C.B. Gutiérrez

Genotyping of Francisella tularensis Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing

ABSTRACT 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.

Value of Indirect Hemagglutination and Coagglutination Tests for Serotyping Haemophilus parasuis

ABSTRACT 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.

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.

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.

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.

Biochemical characterisation of Francisella tularensis strains isolated in Spain

Correspondence to Dr Rodriguez Ferri BRAUN, U., AMREIN, E., ESTERMANN, U., EGLI, J., SCHWEIZER, T., LUTZ, H., EHRENSPERGER, F., VANDEVELDE, M. & KIHM, U. (1998) Untersuchungen an 182 Nachkommen von an boviner spongiformer Enzephalopathie (BSE) erkrankten Kuhen in der Schweiz. Teil 1: Klinische Befunde. Schweizer Archivfiir Tierheilkunde 140, 241-249 BRAUN, U., FLOCKIGER, M., GERSPACH, C. & GREST, P. (2003a) Clinical and radiographic findings in six cattle with cervical diskospondylitis. Veterinary Record 152, 630-632 BRAUN, U., GERSPACH, C., SALIS, F. & FEIGE, K. (2003b) Klinische Befunde bei 4 Rindern mit Abszess in der Halswirbelsaule. Schweizer Archiv fur Tierheilkunde 145, 124-128 BRAUN, U., HERMANN, M. & PABST, B. (1989) Haematological and biochemical findings in cattle with dilatation and torsion of the caecum. Veterinary Record 125, 396-398 BRAUN, U., SCHWEIZER, G., GERSPACH, C. & FEIGE, K. (2003c) Clinical findings in 11 cattle with abscesses in the thoracic vertebrae. Veterinary Record 152, 782-784 COOPER, J. & WALKER, R. D. (1998) Listeriosis. Veterinary Clinics ofNorth America: Food Animal Practice 14, 113-125 DENNIS, S. M. (1993) Listeriosis (circling disease, silage sickness). In Current Veterinary Therapy, Food Animal Practice. Philadelphia, W. B. Saunders. pp 580-583 EHRENSPERGER, F., HILBE, M., SYDLER, T., CORBOZ, L., BRAUN, U. & POSPISCHIL, A. (2001) Zerebrale Listeriose bei Schaf und Ziege: eine histopathologische und immunhistologische Studie. Wiener Tieriirztliche Monatsschrift 88, 219-225 GATES, G. A., BLENDEN, D. C. & KINTNER, L. D. (1967) Listeric myelitis in sheep. Journal of the American Veterinary Medical Association 150, 200-204 KLEE, W. (1987) Diagnose und Therapie der Listeriose beim Rind. Praktischer Tierarzt 68, 53-54 STOBER, M. & GRUNDER, H-D. (1990) Die klinische Untersuchung des Rindes. 3rd edn. Eds G. Dirksen, H-D. Grunder, M. Stober. Berlin, Munchen, Paul PareyVerlag. pp 203-230 SEAMAN, J. T., CARTER, G. I., CARRIGAN, M. J. & COCKRAM, F. A. (1990) An outbreak of listerial myelitis in sheep. Australian Veterinary Journal 67, 142143

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.