Intraoral 3D Scanning or Dental Impressions for the Assessment of Dental Arch Relationships in Cleft Care: Which is Superior?

OBJECTIVE: This study was undertaken to evaluate intraoral 3D scans for assessing dental arch relationships and obtain patient/parent perceptions of impressions and intraoral 3D scanning. MATERIALS & METHODS: Forty-three subjects with nonsyndromic unilateral cleft lip and palate (UCLP) had impressions taken for plaster models. These and the teeth were scanned using the R700 Orthodontic Study Model Scanner and Trios® Digital Impressions Scanner (3Shape A/S, Copenhagen, Denmark) to create indirect and direct digital models. All model formats were scored by three observers on two occasions using the GOSLON and modified Huddart Bodenham (MHB) indices. Participants and parents scored their perceptions of impressions and scanning from 1 (very good) to 5 (very bad). Intra- and interexaminer reliability were tested using GOSLON and MHB data (Cronbach's Alpha >0.9). Bland and Altman plots were created for MHB data, with each model medium (one-sample t tests, P < .05) and questionnaire data (Wilcoxon signed ranks P < .05) tested. RESULTS: Intra- and interexaminer reliability (>0.9) were good for all formats with the direct digital models having the lowest interexaminer differences. Participants had higher ratings for scanning comfort (84.8%) than impressions (44.2%) (P < .05) and for scanning time (56.6%) than impressions (51.2%) (P > .05). None disliked scanning, but 16.3% disliked impressions. Data for parents and children positively correlated (P < .05). CONCLUSIONS: Reliability of scoring dental arch relationships using intraoral 3D scans was superior to indirect digital and to plaster models; Subjects with UCLP preferred intra-oral 3D scanning to dental impressions, mirrored by parents/carers; This study supports the replacement of conventional impressions with intra-oral 3D scans in longitudinal evaluations of the outcomes of cleft care.

RNA splicing and editing modulation of 5-HT(2C) receptor function: relevance to anxiety and aggression in VGV mice.

Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin prevent the expression of proinflammatory chemokines and cytokines in pigs.

The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1 α , IL-1 ß , IL-6, TNF- α , and IL-10) was evaluated by RT-qPCR in colostrum-deprived pigs vaccinated and challenged with Haemophilus parasuis serovar 5. Two vaccines containing native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit) were tested, along with two control groups: one inoculated with PBS instead of antigen (challenge group (CHG)), and another one nonimmunized and noninfected (blank group). The use of NPAPTim and NPAPTit resulted in complete protection against H. parasuis (no clinical signs and/or lesions), and both vaccines were capable of avoiding the expression of the proinflammatory molecules to levels similar to physiological values in blank group. However, overexpression of all proinflammatory molecules was observed in CHG group, mainly in the target infection tissues (brain, lungs, and spleen). High expression of CCL-2, CXCL-8, IL-1 α , IL-1 ß , and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5.

New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer's disease.

Four groups of colostrum-deprived pigs were immunized with Porcilis Glässer® (PG) or with subunit vaccines developed by us (rTbpA, NPAPT(M) or NPAPT(Cp)) against Glässer's disease, and they were challenged with 3×10(8)CFU of Haemophilus parasuis. A strong reduction in CD3(+)γδTCR(+) cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172α(+)CD163(+) cells was detected in all groups but PG, while a reduction in SLAIIDR(+) molecules expression was observed after challenge in control animals. Significant increases in CD3ε(+)CD8α(+)CD8ß(+) and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPT(M) and NPAPT(Cp)) groups. Finally, a greater response in CD4(+)CD8α(-) cells was observed in NPAPT(Cp) compared to NPAPT(M) and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässer's disease.

Comparison of real-time PCR and culture isolation in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis.

