Effect of vaccination of pigs against experimental infection with high and low virulence Mycoplasma hyopneumoniae strains

This study investigated the infection pattern and lung lesion development in pigs caused by a low and highly virulent Mycoplasma hyopneumoniae strain at 4 and 8 weeks (w) post infection (PI). It also determined the efficacy of a commercial inactivated whole-cell vaccine against infection with each one of these M. hyopneumoniae strains. Ninety piglets free of M. hyopneumoniae were selected, and 40 of them were randomly vaccinated during their first week of life. At weaning, all piglets were allocated to 10 different groups and housed in pens with absolute filters. At 4 weeks of age, pigs were inoculated intratracheally with either a highly virulent M. hyopneumoniae strain, a low virulent strain or with sterile culture medium. Half of all animals were euthanized at 4w PI, while the remaining half was euthanized at 8w PI. Coughing was assessed daily, and lung lesions, immunofluorescence (IF), bacteriological analysis and nested PCR were assessed after necropsy. It was demonstrated that contrary to the highly virulent strain, the low virulent strain required more than 4 weeks PI (commonly accepted as the standard infection model) to reach maximum clinical symptoms. Vaccination significantly reduced clinical symptoms, macroscopic and microscopic lung lesions in pigs infected with the highly virulent strain. This effect was more pronounced at 4 than at 8 weeks PI. Protective efficacy was also observed in pigs infected with the low virulent strain, but the effect was less pronounced than on the highly virulent strain.     

Effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia by Mycoplasma hyopneumoniae

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae are frequently isolated pathogens from pigs with respiratory disease. A previous study conducted in our laboratory found that infection with M. hyopneumoniae increased the duration and severity of respiratory disease induced by PRRSV. The purpose of this experiment was to determine whether vaccination against M. hyopneumoniae and/or PRRSV decreased the enhancement of PRRSV-induced pneumonia. Both M. hyopneumoniae bacterin and PRRSV vaccine decreased the severity of clinical respiratory disease. Infection or vaccination with PRRSV appeared to decrease the efficacy of the M. hyopneumoniae bacterin. Vaccination with M. hyopneumoniae bacterin decreased the potentiation of PRRSV-induced pneumonia observed in the dual infected pigs. However, PRRSV vaccination in combination with M. hyopneumoniae bacterin eliminated this benefit and the amount of pneumonia induced by PRRSV increased. PRRSV vaccine alone did not decrease the potentiation of PRRSV pneumonia by M. hyopneumoniae.     

Co-administration of attenuated Mycoplasma hyopneumoniae 168 strain with bacterial DNA enhances the local and systemic immune response after intranasal vaccination in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. M. hyopneumoniae infects pigs at mucosal surfaces of respiratory tract. The aim of the present study was to investigate if the protection rate against M. hyopneumoniae infection following intranasal immunization with attenuated M. hyopneumoniae 168 strain is improved by administration of bacterial DNA containing CpG motifs. Thirty pigs were immunized intranasally or intramuscularly and the levels of local respiratory tract and systemic immune responses were detected. The results showed that the number of intraepithelial lymphocytes in the tracheal fork, the levels of cytokine IL-6, and M. hyopneumoniae specific SIgA in local nasal cavity increased respectively after intranasal vaccination with the attenuated M. hyopneumoniae 168 strain alone. However, the levels of IL-10 and IFN-γ in local nasal cavity, the number of intraepithelial lymphocytes in trachea, CD4(+) and CD8(+) T lymphocytes in the lung and hilar lymph nodes, the specific IgG antibody level in serum on 35 day post immunization were all increased significantly after intranasal vaccination of the attenuated M. hyopneumoniae 168 strain adjuvanted with bacterial DNA. We concluded that intranasal administration of attenuated M. hyopneumoniae 168 strain adjuvanted with bacterial DNA may be effective in evoking the local cellular and humoral immune response in the respiratory tract and the systemic immune response. Intranasal vaccination will be effective in prevention of the transmission and prevalence of MPS.     

