Value of indirect hemagglutination and coagglutination tests for serotyping Haemophilus parasuis.

An indirect hemagglutination test (IHA) and a coagglutination test (CA) were evaluated using saline, boiled, and autoclaved extracts for serotyping Haemophilus parasuis. CA showed several cross-reactions, whereas IHA gave rise to specific reactions, with minor exceptions. IHA was further compared with the immunodiffusion test (the "gold standard") for the serotyping of 67 field isolates. As a conclusion, IHA is recommended as a useful method for sensitive and specific serotyping of H. parasuis.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing.

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

In vitro efficacy of N-duopropenide, a recently developed disinfectant containing quaternary ammonium compounds, against selected gram-positive and gram-negative organisms.

OBJECTIVE: To study the efficacy of N-duopropenide against various gram-positive and gram-negative organisms. SAMPLE POPULATION: One field strain each of Pasteurella multocida subsp multocida, Staphylococcus aureus, Listeria monocytogenes, and L ivanovii, and 2 field strains of Escherichia coli. PROCEDURE: Strains were tested with and without serum as organic matter, using quantitative suspension and carrier tests. Six concentrations of active ingredient (0.005, 0.01, 0.05, 0.11, 0.27, and 0.55%) and 6 contact times (15 and 30 seconds and 1, 3, 5, and 10 minutes) were studied for each. RESULTS: Globally, N-duopropenide was more effective in suspension tests than in carrier tests, and when organisms were suspended in saline solution rather than serum. Under the most disadvantageous conditions (carrier test with serum), a concentration of 0.55% N-duopropenide acting for only 15 seconds was effective in inactivating P multocida subsp multocida and was even more effective against the 2 Listeria species tested. For E coli strains, the same concentration also was effective, but after 10 minutes of contact. On the other hand, N-duopropenide was unable to inactivate the S aureus strain in the carrier test with serum, a concentration of 0.55% for 10 minutes was necessary to inactivate it without organic matter; however, N-duopropenide was highly effective against this organism in the suspension test, even with serum. CONCLUSION: N-duopropenide was highly effective in vitro against 5 of the most commonly encountered organisms in clinical veterinary medicine and, consequently, might be a good choice in control measures against common pathogenic organisms in modern production systems.

Actinobacillus pleuropneumoniae does not require urease activity to produce acute swine pleuropneumonia.

The role in virulence of Actinobacillus pleuropneumoniae urease activity was investigated. A urease-negative mutant was isolated following transposon mutagenesis with a mini-Tn10 derivative. Both the parent strain and the urease-negative mutant exhibited identical LD50 values in a murine infection model. Pig challenge confirmed that the urease-negative mutant was fully virulent, since experimental inoculation with 5 x 10(7) colony forming units resulted in an acute disease indistinguishable from that produced by the wild-type strain at the same dose. Our results demonstrate that urease activity is not required for the development of acute pleuropneumonia.

Efficacy of a variety of disinfectants against Actinobacillus pleuropneumoniae serotype 1.

The efficacy of 23 disinfectants (including the most commonly used chemical groups) and 6 quaternary ammonium compound based commercial formulations against Actinobacillus pleuropneumoniae serotype 1 (ATCC 4074) was studied. The organisms were tested in suspension and carrier tests with serum as the organic matter. Chloramine-T, hydrogen peroxide, glutaraldehyde, and mercurochrome alone, and a quaternary ammonium compound formulation containing 10% benzalkonium chloride, 2.5% glutaraldehyde, 6.8% glyoxal, and 6% formaldehyde were effective in all tests, regardless of the presence or absence of organic load. All but 2 of the nonformulated disinfectants (sodium hypochlorite and an iodophor) caused at least a 3-log10 reduction in colony-forming units in the suspension test. However, most of the disinfectants were not as effective in the carrier test as in the suspension test; this difference ranged from a 1- to 5-log10 reduction in colony-forming units. In addition, the presence of serum considerably reduced the disinfectant capacities of most of the compounds tested, particularly in the carrier test. These results indicate the importance of selecting suitable disinfectants for routine use on surfaces contaminated with this organism, especially in the presence of organic matter. Chloramine-T and the aforementioned commercial formulation were also tested directly under field conditions in pig nurseries, confirming their high effectiveness.

Serological characterisation and antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pigs in Spain.