AIMS: A real-time PCR (RT-PCR) based on the detection of the infB gene of Haemophilus parasuis is compared with culture isolation (Frandoloso et al., (2011) Clin Vaccine Immunol 18, 50-58.), evaluating different subunit or commercial vaccines. METHODS AND RESULTS: Samples from different tissues of 24 experimentally infected and challenged colostrum-deprived piglets were tested. The RT-PCR gave globally a 23·3% more of positive results than culture, and all samples being positive by culture were positive by RT-PCR also. H. parasuis could not be cultured from any of the samples of the piglets included in the three vaccinated groups resulting in a strong protection, but it could be detected by RT-PCR in six samples in the group immunized with the commercial vaccine, in three in that vaccinated with native proteins with affinity to porcine transferrin (NPAPT) administered intramuscularly and in only two in that immunized with NPAPT intratracheally. CONCLUSIONS: The RT-PCR was more sensitive than culture for H. parasuis detection in the organs compared. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR evidenced that NPAPT vaccines were those yielding the best protection results in terms of H. parasuis clearance.

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis.

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5x10(9) CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10(5) CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer's disease.

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis.

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 x 10(9) CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10(5) CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45(+), CD3(+), CD4(+), CD8alpha(+), CD25(+), CD4(+) naïve, alphaIgM(+) and SWC3(+) cells in single-colour fluorescence, and CD4(+)/CD8alpha(+) and CD8alpha(+)/CD8beta(+) combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P<0.05) in the relative proportions of monocytes and granulocytes (SWC3(+)) and B cells (alphaIgM(+)), as well as a significant reduction in CD3(+) cells (P<0.05). These changes were similar for the five groups compared, except for the significant increase of CD25(+) cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.

Antimicrobial resistance of Pasteurella multocida isolates recovered from swine pneumonia in Spain throughout 2017 and 2018.

A total of 32 Pasteurella multocida isolates were obtained from 60 cases of swine pneumonic lungs collected in "Castilla y León" (northwestern Spain) between November 2017 and April 2018. Capsular type A isolates were isolated from 96.9% cases and capsular type D from the remaining 3.1%. All isolates were characterized for their susceptibilities to eight antimicrobial agents and the presence of eight resistance genes. The frequency of susceptibility was lower than 60% in four of the drugs, 84.4% of the isolates showed resistance to at least two compounds, and 46.9% to a combination of three drugs. The resistance patterns suggested that enrofloxacin, chloramphenicol, tetracycline and cefotaxime were the compounds most likely active to P. multocida. The usage of PCR revealed that ermC, bla ROB1, tetB and msrE genes occurred in more than 37.0% isolates, that suggested its putative accountability in the resistance of the strains harbor them. However, most were detected in susceptible strains and only a genetic explanation for the resistance could be linked to erythromycin. Therefore, the resistances to clyndamicin, cotrimoxazol, ß-lactams and tetracyclin observed by phenotypic testing remains genetically unexplained and further investigations are required.

Removal of bacteria from boar semen using a low-density colloid.

Antibiotics are added to semen extenders when preparing commercial semen doses for artificial insemination according to national and international guidelines. However, this addition of antibiotics represents non-therapeutic usage and could be contributing to the development of antibiotic resistance. Colloid centrifugation was shown to reduce the load of bacteria present in boar semen and was capable of removing all bacteria if performed directly after semen collection, albeit with some loss of spermatozoa. The present experiment was conducted with a low density colloid to investigate whether it was possible to separate all of the spermatozoa from seminal plasma i.e. without selection for robust spermatozoa, or whether this would have a detrimental effect on sperm quality. Ejaculates from nine boars were extended in Beltsville Thawing Solution without antibiotics and were transported to the laboratory for Single Layer Centrifugation (SLC) on modified Porcicoll i.e. at a low density (S). A further modification was that a sterile inner tube was included inside some of the 50 mL centrifuge tubes to facilitate harvesting of the sperm pellet (M). Aliquots of all samples (control, S and M) were cultured for bacterial quantification and identification using standard microbiological methods. Sperm quality was evaluated daily. Three of the C and M samples and five of the S samples did not contain any bacteria. Mean bacterial counts for the remaining samples (colony forming units/mL) were as follows: C 259 ±â€¯216; S 30 ±â€¯22; M 33 ±â€¯15 (P < 0.01). Citrobacter spp., Staphylococcus simulans, Klebsiella variicola, Escherichia coli, Myroides odoratimimus, Proteus spp. and Enterococcus faecalis were identified in the control samples. There were marginal differences in sperm quality among treatments, with sperm velocity and linearity being higher in S and M samples than in C at all time points. However, sperm viability, capacitation and acrosome status were marginally better in controls than in S or M on day 0, but these differences disappeared during storage. Conclusions: centrifugation through a low density colloid can remove or reduce bacterial contamination in boar ejaculates without using antibiotics. Furthermore, it is possible to collect boar ejaculates without bacterial contamination by paying strict attention to hygiene.

