RAPID COAGGLUTINATION TEST FOR THE DETECTION AND TYPING OF FOOT-AND-MOUTH-DISEASE VIRUS

Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) O, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV O, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses.

Effects of aerobic endurance training status and specificity on oxygen uptake kinetics during maximal exercise

The main purpose of this study was to analyze the effects of exercise mode, training status and specificity on the oxygen uptake ((V)over dot O-2) kinetics during maximal exercise performed in treadmill running and cycle ergometry. Seven runners (R), nine cyclists (C), nine triathletes (T) and eleven untrained subjects (U), performed the following tests on different days on a motorized treadmill and on a cycle ergometer: (1) incremental tests in order to determine the maximal oxygen uptake ((V)over dot O-2max) and the intensity associated with the achievement of (V)over dot O-2max (I(V)over dot O-2max); and (2) constant work-rate running and cycling exercises to exhaustion at I(V)over dot O-2max to determine the effective time constant of the (V)over dot O-2 response (tau(V)over dot O-2). Values for (V)over dotO(2max) obtained on the treadmill and cycle ergometer [R=68.8 (6.3) and 62.0 (5.0); C=60.5 (8.0) and 67.6 (7.6); T=64.5 (4.8) and 61.0 (4.1); U=43.5 (7.0) and 36.7 (5.6); respectively] were higher for the group with specific training in the modality. The U group showed the lowest values for VO2max, regardless of exercise mode. Differences in tau(V)over dot O-2 (seconds) were found only for the U group in relation to the trained groups [R=31.6 (10.5) and 40.9 (13.6); C=28.5 (5.8) and 32.7 (5.7); T=32.5 (5.6) and 40.7 (7.5); U=52.7 (8.5) and 62.2 (15.3); for the treadmill and cycle ergometer, respectively]; no effects of exercise mode were found in any of the groups. It is concluded that tauVO(2) during the exercise performed at I(V)over dot O-2max is dependent on the training status, but not dependent on the exercise mode and specificity of training. Moreover, the transfer of the training effects on tau(V)over dotO(2) between both exercise modes may be higher compared with (V)over dot O-2max.

Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Validation of an immunohistochemistry assay to detect turkey coronavirus: A rapid and simple screening tool for limited resource settings

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.

The effects of sildenafil on rectal sensitivity and tone in patients with the irritable bowel syndrome

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis

Aims: To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. Methods: Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haemotoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. Results: A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. Conclusions: It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue.

HPLC determination of flumethrin, deltamethrin, cypermethrin, and cyhalothrin residues in the milk and blood of lactating dairy cows

A procedure to determine residue concentrations of synthetic pyrethroid insecticides (flumethrin, deltamethrin, cypermethrin and cyhalothrin) in the milk and blood of lactating dairy cows was developed. Extraction was performed with acetonitrile, n-hexane partitioning, and silica gel column cleanup with n-hexane and diethyl ether. Analysis was carried out by high- performance liquid chromatography and ultraviolet detection. Recovery of the four pyrethroids averaged 78 to 91% with a minimum detectable concentration of 0.001 mg/kg. The method was reproducible and sensitive.

Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with 'apparent cure.' The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.

A comparison of nasopharyngeal endoscopy and lateral cephalometric radiography in the diagnosis of nasopharyngeal airway obstruction

Two auxiliary methods of diagnosing nasopharyngeal airway obstruction were compared. Cephalometric radiography and nasopharyngeal videoendoscopy were evaluated for efficacy in terms of reproducibility and validity. Thirty orthodontic patients (7 to 12 years of age) seeking otorhinolaryngologic treatment for mouth breathing, or mouth and nose breathing, had nasopharyngeal endoscopy and radiographic examinations performed on the same day. Two otorhinolaryngologists analyzed the results. Nasopharyngeal endoscopy was more reliable in identifying all the obstructive nasopharyngeal processes. Endoscopy obtained kappa index scores of almost perfect agreement for diagnosis of posterior nasal septum deviation, of substantial agreement for anterior nasal septum deviation and lower turbinate hypertrophy, and of moderate agreement for middle turbinate hypertrophy. Lateral cephalometric radiography obtained scores of perfect agreement for imaging hypertrophy of the middle turbinate, of almost perfect agreement for imaging hypertrophy of the posterior portion of the inferior turbinate, and of substantial agreement for imaging hypertrophy of the inferior turbinate. Radiographic diagnoses of hypertrophy of the middle and lower turbinates exhibited high sensitivity and low specificity when compared with diagnoses by nasopharyngeal endoscopy.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.