Material and methods. Challenge The pigs were challenged at 16 weeks of age when the challenge control pigs became antibody negative by the

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1 Evaluation of the efficacy of Mycoplasma hyopneumoniae bacterin following immunization of young pigs in the presence of varying levels of maternal antibodies H. Jayappa * MVSc, PhD, R. Davis BS, V. Rapp-Gabrielson PhD and T. Wasmoen PhD, Schering-Plough Animal Health Corporation, Elkhorn, NE E. Thacker DVM, PhD and B. Thacker, DVM, PhD, Iowa State University, Ames, IA Mycoplasma hyopneumoniae causes enzootic pneumonia that primarily affects growing pigs. Although the disease is nonfatal, it causes substantial losses attributable to reduced growth performance, increased susceptibility to secondary bacterial pneumonia, and potentiation of PRRSV-induced pneumonia (1, 2). Both cell-mediated and humoral immune responses appear to be important in providing protection against infection (3). The Tween 20 ELISA and monoclonal antibody blocking ELISA (Commercial DAKO test) have been used for determining antibody response to M. hyopneumoniae (4, 5). Schering-Plough Animal Health Corp. (SPAH) has also developed a competitive ELISA (CE) using rabbit polyclonal serum to measure the M. hyopneumoniae antibody response following vaccination of pigs. A previous study in our laboratory indicated that 4- and 6-week-old antibody positive pigs born to vaccinated sows were significantly protected against lung consolidation following experimental M. hyopneumoniae challenge (6). In that study, conducted in pigs raised in a commercial setting, secondary pathogens contributed to the lesions observed. The purpose of this current study was to expand upon the previous study and to evaluate the relationship between the level of maternally derived antibodies and potential interference with active immunization of young pigs. An additional objective of this study was to compare the antibody response as measured by the DAKO test, the Tween 20 ELISA and SPAH s competitive ELISA (SPAH-CE). Material and methods Animals Pigs were obtained from a large multi-site, all-in all-out, swine production system. The grow-finish units and source of replacement gilts for this system are naturally infected with M.hyopneumoniae. The sows and gilts were vaccinated with a commercial M. hyopneumoniae bacterin a few weeks prior to farrowing. A total of 79 piglets were transported to isolation facilities at weaning (15-17 days of age). Vaccination A routine production serial of Mycoplasma Hyopneumoiae Bacterin (M+ Pac was used to vaccinate the young pigs. The pigs were divided into 5 groups and vaccinated as indicated in Table 1 with 1 ml of M+Pac given intramuscularly. All pigs were re-vaccinated 2 weeks after the initial vaccination. Blood sample collection and antibody determination Blood samples were collected at the time of initial vaccination and at various time points following vaccination and challenge. The serum samples were evaluated for the presence of antibodies by the monoclonal antibody based commercial test (DAKO A/S Glostrup, Denmark) and the competitive ELISA (CE) at SPAH, Elkhorn, NE. The Tween 20 ELISA antibody testing was performed at the Veterinary Medical Research Institute, Iowa State University. The DAKO test is a blocking ELISA and the values are expressed as a percentage of the buffer control; thus, a low percentage indicates a high level of antibodies in the serum. The Tween 20 levels are expressed as an S/P ratio where a value > 0.60 is positive. Suspect sera (50 65% in the DAKO or in the Tween 20 ELISA) were assumed to be negative. The SPAH-CE titers are expressed as the reciprocal of the highest serum dilution showing a value greater than background established by a positive control serum of known titer. Challenge The pigs were challenged at 16 weeks of age when the challenge control pigs became antibody negative by the 237