Effect of multiple vaccinations on transmission and degree of Mycoplasma hyopneumoniae infection in gilts

Mycoplasma hyopneumoniae (M. hyopneumoniae) infections continue to result in significant respiratory challenges in the swine industry worldwide. Vaccination for M. hyopneumoniae is commonly utilized, as reduction in bacterial loads and clinical severity in vaccinated pigs have been shown. However, the effect of M. hyopneumoniae vaccination on transmission across different pig populations has been minimally investigated. The aim of this pilot study was to evaluate the effect of multiple vaccinations on M. hyopneumoniae infection, transmission, and genetic variability in infected and susceptible gilt populations. Thirty-two naïve gilts were allocated to four treatment groups: (1) Vaccinated seeder (VS); (2) Non-vaccinated seeder (NVS); (3) Vaccinated contact (VC); and (4) Non-vaccinated contact (NVC). At 5, 7, and 9 weeks of age, all gilts selected to be vaccinated received a commercial M. hyopneumoniae bacterin for a total of 3 doses. At 11 weeks of age, VS and NVS gilts were inoculated with M. hyopneumoniae to become seeders. At 28 days post-inoculation (dpi), VS and NVS gilts were individually relocated to clean experimental rooms, where they were placed in contact with one age-matched VC or NVC gilt (1:1 ratio) for 14 days. Blood and tracheal samples, bronchial swabs, and lung lesions were collected and/or evaluated for M. hyopneumoniae infection. In this study, a three-dose vaccination strategy against M. hyopneumoniae significantly reduced bacterial load in seeder gilts. Furthermore, a numerical reduction in M. hyopneumoniae lung lesions at 28 dpi was observed in VS gilts. All VC gilts in the VS:VC treatment group pairing remained M. hyopneumoniae negative, compared to other groups in which 1–2 transmission events occurred per treatment group. Results from this investigation provide insight on the potential impact of multiple vaccinations on reducing M. hyopneumoniae transmission and infection. Further research encompassing vaccinations of gilt groups in field settings is necessary to validate findings.     

Trends in slaughter pig production and antimicrobial consumption in Danish slaughter pig herds, 2002-2008

Overuse of antimicrobials in food-animal production is thought to be a major risk factor for the development of resistant bacterial populations. Data on non-human antimicrobial usage is essential for planning of intervention strategies to lower resistance levels at the country, region or herd levels. In this study we evaluated Danish national antimicrobial usage data for five antimicrobial classes used in slaughter pigs in different herd sizes and data on the number of slaughter pigs produced per herd, between 2002 and 2008, in Denmark. The objective was to ascertain if there is an association between herd size and amount of antimicrobials consumed. During this period, the overall number of herds with slaughter pigs decreased by 43%, with larger herds becoming more prevalent. The tetracycline treatment incidence (TI) rate increased from 0·28 to 0·70 animal-defined daily dose (ADD)/100 slaughter pig-days at risk while macrolide TI presented a more moderate increase, from 0·40 to 0·44 ADD/100 slaughter pig-days at risk during the study period. Linear regression analyses revealed a significant association between herd size and TI rates for tetracyclines, macrolides, sulfonamides/trimethoprim and cephalosporins, with small herds presenting significantly higher TI than moderate, large and the largest herds. This study highlights the importance of establishing an antimicrobial consumption monitoring programme, integrated with comprehensive food-animal production surveillance. Further research should be performed to address the potential causes of the detected associations between herd sizes and antimicrobial consumption in pigs.     

Phenotypic characteristics and protective efficacy of an attenuated Mycoplasma hyopneumoniae vaccine by aerosol administration

