Vibeke Sørensen

Mycoplasma hyopneumoniae infection in pigs: Duration of the disease and evaluation of four diagnostic assays

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98-100% and the specificity to 93-100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.

Clinical observations, pathology, bioassay in mice and serological response at slaughter in pigs experimentally infected with Toxoplasma gondii

Experimental infections of a total of 47 pigs with tachyzoites of the Toxoplasma gondii RH-strain, tissue cysts of the SSI-119 and R92 strains as well as oocysts of the SSI-119 strain were performed to determine the sensitivity of an indirect IgG-ELISA, using tachyzoite lysate of the RH-strain as antigen. The infections led to a dose dependent moderate clinical affection (inappetence, fever and poor general condition). Pigs infected with 10000 oocysts or with 1/2 mouse brain containing tissue cysts of the SSI-119 strain showed a significant decrease in weight gain compared to uninoculated pigs during the first 2 weeks p.i., followed, however, by compensatory growth during the next 6 weeks. At slaughter 3 to 4 months after inoculation 39/41 (95.1%) of pigs positive by bioassay in mice were seropositive in ELISA. Tissue cysts were not demonstrable by immunohistochemistry. ELISA OD-values obtained by analysis of meat juice from heart muscle and tongue (diluted 1:40) correlated strongly with OD-values by analysis of serum (diluted 1:400) (r heart juice = 0.942; r tongue juice = 0.915). Thus, meat juice samples were shown to provide a suitable alternative to serum for serological detection of Toxoplasma infection in pigs.

Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies.

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.

Differentiation between serological responses to Brucella suis and Yersinia enterocolitica serotype O:9 after natural or experimental infection in pigs

False-positive serological reactions (FPSR) due to infections with Yersinia enterocolitica serotype Oratio9 (YeOratio9) are a problem in tests for brucellosis. In the present study, FPSR in classical and novel tests for brucellosis following experimental infections of pigs with YeOratio9 were compared with responses of B. suis biovar 2-inoculated pigs. FPSR were limited to 2-9 weeks post-YeOratio9 inoculation, while B. suis-infected pigs were test-positive throughout the 21-week period of investigation. Although YeOratio9-inoculated pigs exhibited FPSR in Brucella tests for a limited period of time, the serological responses in a YeOratio9-purified O-antigen indirect ELISA did not decrease accordingly. Analysis of available cross-sectional serum samples from pig herds naturally infected with YeOratio9 or B. suis biovar 2 confirmed that the observed difference in the duration of the serological responses between the two infections could be used to discriminate between herds infected with B. suis biovar 2 and YeOratio9.

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.

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.

Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotype 12 infection. The blocking ELISA showed no cross-reaction when tested with sera from pigs experimentally infected with 12 other serotypes of Ap biotype 1. The sensitivity and specificity of the blocking ELISA on the herd level was evaluated by testing sera from pig herds naturally infected with Ap serotypes 2 and/or 12 and from herds declared free of infection with Ap. The Ap serotype 12 blocking ELISA showed a herd sensitivity of 0.77 (95% confidence interval, 0.62-0.88) and a herd specificity of 1.00 (0.95-1.00) with a cut-off value at 40% relative absorbance or 60% inhibition. The assay may be used advantageously as a confirmatory test in serological surveillance programmes for Ap infections in SPF systems for pig production.

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89-1.00) and specificity (0.95; 0.88-0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.

Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the antigen contained high molecular weight lipopolysaccharide (LPS) and presumably also capsular polysaccharide (CP). The Ap serotype 5 ELISA was tested using sera from pigs experimentally infected with the 12 different Ap serotypes of biotype 1 and with sera from herds naturally infected with Ap serotypes 5, 6, 7 and 12. Cross-reactions were shown in one pig from a herd naturally infected with Ap serotype 7 and in one pig from a herd naturally infected with Ap serotype 12. The herd sensitivities of the Ap5 ELISA and a complement fixation test (CFT) were both estimated to 1.0, on the basis of serum samples from six herds naturally infected with Ap serotype 5. The herd specificities of both tests were estimated to 0.98, based on serum samples from 123 pig herds (10 samples from each herd) from the Danish specific pathogen-free (SPF) programme for pig production.

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).