Seventy-one isolates of Actinobacillus pleuropneumoniae isolated from the lungs of pigs in outbreaks of pleuropneumonia in Spain were serotyped by indirect haemagglutination. Serotype 4 (42.2 per cent), serotype 7 (22.5 per cent) and serotype 2 (12.8 per cent) were predominant, whereas serotypes 1, 3, 6, 8, 9, 12 and untypable isolates were present only in small numbers. Serotypes 1, 2, 4 and 7 originated mainly from cases of acute pleuropneumonia, whereas serotypes 3, 6, 8, 9 and 12 were associated with chronically infected herds. The susceptibility of the isolates to 20 antimicrobial agents was determined by agar disc diffusion. Most were susceptible to cefuroxime, cefaclor, cefazolin, kanamycin, tobramycin, gentamicin, oxolinic acid, ciprofloxacin, enoxacin, thiamphenicol, colistin and trimethoprim/sulphamethoxazole. Marked resistance was found with amoxicillin, ticarcillin, oxytetracycline, doxycycline and metronidazole. Rifampicin, fosfomycin and tiamulin were the agents most effective against the isolates tested.

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates.

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

[Immunity coverage against measles, rubella and parotiditis viruses in a juvenile population in Leon, Spain]. / Cobertura inmunitaria frente a sarampión, rubéola y parotiditis en una población infantil de León.

BACKGROUND: The immunity coverage, seroprevalence of IgG antibodies against infection by measles, rubella and parotiditis viruses in a juvenile population (50,398 children) was studied. METHODS: Systematic sampling was performed among children from 5-14 years of age who had undergone clinical analysis in the health care area of Leon. The hypothesis of sampling was the most unfavorable (p = q = 0.5) and the size of the sample of 600 children. Information was collected on vaccination state, previous history of disease and place of residence. The presence/absence of IgG antibodies was investigated by a commercial immunoenzymatic technique (EIA-Stat Measles, Mumps, Rubella, Whittaker Bioproducts, USA). RESULTS: Sixty percent of the children studied had IgG antibodies against the three virus, 27% against two and 9% against only one with absence of antibodies against the three virus in 3%. The seroprotection observed was significantly higher (p < 0.05) for measles (87% +/- 3%) that for the other two diseases (rubella 80% +/- 3% and parotiditis 77% +/- 3%). In the group of children with previous history of measles and parotiditis a higher percentage of antibodies (p < 0.01) was observed than in the group with previous history of vaccination (measles: 94% versus 84%, parotiditis: 90% versus 75%). No difference was observed in the case of rubella. CONCLUSIONS: Vaccination is still the principal conditioning factor of the state of immunity of the juvenile population in Leon in relation with the diseases studied (measles, rubella, parotiditis). The place of residence (rural or urban) did not condition different immunity coverage in the sample studied. The susceptibility of infection for some of these virus continues to be high: measles 13%, rubella 20% and parotiditis 23%).

In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents.

Minimal inhibitory concentration of 42 antimicrobial agents was determined against 57 field strains of Actinobacillus pleuropneumoniae isolated from pigs in Spain. Penicillins, aminoglycosides, and tetracyclines had irregular activity; ticarcillin, tobramycin, and doxycycline were the most active of each group, respectively. Macrolides, vancomycin, dapsone, and tiamulin, to which strains had high rate of resistance, were almost ineffective. Thiamphenicol, colistin, rifampin, fosfomycin, mupirocin, and metronidazole had good activity, with resistance ranging between 0 and 8.8%. Finally, cephalosporins (except cephalexin) and quinolones (especially ciprofloxacin, enrofloxacin, and sparfloxacin) were the most active antibiotics against A pleuropneumoniae.

Characterization of V factor-dependent organisms of the family Pasteurellaceae isolated from porcine pneumonic lungs in Spain.

116 V factor (NAD)-dependent strains belonging to the family Pasterurellaceae isolated from porcine pneumonic lungs were collected in Spain over a period of 1 yr and studied using 52 biochemical characters. In addition to Actinobacillus pleuropneumoniae (72 strains), Haemophilus taxon minor group (37 strains) and Taxon D (four strains), other taxon (three strains) were observed. This taxon, provisionally designated as Haemophilus sp. sorbitol+, is closed to A. pleuropneumoniae but differed by some biochemical characteristics. Among A. pleuropneumoniae strains, nine different serotypes were detected, the most frequent being serotypes 4 and 2.