With the improvement of large-scale breeding in pig farms, conventional head-by-head immunization has disadvantages with low efficiency and high cost. Considering that most pathogens leading to pulmonary diseases circulate from the respiratory mucosa, immunization through the respiratory tract route has been a highly attractive vaccine delivery strategy. In this study, to develop an effective Mycoplasma hyopneumoniae (Mhp) aerosol vaccine, a customized ultrasonic atomizer was developed. The aerodynamic diameter, activity, and content of the Mhp aerosol vaccine were measured. In addition, piglets were immunized with the Mhp aerosol vaccine, and the immunity of the animal challenge protection test was evaluated. At the end of nebulization, the mass median aerodynamic diameters (MMAD) and geometric standard deviation (GSD) of the aerosol were 2.98 ± 0.02 μm and 1.51 ± 0.02, respectively. Moreover, 10 min after nebulization, the MMAD and GSD of the aerosol were 2.76 ± 0.02 μm and 1.51 ± 0.01, respectively, which were hardly changed. Compared with theoretical value, the actual titer of aerosol vaccines presented in 50% color changing unit (CCU₅₀) after nebulization decreased 0.6. The shape, size, and uniformity of collected aerosols are relatively stable. The proportion of Mhp in aerosol produced by vaccine stock solution and 10 times diluted vaccine solution was 76.52% and 58.82%, respectively, and the average number of Mhp in a single aerosol was 3.06 and 1.51, respectively. In addition, the aerosol vaccine antigen particles could be transported to the lower respiratory tract, a local mucosal immune response was induced in piglets. The vaccine colonized the respiratory tract and significantly decline the lung lesion index after aerosol vaccination. In conclusion, an effective aerosol vaccine against Mhp infection was developed. And this is the first effective assessment for Mhp live vaccine with aerosolization against infection in piglets.     

Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations

A transmission experiment was performed to quantify the effect of vaccination on the transmission of Mycoplasma hyopneumoniae (M. hyopneumoniae) in nursery piglets by means of an adjusted reproduction ratio (R(n)). Thirty piglets, vaccinated at 1 week of age, and 30 non-vaccinated piglets, free of M. hyopneumoniae, were housed in six separate pens. In each pen, three animals that were intratracheally inoculated with M. hyopneumoniae, were housed together with seven contact piglets during the conventional nursery period of 6 weeks. At the end of the study, the infectious status of the animals was determined based on results of nPCR performed on bronchoalveolar lavage fluid. The R(n)-value in the vaccinated group was 2.38 (1.07-7.53) while in the non-vaccinated group, an R(n)-value of 3.51 (1.51-9.34) was observed, both not significantly different from each other (p=0.77). Under the actual experimental conditions, transmission of M. hyopneumoniae in nursery piglets was only numerically lower in vaccinated groups. In addition, vaccination with a conventional vaccine could not prevent the establishment of M. hyopneumoniae organisms in the lung.     

Oral vaccination with an adenovirus-vectored vaccine protects against botulism

We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (H_C50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1 × 10⁴ to 1 × 10⁷ plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2 × 10⁷ pfu adenovirus, have shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2 × 10⁶ pfu adenovirus or greater were completely protected against challenge with 100 × MLD₅₀ of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism.     

Effects of Mycoplasma hyopneumoniae vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus

The effects of a single-dose Mycoplasma hyopneumoniae vaccine was studied in growing pigs. Each of 24 vaccinated cohorts of approximately 1200 pigs reared in separate barns was matched by time, farm site, and sex with unvaccinated cohorts. Pigs were naturally exposed to M. hyopneumoniae and porcine reproductive respiratory syndrome virus (PRRSv). Daily weight gain was 42 g per pig per day higher and mortality rate was 15.2/1000 pigs lower for vaccinated cohorts. Age at PRRSv onset was associated with mortality rate, but did not modify vaccine effects. M. hyopneumoniae vaccination was effective in promoting growth in spite of concurrent PRRSv infection.     

Characterization of whole blood transcriptome and early-life fecal microbiota in high and low responder pigs before,and after vaccination for Mycoplasma hyopneumoniae

We investigated gene expression patterns in whole blood and fecal microbiota profile as potential predictors of immune response to vaccination, using healthy M. hyopneumoniae infection free piglets (n?=?120). Eighty piglets received a dose of prophylactic antibiotics during the first two days of life, whereas the remaining 40 did not. Blood samples for RNA-Seq analysis were collected on experimental Day 0 (D0; 28?days of age) just prior to vaccination, D2, and D6 post-vaccination. A booster vaccine was given at D24. Fecal samples for microbial 16SrRNA sequencing were collected at 7?days of age, and at D0 and D35 post-vaccination. Pigs were ranked based on the levels of M. hyopneumoniae-specific antibodies in serum samples collected at D35, and groups of ‘high’ (HR) and ‘low’ (LR) responder pigs (n?=?15 each) were selected. Prophylactic antibiotics did not influence antibody titer levels and differential expression analysis did not reveal differences between HR and LR at any time-point (FDR?>?0.05); however, based on functional annotation with Ingenuity Pathway Analysis, D2 post-vaccination, HR pigs were enriched for biological terms relating to increased activation of immune cells. In contrast, the immune activation decreased in HR, 6?days post-vaccination. No significant differences were observed prior to vaccination (D0). Two days post-vaccination, multivariate analysis revealed that ADAM8, PROSER3, B4GALNT1, MAP7D1, SPP1, HTRA4, and ENO3 genes were the most promising potential biomarkers. At D0, OTUs annotated to Prevotella, CF21, Bacteroidales and S24-7 were more abundant in HR, whereas Fibrobacter, Paraprevotella, Anaerovibrio, [Prevotella], YRC22, and Helicobacter positively correlated with the antibody titer as well as MYL1, SPP1, and ENO3 genes. Our study integrates gene differential expression and gut microbiota to predict vaccine response in pigs. The results indicate that post-vaccination gene-expression and early-life gut microbiota profile could potentially predict vaccine response in pigs, and inform a direction for future research.     

