Risk factors for Actinobacillus pleuropneumoniae serotype 2 infection in Danish genetic specific pathogen-free pig herds.

Between 1994 and 1998, 297 genetic specific pathogen-free (spf) pig herds participated in a monthly clinical and serological monitoring programme for infection with Actinobacillus pleuropneumoniae serotype 2 (ap-2). The average annual herd-level incidence was 3.4 per cent but there was a significant decreasing trend. A risk index, summing up the exposure from ap-2-infected neighbouring pigs within a 3 km radius, was derived from a geographical information system. A survival analysis indicates that the risk of ap-2 infection increased in proportion to the risk index, suggesting that local spread of ap-2 from infected neighbours was a significant factor. However, herd-specific purchase policies were not apparently associated with the risk of ap-2 infection.

Isolation of Actinobacillus pleuropneumoniae serotype 2 by immunomagnetic separation.

In Denmark porcine pleuropneumonia is most frequently caused by Actinobacillus pleuropneumoniae serotype 2 (60%). Isolation of A. pleuropneumoniae from nasal cavities or tonsils from carrier animals is complicated due to the mixed bacterial flora present. An immunomagnetic separation technique (IMS) using immunomagnetic beads (Dynabeads((R))) was developed for isolation of A. pleuropneumoniae serotype 2 from pure cultures and from heterogeneous suspensions. Different coating and washing procedures were evaluated in pure and mixed cultures using polyclonal (PAb) and monoclonal antibodies. The highest reisolation yield was achieved when the beads were coated with 1.5 microg PAb IgG/10(7) beads. After washing the beads for four times 9-24% of the bacteria could be reisolated depending on the amount of IgG attached to the beads and the number of beads used. The recovery was increased to 19-61% when only two washing steps were performed. The IMS was further evaluated using dilutions of A. pleuropneumoniae with added Pasteurella multocida (10(9) CFU/ml). After two washing steps 15% of the A. pleuropneumoniae cells and no P. multocida was reisolated. A detection limit of 10 CFU/ml was found in this heterogeneous suspension. No significant difference was observed when comparing the recovery of A. pleuropneumoniae from pure culture, from mixed cultures and from artificially inoculated tonsils. From 12 pigs inoculated with an aerosol of A. pleuropneumoniae serotype 2 the bacterium could not be detected from the nasal cavity or tonsils by cultivation or PCR 6 weeks later. By using IMS A. pleuropneumoniae serotype 2 could be reisolated from the tonsils of three pigs. The IMS method represents a valuable tool for isolation of A. pleuropneumoniae from tissue samples.

Designing serological surveillance programmes to document freedom from disease with special reference to exotic viral diseases of pigs in Denmark.

Surveillance programmes based on laboratory screening tests are increasingly used to document freedom from disease in order to facilitate trade. The following aspects must be considered when designing such programmes: diseases to be selected; epidemiology of the diseases; unit of analysis (animal or herd); target age group (or target farm type); test characteristics and sample size. Issues related to these aspects are discussed and illustrated using the example of serological surveillance for exotic viral diseases in the pig population of Denmark. Sampling designs based on individual animal samples are compared with herd-based sampling (two-stage sampling). While the latter is likely to require a larger sample size, the increased level of information and the reliability of the results obtained are considered to be worth the expense. Issues related to the development of international standards for declaring freedom from disease are discussed. The authors conclude that international standards are desirable, providing that these standards represent scientifically valid principles.

Evaluation of 5' nuclease assay for detection of Actinobacillus pleuropneumoniae.

Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C(t) value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5' nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with C(t) values of