Acute porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains a major clinical problem, particularly in European, Latin American, and Austral-Asian herds; subclinical infections are also common globally. There are 18 recognised APP serotypes, based on their capsule polysaccharide composition.^1,2 These serotype-specific capsule antigens are key factors in the host immune reaction, but without providing significant heterogenous cross-protection.¹ The prevalence of various APP serotypes varies globally, with serotypes 2 and 9 more common in Europe³ and serotypes 5, 7, and 12 more common in North America and Australia.^4,5 Isolates of serotypes 1, 5, and 9 are considered more pathogenic than others due to their greater expression of the tissue-destroying ApxI and ApxII exotoxins.⁶ However, infections with serotype 1 are now less common due to its eradication from many breeding company herds.⁴ Infections with APP serotype 5 and 9 are therefore considered of greatest current concern.

Serology is the preferred method for APP surveillance and detection of subclinical infections in pig herds, with established commercial assays available globally. Currently, there are three established antigen formats for APP serology tests. First, a serotype-specific test based on the individual serotype’s long-chain lipopolysaccharide (LPS) “O” antigen⁷ and second, a test based on the ApxIV exotoxin antigen,⁸ which is APP-specific but cannot differentiate between serotypes. The second test is therefore aimed at screening pigs for their APP status. A further screening test has also been established commercially in the first format, by using a pool of long-chain LPS antigens from various serotypes. A third type of test format based on the ApxI or ApxII toxin antigens has also been established,⁹ but is considered less specific for APP infection, and therefore less useful for herd screening. Previous studies have used these serologic tests for exploration of APP epidemiology, individual herd status, and response to vaccination.^10-12 Blood samples taken from late-stage finisher pigs (such as 16 to 20 weeks of age) are considered the most sensitive for detection of APP antibody responses representative of herd status.^4,10

The pathogenesis of APP consists of the separate stages of colonization, resistance to clearance and damage to lungs.¹³ Current vaccination programs for APP disease fall into different categories.¹⁴ With knowledge of the APP serotype status of the herd, relevant serotype-specific bacterins can be supplied commercially or prepared as an autogenous vaccination program. These aim to prevent colonization. Subunit vaccines based on the major Apx exotoxin antigens have also been commercially developed and utilised. These aim to prevent tissue damage and can provide protection across APP serotypes. Other vaccine strategies for APP have been developed, such as live attenuated vaccines, but are yet to find wide acceptance.¹⁴ One analysis suggested that 90% of global APP vaccination still occurs via the specific bacterin programs.¹⁵

In this study, we aimed to further characterize the serologic response for pig herds infected with major serotypes of APP and the response of pigs within a vaccination program for APP serotype 5.

Materials and methods

The Animal Ethics Care Committee of the State Government of Victoria approved the animal use and sampling protocols used in this study.

Case farms and APP status

Twelve separate pig finisher units A to L located across eastern Australia were selected for APP testing. These grower-finisher herds were characterised by their intake of 10-week-old grower pigs per week (Table 1). These herds were each derived from separate breeding/nursery herds either on the same site or under the same management system. The herds A to K had all suffered occasional outbreaks of clinical APP disease, but mortality in these herds was consistently below 2% in the intake to slaughter interval over the study period. All herds incorporated routine vaccination programs for Mycoplasma hyopneumoniae infection, but no APP vaccination. The herds had been free of clinical signs associated with porcine reproductive and respiratory syndrome virus and pathogenic porcine circovirus type 2 for 5 years preceding and throughout this study, as monitored by on-going necropsy, specific serology, and immunohistochemistry studies.

In addition to historical diagnostic results data, five fresh lung samples, some with noticeable lesions of pleuropneumonia, were collected from each farm either at on-farm necropsy or at scheduled slaughter (23 weeks of age). Bacteriologic culture, biochemical identification, and capsule serotyping for APP were performed on each lung sample using established methods ⁵ to confirm each herd’s APP status immediately prior to the study period.

Blood samples were then collected from at least 13 pigs from each farm at scheduled slaughter (Table 1). Lungs from each pig were also examined visually for pleurisy lesions. Serum derived from each blood sample was stored in aliquots at -20°C then thawed and subjected to 4 separate commercial APP serologic assays: 1) indirect ELISA based on recombinant ApxIV antigen (Idexx APP ApxIV ab test; Idexx Laboratories Inc); 2) indirect ELISA based on extract of long-chain LPS antigen for APP serotypes 5 (Swinecheck APP 5a, 5b; Biovet Inc), 4, or 7 (Swinecheck APP 4, 7; Biovet Inc); 3) indirect ELISA based on a mix set of long-chain LPS antigens derived from pools of APP serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15 (Swinecheck Mix APP 1-9-11, 2, 3-6-8-15, 4-7, 5, and 10-12; Biovet Inc).

