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.

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.

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%.

Clostridium difficile has emerged as a major cause of neonatal colitis in piglets, displacing classic bacterial pathogens. However, there is no information regarding the distribution of this microorganism in pig farms in Brazil. In the present study, the presence of toxins A/B and of C. difficile strains in stool samples from 60 diarrheic or non-diarrheic newborn piglets (one to seven days old), from 15 different farms, was studied. The presence of toxins A/B was detected by ELISA and PCR was used to identify toxin A, toxin B and binary toxin gene in each isolated strain. C. difficile A/B toxins were detected in ten samples (16.7%). Of these, seven were from diarrheic and three were from non-diarrheic piglets. C. difficile was recovered from 12 out of 60 (20%) fecal samples. Of those, three strains were non-toxigenic (A-B-) and nine were toxigenic. Of the nine toxigenic strains, four were A+B+ strains and five were A-B+ strains. The presence of binary toxin observed in the present study was much higher (50%) than in previously reported studies. All three non-toxigenic strains were isolated from otherwise healthy piglets. The results suggest the occurrence of neonatal diarrhea by C. difficile in farms in Brazil.