Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain.

A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.

The effect of N-duopropenide (a new disinfectant with quaternary ammonium iodides) and formaldehyde on survival of organisms of sanitary interest in pig slurry.

The counts of different groups of organisms (mesophilic total aerobic and anaerobic bacteria, enterobacteriaceae, faecal coliform bacteria and faecal streptococci type D), as well as the presence or absence of Pseudomonas spp. and Salmonella spp., were recorded in both unfiltered and filtered pig slurry samples at 1, 8 and 29 days of storage, before and after applying N-duopropenide (a new disinfectant with a quaternary ammonium structure) at 1, 2 x 1, 5 and 2 x 5% concentrations of the commercial product (equivalent to 0.11, 2 x 0.11, 0.55 and 2 x 0.55%, respectively, of active ingredient) for 1 h, or 0.5% commercial formaldehyde for 6 days. Before disinfection, anaerobic and aerobic organisms resulted in the highest counts (between 6 and 7 log units/ml), followed by enterobacteriaceae, faecal coliforms and streptococci (4-5 log units/ml). The unfiltered slurry showed higher bacterial counts than the filtered slurry. The variation in counts was similar for all the groups studied, with the highest count on day 1 of storage with a continuous decrease over the 3 days studied. N-duopropenide efficacy depended on the dose used, but a 2 x 5% concentration resulted in total inactivation (100% reduction) of all the bacteria studied, except for the total aerobic and anaerobic organisms present in unfiltered slurry. Formaldehyde efficacy was much lower. In respect of Pseudomonas spp., more isolates were obtained after using N-duopropenide at lower concentrations (1 and 2 x 1%) than before treatment, thus suggesting its suitability for selective isolation of this genus. Finally, no Salmonella spp. strains were isolated in any of the cases considered in this study.