The ELISA procedures were performed according to the manufacturer’s instructions and were similar to those described previously.^7,8 Briefly, the appropriate dilution of the LPS or ApxIV antigen was determined by checkerboard titration in microtiter plates using 0.5M carbonate buffer. Plates coated with each antigen (50 µL/well) were incubated overnight at 4°C. Plates were then washed with phosphate buffered saline-0.05% Tween 20 (PBST) and blocked with PBST-1% bovine serum albumin (BSA) for 1 hour at 20°C. After washing with PBST, serum samples (diluted 1:100 with PBST-1% BSA) were added; plates were then incubated for 30 minutes at 37°C. After washing with PBST, peroxidase-conjugated rabbit anti-swine IgG (1:10,000; Rockland Immunochemicals Inc) was added and allowed to react for 30 minutes at 37°C. The plates were washed twice with PBST and a chromogenic solution was allowed to react for 30 minutes at 30°C. The optical density at 490 nm was measured with 650 nm as the reference. The ELISA titer = (sample value absorbance − negative reference absorbance) ÷ (positive reference absorbance − negative reference absorbance). Typical absorbances of the negative and positive reference sera were 0.00 to 0.07 and 1.0, respectively. Each ELISA was performed in batches incorporating identical reagents.

Vaccination programs and monitoring   

Growing pigs selected for gilt development at 10 weeks of age (n = 164) were assembled and individually tagged on farm F (APP serotype 7 positive; Table 1) for premovement isolation and were later moved to farm K (APP serotype 5 positive; Table 1).

Seven groups, each with 20 to 30 pigs, within the farm F cohort of pigs were enrolled to assess serologic response to two serotype 5 bacterin vaccines and a commercial APP vaccine. The inoculation protocol for groups 1 to 6 (vaccinated) and group 7 (non-vaccinated controls) is outlined in Table 2. All 164 pigs remained in pens in one large barn enclosure, with ad libitum feed, water, and bedding. No antibiotics were administered to any pigs during the study period. While pigs remained healthy throughout, occasional pigs were removed during the study for non-study purposes.

For autogenous bacterin production, two APP serotype 5 cultures (V1 and V2) derived from farm K had been expanded and finally grown separately for 6 hours in 2 L culture vessels containing Tryptone yeast extract broth, with added nicotinamide adenine dinucleotide (10 µg/mL) and 5% vol/vol inactivated bovine serum. Each batch was then tested for potency (colony forming units/mL) and purity by cultures titrated onto routine aerobic and anaerobic plates. Each batch of pure culture was then inactivated with a final 0.2% vol/vol formalin, blended into the final vaccine strain and a commercial aluminum hydroxide gel adjuvant added at 500 µg/mL. Aliquots from each selected final batch were decanted into 100 mL bottles for use as specific autogenous bacterin vaccines in the assembled pigs.

A commercial, whole-cell, killed APP vaccine (V3; Porcilis APPvac; Intervet Co), stated to contain APP serotypes 1, 7, and 15 and commercial adjuvant, was purchased and used according to the manufacturer’s recommendations.

Pigs in each group were dosed intramuscularly behind the ear with 2 mL of V3 vaccine (> 5 × 10⁸ APP/mL) or either 1 mL (single dose) or 2 mL (double dose) of V1 or V2 vaccine (approximately 1 × 10⁹ APP/mL). Doses were given either twice or thrice at 3-week intervals, commencing at 10 weeks of age (Day 0 of the study period; Table 2).