Herd factors associated with the serological Yersinia prevalence in fattening pig herds

Recent epidemiological evidence has demonstrated that pork is an important source of yersiniosis in humans. Identifying risk factors and potential interventions in swine production that may decrease the risk of pork production contamination during harvest and processing is an important step before controlling Yersinia spp. Therefore, management strategies and production processes that might be associated with fattening pigs testing seropositive for pathogenic Yersinia spp. were investigated in 80 fattening pig farms. Although >70 farm characteristics were included in the risk assessment, there were only a few that seemed to be connected with serological prevalence: housing on a fully slatted floor and the use of municipal water were observed more often in herds with low serological Yersinia prevalence, whereas recurring health problems and a low daily weight gain compared with the mean of the herds included in the study were found in herds with a high prevalence. Besides, the Yersinia prevalence seemed to be inversely proportional to the herds' serological Salmonella status collected in accordance with German legislation. Additionally, the development of the serological Yersinia status of selected herds was assessed over a period of a year to gain knowledge of the dynamics of Yersinia infections in fattening pig herds. Three out of four serological negative herds maintained a low level of Yersinia prevalence, whereas one herd shifted between negative status and a prevalence of 100%. The reason for these considerable fluctuations could not be explained, and there was no direct association with the analyzed risk factors. Further research should be carried out to prove the given risk factors, especially the possible relation to the Salmonella prevalence before implementing a combined zoonoses surveillance and control program.     

Preventive and therapeutic DNA vaccination partially protect dogs against an infectious challenge with Trypanosoma cruzi

American trypanosomiasis, or Chagas disease, is caused by Trypanosoma cruzi, and a vaccine would greatly improve disease control. While some studies in mice suggest that a vaccine is feasible, limited efficacy has been observed in dogs. We evaluated here the safety and efficacy of a DNA vaccine encoding TSA-1 and Tc24 antigens in a dog model of acute T. cruzi infection. Mongrel dogs were immunized with two doses of 500 μg of DNA vaccine, two weeks apart, and infected with T. cruzi (SylvioX10/4 strain) two weeks after the second vaccine dose. Another group of dogs was infected first and treated with the vaccine. Disease progression was monitored for up to 70 days post-infection. The vaccine did not induce any critical change in blood parameters, nor exacerbation of disease in vaccinated animals. On the contrary, it prevented anemia and a decrease in lymphocyte counts following T. cruzi infection in vaccinated dogs. Both preventive and therapeutic vaccination significantly reduced parasitemia, cardiac inflammation and cardiac parasite burden, and tended to reduce the development of cardiac arrhythmias. These results indicate that a preventive or therapeutic DNA vaccine encoding TSA-1 and Tc24 antigens is safe and may reduce both parasite transmission and the clinical progression of Chagas disease in vaccinated dogs. This DNA vaccine may thus be an excellent veterinary vaccine candidate. These data also further strengthen the feasibility of a Chagas disease vaccine for humans.     

