Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes.

The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions with M. flocculare and M. hyorhinis reduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M. hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection of M. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.

Multiplex PCR for detection and typing of porcine circoviruses.

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.

Decrease of bradykinin-induced glomerular contraction in diabetic rat: a new cellular interpretation.

The contractile response to bradykinin (BK), measured by the reduction of the planar surface area, was studied in glomeruli and mesangial cells (MC) isolated from diabetic rats (D) one week after diabetes induction with injection of streptozotocin (STZ; 60 mg kg-1, i.p.). Results were compared with age and weight-matched untreated rats (N) and were expressed by two parameters of cell activity, the mean maximum contraction (MMC) and the proportion of contractile cells (PCC). Glomerular and mesangial contraction were found to be clearly reduced in diabetic rats in response to 100 nM BK. The lower contractile response was associated with a decrease of both glomerular calcium uptake and mesangial cell intracellular calcium mobilization. The fact that cell pretreatment with two protein kinase C (PKC) inhibitors, phorbol 12-13 myristate acetate and calphostin, lowered normal cell contraction at the level of that found in diabetic MC without any significant effect in the latter, suggests the involvement of a PKC pathway, perhaps by a decrease of activatable PKC in diabetes. In addition, our results led to the first description of a possible role of the kallikrein-kinin system in the early glomerular hemodynamic changes occurring in diabetes. Insulin (1-200 nM) increased the contractile response of cultured diabetic cells (MMC), and in this case, it also increased the PCC. It must be stressed that the effect of 1 nM insulin on the former (88% increase) was very much smaller than its effect on the latter (103% increase). The combination of the two parameters (contraction index, CI) provided a realistic evaluation of the contractile capacities of the cell population of the cultures as a whole. The differences in this index between normal and diabetic cell populations, in the absence or presence of insulin, were strictly parallel to those found in intact glomeruli. Finally, our results further confirm (Ouardani et al., Biol. Cell 86, 127, (1996)) the limit of the first five cell passages within which cultured MC can be reasonably used for the study of contractile abnormalities occurring in the early steps of diabetic state.

Loss of differences in mesangial cell phenotype between diabetic and normal rats: role of culture passages.

In mesangial cells (MC) isolated from streptozotocin (STZ)-induced diabetic rat kidneys, sensitivity to bradykinin (BK) for the induction of cell division and collagen synthesis, was found to be lower than in normal MC. Nevertheless, decreased activities could be reverted in vitro by insulin, at non-proliferative concentration (Girolami et al (1995), Can J Physiol Pharmacol 73, 848-853). The aim of the present study was to determine whether differences in the properties of diabetic MC could be ascribed to the diabetic state per se, and/or to experimental conditions, ie culture replating. Through successive cell replating, normal and diabetic types of MC were compared in terms of proliferation, contraction, free calcium concentration in response to KCl depolarization, and in relation to the expression of two cytoskeleton proteins specific to muscle cells, myosin and dystrophin. Studies of proliferation, contraction and free calcium concentration consistently showed that passage 5 was a limit beyond which differences between the two MC types were very small and sometimes non-significant. We found that the mean maximum contraction (MMC) and especially the proportion of contractile cells (PCC) among diabetic cells was lower than in normal MC. In addition, loss in proliferation activity and in [Ca2+]i concentrations were also found to occur during these five early passages. Dystrophin, a new marker of contractile phenotype recently described in smooth muscle cell (Leis et al (1994) Cell Biol Toxicol 10, 305), was first localized in MC and was compared with myosin also expressed in MC. However, during the course of cell replating and/or with the diabetic state, no visible quantitative changes were detected in the expression of the two contractile proteins. We conclude that cultured mesangial cells undergo phenotype modulations, as observed in other cells, in particular smooth muscle cells and consequently, comparative studies between normal and diabetic MC should not be carried out after the 5th passage.

Comparison of B1 and B2 receptor activation on intracellular calcium, cell proliferation, and extracellular collagen secretion in mesangial cells from normal and diabetic rats.

The mesangial cell is a contractile secreting cell found in a key position in the renal glomerulus. Several kidney and systemic diseases are associated with dysfunctions of the mesangial cells. We compared the effect of bradykinin (BK; B2 agonist) and des-Arg9-bradykinin (DBK; B1 agonist) on intracellular calcium mobilization, cell proliferation, and collagen secretion of mesangial cells from normal and streptozotocin-induced diabetic rats. Stimulation of mesangial cells with BK and DBK caused an increase in intracellular calcium (Ca2+). However, the patterns of the Ca2+ increases induced by BK and DBK were different, indicating that DBK induced a major Ca2+ influx, whereas BK preferentially released Ca2+ from intracellular pools. Stimulation with BK and DBK did not show any heterologous desensitization, thus indicating the presence of two distinct binding sites. In normal cells, DBK stimulated cell proliferation more than BK, and this action was potentiated by insulin. No effect of BK or DBK was found in cells harvested from diabetic rats. The proliferation effect of BK and DBK was restored by insulin. DBK stimulated more collagen synthesis than BK in normal cells. In cells harvested from diabetic rats the collagen secretion was increased, but BK and DBK no longer had any effect. Insulin reduced basal collagen secretion in normal cells and cells harvested from diabetic rats. Insulin also blunted stimulation by BK and DBK in normal cells but did not restore the response to BK and DBK in cells harvested from diabetic rats. Our results show that the sensitivity to DBK and BK decreases during the course of insulin-dependent diabetes, indicating modulation by insulin.