Evaluation of an immunoperoxidase technique using an only biotin-labeled antibody for the demonstration of Actinobacillus pleuropneumoniae in tissue sections.

In this report an immunoperoxidase technique (IPB) using an only biotin-labeled antibody is compared to culture isolation method (CIM) for the demonstration of Actinobacillus pleuropneumoniae serotype 1 in tissues of mice inoculated intravenously. The organism was isolated in 86.7% of mice and detected by IPB in 100% of cases. A. pleuropneumoniae antigen was mainly demonstrated in the liver, spleen and lungs, but also in the kidney and brain, being especially located in the cytoplasm of macrophages. Significant differences were observed between the results obtained by both tests (P < 0.001). Cross-reactions occurred by IPB when a rabbit anti-A. pleuropneumoniae serotype 11 serum was tested but never when an anti-serotype 9 serum was used. The immunoperoxidase test here described yielded much better results than CIM and it could be useful in routine diagnosis of A. pleuropneumoniae infections.

Quantifying by monoclonal antibodies of specific IgG, IgM and IgA in the serum of minipigs experimentally infected with Actinobacillus pleuropneumoniae.

Fifteen minipigs were infected intratracheally with three different doses (10(8), 10(5) or 5 x 10(3) colony forming units) of the reference strains of serotypes 2 or 4 of Actinobacillus pleuropneumoniae, and three remained as controls. The titre of specific IgG, IgM and IgA in the serum was measured weekly for 15 weeks with an indirect ELISA using monoclonal antibodies specific for each isotype. IgG attained the highest titres, IgM lower and IgA the lowest, being only detected in four animals. Serotype 4 evoked significantly higher titres than serotype 2 (P < or = 0.01). In general the highest IgG titres were attained at four to six weeks after infection. Some of the minipigs were reinfected after seven weeks but this evoked an increased titre in only two instances.

Viability of Actinobacillus pleuropneumoniae in frozen pig lung samples and comparison of different methods of direct diagnosis in fresh samples.

A comparative study on different methods of diagnosis of Actinobacillus pleuropneumoniae from both fresh and frozen pig lungs is described. A total of 196 lung tissues with pneumonic lesions were examined for culture isolation on chocolate blood agar, as well as for antigen detection by means of the coagglutination test, the immunodiffusion test and the indirect ELISA. These samples were subsequently frozen for 1 yr and then they were recultured. A. pleuropneumoniae was recovered from fresh lung specimens in 30 cases (15.3%) and from frozen samples in only two cases (0.9%). Such a different degree of isolation demonstrates that long freezing had an adverse effect on the viability of this organism in lung samples. A pleuropneumoniae detection was positive in 134 samples (68.4%) by at least one of the immunological techniques examined. The indirect ELISA was the most sensitive and specific test, with antigen detected in 125 lungs (63.8%). In comparison with the coagglutination and immunodiffusion tests, the sensitivities of the indirect ELISA were 95.8 and 93.7%, and the specificities were 67.0 and 63.4%, respectively.

Epidemiologic investigation of a silage-associated epizootic of ovine listeric encephalitis, using a new Listeria-selective enumeration medium and phage typing.

The role of silage feeding in the origin of an epizootic of encephalitic listeriosis in a sheep flock was investigated by use of a new direct Listeria-selective isolation and enumeration medium, in combination with serotyping and phage typing. The silage contained high numbers (about 10(6) cells/g) of a L monocytogenes strain indistinguishable with respect to serovar and phagovar from that isolated from the brains of sick sheep. These results provided unambiguous bacteriologic evidence of the epidemiologic link between silage consumption and listeriosis in ruminants.

Cross-reactivity between Actinobacillus pleuropneumoniae serotypes comparing different antigens and serological tests.

Several antigens were prepared from suspensions of reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae (saline extract [SE], capsular extract [CE], whole cell suspension [WCS], boiled extract [BE], and autoclaved cell antigen [ACA]). The cross reactions between each antigen and the antisera against reference strains of the other 11 serotypes were compared by using the complement fixation test, ELISA and the indirect haemagglutination test. ACA produced the most cross reactions, which, in some serotypes, took place in all the antisera tested. BE produced fewer cross reactions, but these were more abundant than those obtained with SE, CE or WCS. The least cross reactivity occurred with SE in the indirect haemagglutination test. This test is, therefore, the most reliable method for serotyping field strains of A pleuropneumoniae.