Serologic evaluation of the vaccine study was conducted via response to APP serotype 5 LPS, and also to Apx toxins, as APP serotypes 1 and 5 are known to contain ApxI and II, whereas serotype 7 only contains ApxII.⁵ Blood samples were collected from each pig at 10, 13, 16, and 19 weeks of age (Days 0, 23, 42, and 64). Serum from each sample was stored in aliquots at -20°C and incorporated into: 1) the indirect ELISA based on extract of long-chain LPS for APP serotype 5, 2) the indirect ELISA based on ApxI toxin antigen, and 3) the indirect ELISA based on ApxII toxin antigen. The procedures for the ApxI and II ELISAs were performed according to methods described previously.^9,16 Recombinant Apx toxin antigens were kindly provided by Dr Han Sang Yoo, of Seoul National University. Briefly, the plates were coated with respective antigen at 4°C overnight. Preliminary checkerboard titration results indicated that the optimal concentration of recombinant ApxI and ApxII antigen was 625 and 100 ng/well, respectively. Plates were washed with PBST after antigen coating and blocked with 10% horse serum for 2 hours at 37°C. After washing with PBST, serum samples (diluted 1:100 with PBST-1% BSA) were added, incubated for 2 hours at 37°C, followed by washing and incubation with peroxidase-conjugated goat anti-pig IgG (Rockland Immunochemicals Inc) for 30 minutes at 37°C. The plates were washed twice with PBST and a chromogenic solution was allowed to react for 30 minutes at 30°C. The optical density at 405 nm was measured with 650 nm as the reference. The largest differences between positive and negative controls were found with anti-pig IgG at a 1:1000 dilution (ApxI) or a 1:2000 dilution (ApxII). Each ELISA titer = (sample value absorbance − negative reference absorbance) ÷ (positive reference absorbance − negative reference absorbance). Typical absorbances of the negative and positive reference sera were 0.05 to 0.1 and 0.1 to 0.15, respectively. Each ELISA was performed in batches incorporating identical reagents.

Differences between serum titers at each blood collection point for each group of vaccinate or control pigs were compared using an analysis of variance.

Results

Serologic analysis of APP herds

The results of lung culture for APP and identification of APP serotypes identified in pigs on farms A to K are shown in Table 1, with the on-farm presence of APP serotypes 5, 7, 12, and 15 identified. These herd designations confirmed historical diagnostic sample results (data not shown). Only one herd (D) had an apparent dual serotype infection with serotypes 7 and 15. One herd (L) was apparently free of APP infection during the study period.

The results of testing with four separate APP ELISAs of blood samples collected from slaughter pigs from farms A to L are shown in Table 1. The results of lung examinations for visible lesions of pleurisy in these sampled pigs are also shown in Table 1. A greater proportion of sampled pigs with pleurisy was noted in herds positive for APP serotype 5 compared to those infected with other serotypes.

Both the ApxIV ELISA and the APP mix-LPS ELISA regularly detected all apparent herd infections with 5, 7, and 15 serotypes, albeit with a variable ratio of 14% to 100% of blood samples analyzed. However, while the 2 farms identified as having APP serotype 12 infections were detected in the mix-LPS ELISA screen, these reactions were not detected in the ApxIV screen assays performed on the same sets of sera (Table 1). The ELISA employing LPS antigen specific to serotypes 5 or 7 regularly detected herd infections with the relevant serotype, albeit with a ratio of 15% to 100% of blood samples analyzed. Occasional single cross-reactions were detected with these assays in samples taken from pigs in uninfected herds or those herds infected with other serotypes (Table 1).

Vaccination monitoring

A summary of the results of ELISA testing for APP serotype 5 status and Apx toxin antibody status of each group of vaccinated pigs and control pigs from day 0 through day 64 is shown in Figures 1 and 2, respectively. The specific LPS-antigen titers of pigs vaccinated three times with autogenous APP serotype 5 were noticeably higher at day 64 than those dosed only twice with the equivalent 1 mL or 2 mL dose sizes (Figure 1). The analysis of variance indicated a significant difference at day 64 between the groups vaccinated three times with either autogenous serotype 5 strain V1 or V2 (groups 3, 5, and 6) and their titers at day 0 and the control group at day 64 (P < .05). The analyses of other vaccinated groups at day 64 and of all groups at other blood collection points indicated no significant difference in the ELISA titers detected from those at day 0 or from the control group.

The ApxI toxin antibody analysis indicated a noticeable anamnestic response within each group of the serotype 5 bacterin vaccine program (Figure 2; groups 2-6). The ApxII toxin antibody analysis indicated a limited response to all bacterin vaccines (Figure 2). Following subsequent movement of pigs to farm K, APP was not detected clinically or at post-mortem examinations of exposed vaccinates for 6 months.

