Roberto M.C. Guedes

Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis

Little is known about the humoral and, especially, cell-mediated immune response in pigs exposed to Lawsonia intracellularis. The objectives of this study were to investigate the onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or a commercial live vaccine strain of L. intracellularis. Twenty-four 5-week-old pigs were exposed to 4.4x10(9) organisms of a pathogenic L. intracellularis isolate PHE/MN1-00 (10 pigs), a L. intracellularis live attenuated vaccine strain (10 pigs) or sham inoculum (4 pigs). Fecal, serum and whole blood samples were collected from all animals before exposure and weekly up to 13 weeks post inoculation and tested by PCR, immunoperoxidase monolayer assay serology and an interferon-gamma assay, respectively. One animal from each group was euthanized on day 22 post exposure to confirm infection. Humoral and cell-mediated immune responses were initially detected 2 weeks after exposure in pigs challenged with the pathogenic isolate, and 5 and 4 weeks, respectively, in pigs exposed to the modified-live vaccine group. Humoral and cell-mediated immune responses were still detected in some pigs from both L. intracellularis exposed groups 13 weeks after exposure. Fecal shedding was initially detected 1 week and lasted, intermittently, 12 weeks post exposure in pigs challenged with the pathogenic isolate, while fecal shedding was first detected 2 weeks and lasted, also intermittently, 9 weeks after exposure to the vaccine. In summary, both pathogenic isolate challenged and vaccine exposed pigs demonstrated long-term shedding of and immune responses to L. intracellularis.

Validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy

The sensitivity and specificity of an immunoperoxidase monolayer assay (IPMA) was evaluated in a blind serologic study of a group of disease-free pigs and a group of pigs experimentally infected with intestinal homogenate containing Lawsonia intracellulars organisms. Sixty pigs from the control group were kept in the source farm, and another 60 animals were transferred to an isolation unit and challenged intragastrically. All animals were bled before and 21 days after challenge. Fecal samples were collected on the same dates. The IPMA results were tested for sensitivity and specificity in a 2 × 2 table using the challenged and nonchallenged status as gold standard. Sensitivity and specificity were evaluated for different cutoff points (serum dilutions). Specificities of 100% were obtained for all the serum dilutions tested (1:15, 1:30, 1:60, and 1:120). The sensitivity levels were 90.7%, 88.9%, 81.5%, and 75.9% for the serum dilutions 1:15, 1:30, 1:60, and 1:120, respectively. The sensitivity of the dilution 1:15 was slightly, but not significantly, higher than the dilution currently used as the cutoff point (1:30). Cross-reactivity of the IPMA test was evaluated using sera from pigs experimentally inoculated with Brachyspira pilosicoli and various Campylobacter species. All these samples were negative. Sera samples from 3 porcine proliferative enteropathy known negative populations, 40 growing pigs from 2 commercial farms and a group of 6 cesarean-derived and colostrum-deprived pigs, also tested negative by IPMA. The IPMA serologic test with the cutoff point of 1:30 showed specificity of 100% and sensitivity close to 90% and, therefore, is an appropriate diagnostic test for herd screening but not for diagnosing PPE on the individual level.

Preparation and Characterization of Polyclonal and Monoclonal Antibodies against Lawsonia Intracellularis

Proliferative enteropathy is an intestinal infectious disease caused by the obligate intracellular bacterium Lawsonia intracellularis. Immunohistochemistry staining has superior sensitivity over hematoxylin and eosin and silver staining for detecting L. intracellularis in histological sections. A L. intracellularis—specific monoclonal antibody (MAb) produced in the UK (IG4 MAb) has been described in the literature. However, no monoclonal or polyclonal antibodies are commercially available. Therefore, the objective of this study was to produce and characterize new polyclonal and monoclonal antibodies against L. intracellularis that are suitable for diagnostic use. The new monoclonal (2001 MAb) and polyclonal antibodies (1999 PAb) were compared with the IG4 MAb using Western blot analysis of outer membrane proteins (OMPs) of 6 L. intracellularis isolates, Bilophila wadsworthia and Brachyspira hyodysenteriae and using immunohistochemistry of known positive and negative histologic samples and pure cultures of L. intracellularis, B. wadsworthia, B. hyodysenteriae, Salmonella choleraesuis, S. typhimurium, and Escherichia coli K88. Immunogold staining using 2001 MAb was performed to show the specificity of the antibody against an L. intracellularis surface protein. Western blot analysis showed that the 2001 MAb targeted an OMP of 77 kD, which made it different from the IG4 MAb that targeted an 18-kD OMP. The immunogold stain demonstrated the specificity of the 2001 MAb to a surface protein of L. intracellularis. The polyclonal antibody (1999 PAb) targeted 5 OMPs (77, 69, 54, 42, and 36 kD). Both the 2001 MAb and 1999 PAb stained known positive, but not negative, histologic samples. Both the 2001 MAb and 1999 PAb reacted with a pure culture of L. intracellularis but not with any other common enteric pathogens. These two new antibodies will be useful for immunodiagnosis of L. intracellularis.

Comparison of intestinal mucosa homogenate and pure culture of the homologous Lawsonia intracellularis isolate in reproducing proliferative enteropathy in swine

Little information is available on reproduction of proliferative enteropathy (PE) using a virulent pure culture of Lawsonia intracellularis. Reproduction of the disease using PE-diseased mucosa homogenates, however, is well-characterized. The aims of this study were to evaluate and compare clinical signs, growth performance and the severity of lesions in pigs inoculated with intestinal mucosa homogenate or pure culture of the homologous L. intracellularis isolate. Five-week-old pigs were inoculated with pure culture of L. intracellularis (isolate PHE/MN1-00; n=10), PE-diseased mucosa (n=10), or control media (n=4). The L. intracellularis isolate PHE/MN1-00 used in the pure culture inoculum was extracted from a fragment of the same intestine used to prepare the mucosa homogenate. Clinical signs and growth performance were evaluated throughout the study. Fecal shedding was evaluated in all animals weekly during the experiment. All animals were euthanized 22 days post-inoculation, the intestines were examined grossly and histologically. Results showed that both the infection procedures reproduced clinical disease, macroscopic and histologic lesions typical of PE. Fecal shedding was detected in animals in both challenge groups. In conclusion, the L. intracellularis isolate PHE/MN1-00 reproduces typical clinical signs and lesions of PE similar to the homologous infection with an intestinal mucosa homogenate.

A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy.

The currently used indirect fluorescent antibody test (IFAT) for the detection of antibodies against porcine proliferative enteropathy (PPE) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used in this comparison were collected from 5-week-old pigs on day 0 (pre-experimental challenge) and on days 7, 14, 21, and 28 after oral inoculation with intestinal homogenate from pigs affected by PPE (28 challenged pigs) and sucrose phosphate glutamate solution (2 control pigs). All animals were euthanized 4 weeks after inoculation. Immunohistochemistry staining was applied to formalin-fixed, paraffin-embedded sections of ileum for the detection of Lawsonia intracellularis antigen. The serology results with each method agreed in all samples, except on days 0 and 7 in 1 control animal, which was positive by IPMA, but negative by IFAT. The percentage of agreement between IFAT and IPMA was 98.6%.