Evaluation of different API systems for identification of porcine Pasteurella multocida isolates.

An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.

Antimicrobial susceptibility patterns of Haemophilus parasuis from pigs in the United Kingdom and Spain.

A total of 30 British and 30 Spanish Haemophilus parasuis isolates were tested for their susceptibility to 19 of the antimicrobials currently used in swine practice with a broth microdilution method in order to know the emergence of resistance against these compounds in this porcine pathogen. All the British isolates were susceptible to penicillin, ceftiofur, erythromycin, tilmicosin, enrofloxacin, and florfenicol, and most of them were susceptible to the remaining antimicrobials (the highest resistance rate found was of 20% to neomycin). In contrast, all the Spanish isolates were susceptible exclusively to florfenicol, and high proportions of resistance were encountered for penicillin, ampicillin, oxytetracycline, erythromycin, tilmicosin, tiamulin and trimethoprim+sulphamethoxazole; in addition, a bimodal or multimodal distribution, or tailing of Spanish isolates over the MIC range was observed for clindamycin, sulphonamides and tylosine tartrate, suggesting the development of acquired resistance. In addition, several multiresistance patterns were found among the Spanish isolates, 23.3% of them being resistant to at least eight antimicrobials, the same rate as that encountered for those being susceptible to all antimicrobials tested. This study showed that in general British H. parasuis isolates are susceptible to antimicrobial agents routinely used for treatment of porcine respiratory diseases; however, the Spanish isolates need a more continuous surveillance of their susceptibility patterns.

aroA gene PCR-RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species.

The Haemophilus parasuis aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase and participates in the aromatic amino acids and the folic acid universal metabolic pathway of bacteria. The application of aroA-based PCR-RFLP methodology yields a significant degree of diversity in H. parasuis and Actinobacillus species. PCR amplification of the aroA gene rendered a 1,067-bp fragment in all 15 H. parasuis serovars, and also in Actinobacillus pleuropneumoniae serotypes 1-12, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus suis, Actinobacillus ureae, Actinobacillus minor and Actinobacillus indolicus. Sau3AI and RsaI digestions of the aroA PCR products rendered seven different restriction fragment length polymorphism (RFLP) patterns: group I (H. parasuis serovars 1, 2, 4-6, and 8-15, A. porcinus and A. ureae), group II (H. parasuis serovars 3 and 7, and A. pleuropneumoniae serotypes 1, 4, 5, 9, 11 and 12), group III (A. lignieresii), group IV (A. pleuropneumoniae serotype 7), group V (A. pleuropneumoniae serotypes 2, 3, 6 and 8, A. equuli, A. rossii, A. minor and A. indolicus), group VI (A. suis) and group VII (A. pleuropneumoniae serotype 10). This is the first report describing the presence of aroA gene in H. parasuis, A. lignieresii, A. porcinus, A. rossii, A. suis, A. ureae, A. minor and A. indolicus and the data presented here demonstrates a significant degree of aroA genetic diversity in H. parasuis and species of the genus Actinobacillus.

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.

TC21 and Ras share indistinguishable transforming and differentiating activities.

Constitutively activated mutants of the Ras-related protein TC21/R-Ras2 cause tumorigenic transformation of NIH3T3 cells. However, unlike Ras, TC21 fails to bind to and activate the Raf-1 serine-threonine kinase. Thus, whereas Ras transformation is critically dependent on Raf-1 TC21 activity is promoted by activation of Raf-independent signaling pathways. In the present study, we have further compared the functions of Ras and TC21. First we determined the basis for the inability of TC21 to activate Raf-1. Whereas Ras can interact with the two distinct Ras-binding sequences in NH2-terminus of Raf-1, designated RBS1 and Raf-Cys, TC21 could only bind Raf-Cys. Thus, the inability of TC21 to bind to RBS1 may prevent it from promoting the translocation of Raf-1 to the plasma membrane. Second, we found that TC21 is an activator of the JNK and p38, but not ERK, mitogen-activated protein kinase cascades and that TC21 transforming activity was dependent on Rac function. Thus, like Ras, TC21 may activate a Rac/JNK pathway. Third, we determined if TC21 could cause the same biological consequences as Ras in three distinct cell types. Like Ras, activated TC21 caused transformation of RIE-1 rat intestinal epithelial cells and terminal differentiation of PC12 pheochromocytoma cells. Finally, activated TC21 blocked serum starvation-induced differentiation of C2 myoblasts, whereas dominant negative TC21 greatly accelerated this differentiation process. Therefore, TC21 and Ras share indistinguishable biological activities in all cell types that we have evaluated. These results support the importance of Raf-independent pathways in mediating the actions of Ras and TC21.

