

Effect of passive stretching on the immobilized soleus muscle fiber morphology

The aim of the present study was to determine the effect of stretching applied every 3 days to the soleus muscle immobilized in the shortened position on muscle fiber morphology. Eighteen 16-week-old Wistar rats were used and divided into three groups of 6 animals each: a) the left soleus muscle was immobilized in the shortened position for 3 weeks; b) during immobilization, the soleus was stretched for 40 min every 3 days; c) the non-immobilized soleus was only stretched. Left and right soleus muscles were examined. One portion of the soleus was frozen for histology and muscle fiber area evaluation, while the other portion was used to identify the number and length of serial sarcomeres. Immobilized muscles (group A) showed a significant decrease in weight (44 ± 6%), length (19 ± 7%), serial sarcomere number (23 ± 15%), and fiber area (37 ± 31%) compared to the contralateral muscles (P < 0.05, paired Student t-test). The immobilized and stretched soleus (group B) showed a similar reduction but milder muscle fiber atrophy compared to the only immobilized group (22 ± 40 vs 37 ± 31%, respectively; P < 0.001, ANOVA test). Muscles submitted only to stretching (group C) significantly increased the length (5 ± 2%), serial sarcomere number (4 ± 4%), and fiber area (16 ± 44%) compared to the contralateral muscles (P < 0.05, paired Student t-test). In conclusion, stretching applied every 3 days to immobilized muscles did not prevent the muscle shortening, but reduced muscle atrophy. Stretching sessions induced hypertrophic effects in the control muscles. These results support the use of muscle stretching in sports and rehabilitation.

Cyclosporin A preferentially attenuates skeletal slow-twitch muscle regeneration

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, is associated with muscle regeneration via NFATc1/GATA2-dependent pathways. However, it is not clear whether calcineurin preferentially affects the regeneration of slow- or fast-twitch muscles. We investigated the effect of a calcineurin inhibitor, cyclosporin A (CsA), on the morphology and fiber diameter of regenerating slow- and fast-twitch muscles. Adult Wistar rats (259.5 ± 9 g) maintained under standard conditions were treated with CsA (20 mg/kg body weight, ip) for 5 days, submitted to cryolesion of soleus and tibialis anterior (TA) muscles on the 6th day, and then treated with CsA for an additional 21 days. The muscles were removed, weighed, frozen, and stored in liquid nitrogen. Cryolesion did not alter the body weight gain of the animals after 21 days of regeneration (P = 0.001) and CsA significantly reduced the body weight gain (15.5%; P = 0.01) during the same period. All treated TA and soleus muscles showed decreased weights (17 and 29%, respectively, P < 0.05). CsA treatment decreased the cross-sectional area of both soleus and TA muscles of cryoinjured animals (TA: 2108 ± 930 vs 792 ± 640 µm²; soleus: 2209 ± 322 vs 764 ± 439 m²; P < 0.001). Histological sections of both muscles stained with Toluidine blue revealed similar regenerative responses after cryolesion. In addition, CsA was able to minimize these responses, i.e., centralized nuclei and split fibers, more efficiently so in TA muscle. These results indicate that calcineurin preferentially plays a role in regeneration of slow-twitch muscle.

Effect of salbutamol on innervated and denervated rat soleus muscle

The objective of the present investigation was to perform a 14-day time-course study of treatment with salbutamol, a ß2 adrenoceptor agonist, on rat soleus muscle in order to assess fiber type selectivity in the hypertrophic response and fiber type composition. Male Wistar rats were divided into four groups: control (N = 10), treated with salbutamol (N = 30), denervated (N = 30), and treated with salbutamol after denervation (N = 30). Salbutamol was injected intraperitoneally in the rats of the 2nd and 4th groups at a concentration of 0.3 mg/kg twice a day for 2 weeks. The muscles were denervated using the crush method with pean. The animals were sacrificed 3, 6, 9, 12, and 14 days after treatment. Frozen cross-sections of soleus muscle were stained for myosin ATPase, pH 9.4. Cross-sectional area and percent of muscle fibers were analyzed morphometrically by computerized image analysis. Treatment with salbutamol induced hypertrophy of all fiber types and a higher percentage of type II fibers (21%) in the healthy rat soleus muscle. Denervation caused marked atrophy of all fibers and conversion from type I to type II muscle fibers. Denervated muscles treated with salbutamol showed a significantly larger cross-sectional area of type I muscle fibers, 28.2% compared to the denervated untreated muscle. Moreover, the number of type I fibers was increased. These results indicate that administration of salbutamol is able to induce changes in cross-sectional area and fiber type distribution in the early phase of treatment. Since denervation-induced atrophy and conversion from type I to type II fibers were improved by salbutamol treatment we propose that salbutamol, like other ß2 adrenoceptor agonists, may have a therapeutic potential in improving the condition of skeletal muscle after denervation.