2 DAKO test. A virulent, heterologous M. hyopneumoniae challenge culture (a derivative of Strain 11) was used. The challenge culture was stored at 50 C or colder until immediately prior to challenge. The pigs were anesthetized before challenge and challenged intratracheally with 20 ml of challenge inoculum containing approximately 10 5 Color Changing Units /ml. The pigs were euthanized 5 weeks following challenge and lungs were examined grossly for consolidation typical of M. hyopneumoniae. The percent lung consolidation was evaluated as follows: % Consolidation = A (0.04) + B (0.09) + C (0.25) + D (0.07) + E (0.15) + F (0.35) + G (0.05) Where A, B and C are the left apical, cardiac and diaphragmatic lobes and D, E, and F are the right apical cardiac and diaphragmatic lobes, respectively. G represents intermediate lobe. Statistical analysis Percent lung consolidation scores were transformed using arcsine-square root transformation and analyzed by linear models procedures. Results Lung consolidation The nonvaccinated, challenged pigs had a mean lung consolidation score of 5.0% at necropsy (Figure 1). This low percent consolidation is typical in experimental M. hyopneumoniae challenges that are not complicated by the presence of secondary bacterial or viral pathogens. The lung scores in pigs vaccinated at 1 week and 3 weeks of age (3.2 and 2.8, respectively) were not significantly lower than the score of the nonvaccinated controls. However, pigs vaccinated at 6 weeks of age were significantly protected from challenge (0.9% consolidation; P = ). None of the non-challenged pigs showed any lung consolidation. Antibody determinations The high antibody levels in these pigs were likely a result of both natural exposure of the replacement gilts in the grow-finish units and the standard practice of vaccinating all sows and gilts a few weeks prior to each farrowing. Only at 15 weeks of age did 100% of the nonvaccinated pigs become serologically negative by the DAKO test (Tables 2 and 3). At both 1-week and 3-weeks of age, nearly 100% of the pigs were serologically positive with 238 very high maternal antibody levels apparent in all three serological tests. Pigs vaccinated at 1 week and 3 week of age did not appear to respond to vaccination serologically since neither the magnitude of the antibody response or the percentage of positive pigs returned to the prevaccination levels. The group of 6-week-old pigs still had high antibody titers at the time of first vaccination (82.4% of pigs positive; Tween 20 mean S/P ratio of 1.42; and DAKO mean 26.8% of control). After vaccination, both the antibody titers and the percentage of positive pigs was higher than in Groups 1 and 2, indicating that the pigs did respond to the vaccine. Following challenge, nearly 100% of the pigs in all groups became serologically positive by all three tests. Discussion The extremely high levels of maternal antibody seen in these young pigs were likely a result of routine prefarrowing vaccination of previously exposed sows. In the groups with Tween 20 S/P ratios greater than 2.0 (and DAKO values less than 10% of the control) at the time of vaccination, interference did affect active immunization of young pigs. However, Group 3 pigs were successfully vaccinated in the face of somewhat lower antibody levels (84.2% of pigs positive; a mean Tween 20 S/P ratio of 1.4 and a mean DAKO level of 26.8%). In a previous study, maternal antibodies did not interfere with vaccination of 4 or 6 week old pigs, but the antibody levels in these pigs were lower than those seen in the current study (6). The results of both studies are summarized in Table 4. The data indicate that the antibody level is more important than the age of pig when evaluating whether or not maternal antibodies might interfere with active immunization. If pre-farrowing vaccination is a routine practice, vaccination of piglets should be delayed until maternal antibody titers drop to a level where they will not interfere. However, unlike the situation with some other vaccines, pigs do not have to be maternal antibody negative for M. hyopneumoniae vaccination to be successful. When evaluating pigs for the presence of maternal antibodies and for antibodies following challenge, there is good correlation between the DAKO test, Tween 20 ELISA and SPAH competitive ELISA.

3 References 1.Thacker E.L., P. G. Halber, R.F. Ross, R. Thanawongnuwech, B. J. Thacker Mycoplasma hyopneumoniae potentiataion of porcine reproductive and respiratory syndrome virus-induced pneumonia (37) J.Clin Microbiol Amass, S.F., L. K. Clark, W. G. Van Alstine Interaction of Mycoplasma hyopneumoniae and Pasteurella multocida infections in swine. JAVMA (204) Thacker E, B. Thacker, T.B. Boettcher, H. Jayappa Comparison of antibody production, lymphocyte stimulation, and protection induced by four commercial Mycoplasma hyopneumoniae bacterins. Swine Health and Production (6), Bereiter M. T.F. Young, H.S. Joo, R. F. Ross Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum Vet Microbiol (25) Fred N.C., P. Qvist, P. Ahrens, N.F. Friis, A. Meyling A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae Vet Microbiology (30) Wasmoen T, V. Rapp-Gabrielson, R. Davis, H. Jayappa Proceedings of the 81 st Conference of Research Workers in Animal Disease, Chicago, IL, Abstract No. 111 Table 1: Treatment group assignments Table 2: Antibody levels as measured by the Tween 20, DAKO, and SPAH-Competitive ELISA tests. 1 Tween 20 antibody titers expressed as S/P ratios (> 0.60 is positive) DAKO titers expressed as % of control (< 50% is positive) SPAH Competitive ELISA expressed as geometric mean of endpoint titers (>8 is positive) 2 1 week pre-challenge not done 239

4 Table 3: Percentage of pigs positive by the Tween 20, DAKO, and SPAH-Competitive ELISA tests. 1 Tween 20 antibody titer >0.60 is positive DAKO % of control < 50% is positive SPAH Competitive ELISA titer >8 is positive 2 1 week pre-challenge not done Table 4: Relationship of maternal antibody levels and interference with vaccination 1-- = not done. 240

5 Figure1: Efficacy Following Vaccination At 1,3 and 6 Weeks of Age (Mean Lung Consolidation) 241