Diurnal variations in maternal and fetal steroids in pregnant rhesus monkeys.

To examine diurnal changes in maternal and fetal steroids in chronically catheterized pregnant rhesus monkeys, we have measured cortisol (F), progesterone (P4), estone, and estradiol in maternal blood and F, P4, and dehydroepiandrosterone sulphate (DHAS) in simultaneous samples of fetal blood taken in the morning (0700--1000 h) and in the evening (1700--2100 h). In the mother, the concentrations of F, estradiol, and estrone were higher in the morning than in the evening. In contrast, progesterone was higher in evening than in morning samples. In fetal blood, there was no significant morning-evening difference for F; however, the concentrations of P4 and DHAS were higher in the evening than in the morning samples. In three individual monkeys, the concentrations of P4 and DHAS in serial samples of fetal blood were correlated. We conclude 1) that there are marked diurnal changes in the concentration of several steroids in samples of maternal and fetal blood and 2) that the concentration of F in maternal blood shows the opposite pattern to that of possible fetal adrenal steroids in fetal blood.

Effect of thyrotropin-releasing hormone on secretion of thyrotropin, prolactin, thyroxine, and triiodothyronine in pregnant and fetal rhesus monkeys.

The effect of thyrotropin-releasing hormone (TRH) on the pituitary-thyroid axis and on prolactin secretion was studied in pregnant Rhesus monkeys during the latter period of gestation and in non-pregnant female controls. The baseline plasma concentrations of TSH, T3, T4, and prolactin (PRL) of pregnant monkeys did not differ from those of non-pregnant monkeys. After administration of TRH, plasma prolactin rose to higher levels in pregnant monkeys than in non-pregnant monkeys whereas there was a similar response of plasma TSH, T4 and T3 in both groups. The baseline plasma TSH was elevated and plasma T3 was decreased in the fetus compared with the mother. Administration of TRH iv to the maternal monkey caused a larger response in the fetal plasma TSH than in that of the mother and was followed by larger increments in plasma T4 and T3 concentrations in the fetuses than in the mothers. The larger increments of plasma TSH and thyroid hormones in the fetus compared with the mother also occurred when TRH was given iv to the fetus. There was a significant rise of plasma prolactin in both mother and fetus after administration of TRH to mother or fetus; the increase of plasma PRL was much higher in the mother than in the fetus. The data show that TRH can cross the primate placenta in either the maternal to fetal or fetal to maternal direction. The fetal thyroid of the Rhesus monkey during the latter period of gestation can release both T4 and T3 in response to TSH.

Detection and subtyping of Actinobacillus pleuropneumoniae strains by PCR-RFLP analysis of the tbpA and tbpB genes.

A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5-50 and 3 x 10(2)-3 x 10(3) CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.

Reversible fetal hydrops associated with indomethacin use.

BACKGROUND: Ductus arteriosus constriction is a known complication of exposure to indomethacin in utero. Nonimmune hydrops associated with indomethacin use has been reported in only six cases, all twins over 32 weeks' gestation. CASE: We present a case of fetal hydrops associated with tricuspid regurgitation and ductal constriction developing within 30 hours of instituting indomethacin tocolysis in a 28-week singleton gestation. Discontinuation of indomethacin resulted in partial resolution of these findings 72 hours later. A normal infant was delivered subsequently. CONCLUSION: Ultrasonographic screening for signs of constriction of the ductus arteriosus should be done within 48 hours of instituting indomethacin therapy at any gestational age.

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae.

Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognized serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG2a, IgG2b, and IgG3 were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group 1 (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.