Discussion

The current diagnosis of individual and herd status for APP is established via culture of lungs and serotyping of APP cultures and ELISA serology. Our study confirms the generally good diagnostic relationship between pleurisy lesions, lung cultures, and current ELISA serology techniques for a range of on-farm APP serotype infections. The long-chain LPS antigen ELISA methodology allowed accurate identification of herd status in all 12 farms examined. Analysis of sufficient samples is indicated to account for occasional cross-reactions. We established that the ApxIV antigen ELISA methodology also accurately predicted herd status, except for the two herds known to be infected with APP serotype 12. In contrast, a previous herd study¹² found some false positives with the ApxIV assay (case No. 7). Use of both the LPS and ApxIV antigen assays may be required to fully clarify the status of herds with APP serotype 12. While some APP serology studies have failed to accurately determine herd status with limited sample numbers,¹⁰ other studies found that the combined use of LPS and ApxIV assays on sufficient sample sets was associated with accurate investigations of herd status.^4,12

The pathogenesis of APP consists of the three separate stages of colonization, resistance to clearance, and damage to lungs.¹³ Although all serotypes of APP are considered pathogenic, the lung examinations for pleurisy lesions supplemented the diagnostic information from culture and ELISA results confirming the greater extent of lesions likely to be seen with APP serotype 5 infections. This is considered to be due to greater expression of the Apx exotoxins, particularly ApxI and ApxII.⁶ While the pathogenicity of APP in terms of tissue damage varies according to serotype and Apx content, the number of animals infected within any particular herd may also vary according to serotype. Our study confirms that serologic monitoring of APP herd status is best achieved via approximately 30 samples, of which 3 or more clear positives leads to an accurate indication of APP status, as also suggested previously.⁴

Prevention or eradication of APP infection across serotypes has remained elusive, with novel strategies, such as live attenuated vaccines or outer membrane vesicle subunit toxoids, found to be ineffective.¹⁴ Commercial subunit vaccines based on the Apx toxoids are effective at reducing the tissue damage phase, but do not appear to prevent the initial colonization stage. Their use is therefore considered ineffective at controlling initial APP infections leading to the possible presence of carrier pigs among infected herds.^14,17 In our study, serologic evaluation of bacterin vaccines was conducted via response to APP serotype 5 LPS and Apx toxins, as APP serotypes 1 and 5 are known to contain ApxI and II, whereas serotype 7 only contains ApxII.⁶ We confirmed that noticeable antibody production to the ApxI and II toxins occurred in vaccinated pigs, particularly those given APP serotype 5 bacterin. In our study and others,⁴ the interpretation of Apx ELISAs was considered more problematic due to less certain cut-off values and its limited availability. Although our results indicated that repeated dosage of APP serotype-specific vaccines can be monitored successfully by LPS ELISA serology, these assays based on APP LPS antigen are aimed at detection of infection status rather than vaccine responses. It is possible that vaccinated pigs may develop reactions to other protective antigens, unrelated to any detectable LPS response.

The long-standing use of serotype-specific bacterin vaccines remains the most popular form of APP vaccination, despite the considerable time and effort required for their autogenous preparation. It is possible that the use of bacterin vaccines may confer both some protection against colonization and some protection against Apx related tissue damage. Our results indicated that repeated dosage of APP serotype-specific vaccines can be monitored successfully by LPS ELISA serology, with an anamnestic response to LPS antigen (note titers at days 0, 42, and 64 presented in Figure 1) similar to vaccinated pigs in previous studies of the administration of APP serotype 5 bacterin vaccines.¹⁸ It is possible that the preparation and use of whole-cell, unwashed bacterial material for production of the autogenous vaccines in our study may have conferred some protective advantage in comparison to other vaccine substrates. These previous studies also confirmed that repeated doses of APP bacterins are required for a measurable response and that there is little difference in the measured LPS antigen titer response to pigs given either 1 mL or 2 mL doses.¹⁸ In our study, we found that a greater response occurred with three doses of APP serotype 5 bacterin compared to only two doses. Whether this may be useful for all APP bacterins, or merely this example of pathogenic APP serotype 5 infection, is not clear.

Implications

Under the conditions of this study:

• Commercial and serotype-specific ELISAs were used to identify herd APP status.
• Repeated autogenous APP serotype 5 vaccine doses provided strong antibody responses.

