International audience | OBJECTIVE: To determine the intra- and interobserver variability of systolic arterial pressure (SAP) and diastolic arterial pressure (DAP) measurements obtained with 2 indirect methods in awake dogs and percentage of successful measurements. ANIMALS: 6 healthy conscious adult dogs. PROCEDURES: 4 observers with different levels of training measured SAP and DAP on 4 days by use of Doppler ultrasonography (DU) and high-definition oscillometry (HDO). The examinations were randomized. Measurements for each technique were recorded 5 consecutive times, and mean values (total, 720 measurements) were used for statistical analysis. RESULTS: All within- and between-day coefficients of variation (CVs) for SAP were < 15% irrespective of the observer or method (HDO, 3.6% to 14.1%; DU, 4.1% to 12.4%). Conversely, half the CVs for DAP were > 15% with the highest within- and between-day CVs obtained by the least experienced observer by use of DU (19.5% and 25.9%, respectively). All attempts with HDO were successful, whereas DAP could not be measured by use of DU by the least experienced observer in 17% of attempts. CONCLUSIONS AND CLINICAL RELEVANCE: SAP may be assessed in healthy dogs by use of DU and HDO with good repeatability and reproducibility after a short period of training. Conversely, the variability of DAP is higher and longer training is required to assess DAP via DU than via HDO.

The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR) method to detect African swine fever virus (ASFV). The methods of ASFV real-time PCR and ddPCR were established and optimal reaction conditions were confirmed. Each method was evaluated for linearity, limit of detection, and specificity. The results indicated that ASFV ddPCR had a high degree of linearity (R2 ≥ 0.998) and specificity. The detection limit was 10 copies/reaction, which was approximately a 10-fold greater sensitivity than real-time PCR. This sensitive method could be used as an efficient molecular biology tool to diagnose ASFV, which is very important for preventing the spread of diseases across borders.

The purpose of this study was to establish a rapid, reliable diagnostic method detecting Encephalomyocarditis virus(EMCV) RNA in formalin-fixed, paraffin-embedded tissues of EMCV naturally infected pigs by cDNA probe of EMC K3, the EMCV strain isolated from Korea. Using a biotin-labelled nick translated probe for the cDNA marker. We made up for some defects of radiolabeled method. In situ hybridization(ISH) technique, differently from theother nucleic acid hybridization methods, is able to detect the virus genome specifically in the state of the intact shapes of cells and/or tissues. We succeeded in performing the experiment to detect the EMCV within 1~2 hours usign the MicroProbeTM capaillary action system. In this study, we ovserved highly specific positive sighals of red color by staining the paraffinembedded tissue sections of naturally EMCV-infected pig organs or tissues, including brain, heart, kidney and lacrimal gland with the Fast Red TR salt/Naphtol phosphate chromogen. The results suggested that this ISH method is considered as a highly sensitive and reliable tool for molecular biologic diagnosis of the EMC viral disease.

Plasmid profiles were compared between nonpiliated and piliated forms of Moraxella bovis isolates. The piliated form of M bovis isolate IBH64 contained 1 fewer plasmid than did the nonpiliated form. Piliated and nonpiliated cells of IBH64 contained plasmids having molecular size of 45, 32.8, 4.9, and 4.6 kilobases (kb). Single- and double-restriction endonuclease digestion by Ava I and Nde I indicated that the size of the additional plasmid carried by the nonpiliated form of IBH64 was approximately 43.6 kb. The M bovis isolates, Newport and GRS, contained the same number of plasmids in either their piliated or nonpiliated form.

While the C-terminal cytoplasmic tail of anion exchanger 1 (AE1, band 3) has been reported to possess important physiological roles, including one for proper membrane trafficking, its precise characteristics remain unclear. To clarify the overall structural consequences of the conserved sequence EL(K/Q)(L/C)LD(A/G)DD, containing the core binding sequence LDADD for carbonic anhydrase II, in the C-terminal region, we analyzed the membrane expression and turnover of bovine AE1 with a series of truncation and substitution mutations in HEK293 cells. Immunofluorescence microscopy and cell-surface biotinylation demonstrated that truncation mutants missing 18 C-terminal residues targeted the plasma membrane, but the one lacking the conserved region, by truncation of 28 amino acid residues, was retained inside the cells. Substitutions of Ala for Glusup(901), Leusup(902), Leusup(905), and Aspsup(906) in the sequence E901L(K/Q)(L/C)LDADD909 of bovine AE1 or those in the corresponding murine sequence also caused intracellular retention, though these mutants had half-lives comparable to that for wild-type AE1. These data demonstrate that the conserved amino acid residues Glusup(1), Leusup(2), Leusup(5), and Aspsup(6) in the EL(K/Q)(L/C)LD(A/G)DD region have essential structural consequences in stable expression of AE1 at the plasma membrane regardless of the ability in binding to carbonic anhydrase II of this region.

In MRJ/MpJ mice, there is a genetic mutation of exonuclease 1 (Exol), in which the exon 9 is sometimes deleted. In the present study, to check the gen-eration of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/ Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B , but was present little or very weakly in pCX/Ex/EIE/M . Next, the same spliced band was demonstrated in pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the del1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-l and tr-2 Exol).

A total of 253 samples from cattle (52), buffalo (51), sheep (50), goat (50) and chicken (50) were screened for the presence of Pasteurella multocida. Out of these, 17 samples were found positive by species specific PCR (PM-PCR) with ~460 bp amplified product. Overall prevalence of P. multocida was found to be 6.7%. Out of 17 PM-PCR positive samples, 12 (70%) were found positive by capsular type-B specific (HSB-PCR) and multiplex PCR assays with amplification of ~620 bp product in both the cases. A total of 5 isolates of P. multocida were obtained in 17 PM-PCR positive samples of which 4 were of serotype B:2 and 1 was of serotype A:3. The PM-PCR and HSB-PCR assays can be performed directly on clinical samples from animals with reproducible results without any non-specific amplification. Multiplex PCR further reduces the time for the species and type specific detection of P. multocida.

Thirty five human sputum collected from TB hospital Bareilly were investigated for Mycobacteria based on direct microscopy, culture and by multiplex peR targeting 12.7 kb fragment and IS 611O. DNA was isolated directly forms putums amples. Outof35 samples,25 were smear positive and 18 yielded culture and 16 were positive by the multiplex PeR. 10 samples were negative on smear mircoscopy, culture and PCR.