Acknowledgments

We thank the diligent farm and slaughterhouse managers for assistance with sample collections. We thank the staff of ACE laboratories (Bendigo East, Australia) for assistance in performing some of the culture and ELISAs. We thank the Pork Research Council of Australia for providing financial assistance for the study.

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

References

1. Dubreuil JD, Jacques M, Mittal KR, Gottschalk M. Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity. Anim Health Res Rev. 2000;1:73-93.

2. Bossé JT, Li Y, Sárközi R, Fodor L, Lacouture S, Gottschalk M, Amoribieta MC, Angene Ø, Nedbalcova K, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR, BRaDP1T consortium. Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis. Vet Microbiol. 2018;217:1-6.

3. Šatrán P, Nedbalcová K. Prevalence of serotypes, production of Apx toxins, and antibiotic resistance in strains of Actinobacillus pleuropneumoniae isolated in the Czech Republic. Vet Med (Praha). 2002;47:92-98.

*4. Gottschalk M. Detection/interpretation of Actinobacillus pleuropneumoniae antibodies. Proc ISU Swine Disease Conf . 2012;20:125-132.

5. Blackall PJ, Bowles R, Pahoff JL, Smith BN. Serological characterisation of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996. Aust Vet J. 1999;77:39-43.

6. Beck M, van den Bosch JF, Jongenelen IMCA, Loeffen PLW, Nielsen R, Nicolet J, Frey J. RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains. J Clin Microbiol. 1994;32:2749-2754.

7. Gottschalk M, Altman E, Charland N, De Lasalle F, Dubreuil JD. Evaluation of a saline boiled extract, capsular polysaccharides and long-chain lipopolysaccharides of Actinobacillus pleuropneumoniae serotype 1 as antigens for the serodiagnosis of swine pleuropneumonia. Vet Microbiol. 1994;42:91-104.

8. Dreyfus A, Schaller A, Nivollet S, Segers RPAM, Kobisch M, Mieli L, Soerensen V, Hüssy D, Miserez R, Zimmermann W, Inderbitzin F, Frey J. Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA. Vet Microbiol. 2004;99:227-238.

9. Shin M, Kang ML, Cha SB, Lee W, Sung JH, Yoo HS. An immunosorbent assay based on the recombinant ApxIa, ApxIIa, and ApxIIIa toxins of Actinobacillus pleuropneumoniae and its application to field sera. J Vet Diagn Invest. 2011;23:736-742.

10. Opriessnig T, Hemann M, Johnson JK, Heinen S, Giménez-Lirola LG, O’Neill KC, Hoang H, Yoon K, Gottschalk M, Halbur PG. Evaluation of diagnostic assays for the serological detection of Actinobacillus pleuropneumoniae on samples of known or unknown exposure. J Vet Diagn Invest. 2013;25:61-71.

11. Jung M, Won H, Shin M, Oh MW, Shim S, Yoon I, Yoo HS. Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency. J Vet Sci. 2019;20(2):e2. doi:10.4142/jvs.2019.20.e2

12. Broes A, Martineau GP, Gottschalk M. Dealing with unexpected Actinobacillus pleuropneumoniae serological results. J Swine Health Prod. 2007;15:264-269.

13. Bossé JT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, Kroll JS, Langford PR. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect. 2002;4:225-235.

14. Antenucci F, Fougeroux C, Deeney A, Ørskov C, Rycroft A, Holst PJ, Bojesen AM. In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae. Vet Res. 2018;49:4. doi:10.1186/s13567-017-0502-x

15. Gottschalk M. Actinobacillosis. In: Zimmerman JJ, Karriker L, Ramirez A, Schwartz KJ, Stevenson GW, eds. Diseases of Swine. 10^th ed. Ames, IA: Wiley-Blackwell; 2012:653-669.

16. Teshima K, Lee J, To H, Kamada T, Tazumi A, Hirano H, Maruyama M, Ogawa T, Nagai S, Turni C, Tsutsumi N. Application of an enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15 in pig sera. J Vet Med Sci. 2017;79:1968-1972.

17. Sjölund M, Wallgren P. Field experience with two different vaccination strategies aiming to control infections with Actinobacillus pleuropneumoniae in a fattening pig herd. Acta Vet Scand. 2010;52:23. doi:10.1186/1751-0147-52-23

18. Rousseau P, Assaf R, Boulay G, Désy M. Immune response to an Actinobacillus pleuropneumoniae vaccine in swine. Can Vet J. 1988;29:989-992.

* Non-refereed reference.