Comparative efficacy of commercial Mycoplasma hyopneumoniae and porcine circovirus 2 (PCV2) vaccines in pigs experimentally infected with M. hyopneumoniae and PCV2

The efficacies of two commercial Mycoplasma hyopneumoniae bacterins and porcine circovirus 2 (PCV2) vaccines were compared in conventional pigs immunized at different ages based on humoral response, pathological observation, and growth performance from birth to finishing (175 days of age) using a M. hyopneumoniae and PCV2 co-infection challenge model. One-week-old pigs (n = 110) were randomly assigned to five groups: three vaccinated and challenged (VC), and one each of non-vaccinated and challenged (NVC) and negative control. A significant difference was found in the number of genomic copies of M. hyopneumoniae in nasal swabs and PCV2 in serum samples, the average daily weight gain (gram/pig/day) between 63 and 133 dpi, gross and histopathological lung lesion scores, histopathological lymph node lesion scores, and the immunohistochemical analysis of PCV2 among the three VC groups. The single dose schedule for M. hyopneumoniae bacterins and PCV2 vaccines have the advantages of (i) improving daily weight gain (122.4%) and slaughter weight (120.5%), and (ii) reducing the incidence of clinical signs and lung and lymph node lesions.     

A candidate multi-epitope vaccine against porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae induces robust humoral and cellular response in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) are two of the most common pathogens involved in the porcine respiratory disease complex (PRDC) resulting in significant economic losses worldwide. Vaccination is the most effective approach to disease prevention. Since PRRSV and Mhp co-infections are very common, an efficient dual vaccine against these pathogens is required for the global swine industry. Compared with traditional vaccines, multi-epitope vaccines have several advantages, they are comparatively easy to produce and construct, are chemically stable, and do not have an infectious potential. In this study, to develop a safe and effective vaccine, B cell and T cell epitopes of PRRSV-GP5, PRRSV-M, Mhp-P46, and Mhp-P65 protein had been screened to construct a recombinant epitope protein rEP-PM that has good hydrophilicity, strong antigenicity, and high surface accessibility, and each epitope is independent and complete. After immunization in mice, rEP-PM could induce the production of high levels of antibodies, and it had good immunoreactivity with anti-rEP-PM, anti-PRRSV, and anti-Mhp antibodies. The anti-rEP-PM antibody specifically recognizes proteins from PRRSV and Mhp. Moreover, rEP-PM induced a Th1-dominant cellular immune response in mice. Our results showed that the rEP-PM protein could be a potential candidate for the development of a safe and effective multi-epitope peptide combined vaccine to control PRRSV and Mhp infections.     

The behaviour of porcine cytomegalovirus in commercial pig herds.

A longitudinal, virological and serological study of pigs in two herds with respiratory disease showed that infection by porcine cytomegalovirus (PCMV) was universal in both. Virus excretion usually began when piglets were between 3 and 6 weeks of age and reached a maximum between 5 and 8 weeks; it was usually no longer detectable at 11-12 weeks. Antibody demonstrable in indirect immunofluorescence (IIF) tests was present to moderate or high titre in all piglets at 2-3 weeks. This was presumed to be maternal in origin as it declined in titre between 2-3 and 5-6 weeks. After this fall the majority of piglets showed seroconversion as a result of virus infection. One group of 12 pigs in which infection occurred earlier than usual showed a very poor antibody response, which, nevertheless, persisted through to week 27. The findings are discussed with relation to porcine atrophic rhinitis and cytomegalovirus infection in other species.     

Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old

The continuous outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks demonstrate that current vaccination strategies are not fully efficacious and should be improved by new generation of vaccines. In this context, maternally immune conventional layer chickens were vaccinated in ovo with a turkey herpesvirus recombinant expressing the fusion (F) gene of NDV (rHVT-ND) and/or at day-old with an apathogenic enterotropic live ND vaccine co-administrated or not with chitosan by oculo-nasal route. The induced vaccinal immune responses and conferred protection against a challenge with a circulating NDV velogenic viscerotropic strain were evaluated. The innovative rHVT-ND/live ND-chitosan vaccination regimen provided the best protection against mortality and morbidity as well as the strongest reduction of virus shedding that could be related to the higher measured cellular immune response and digestive antibody-mediated immunity.     

Reproductive performance of high producing lactating cows in Coxiella-infected herds following vaccination with phase-I Coxiella burnetii vaccine during advanced pregnancy

This study was designed to assess the safety of phase I vaccination against Coxiella burnetii in advanced pregnancy and the effect of vaccination on subsequent reproductive performance of high producing dairy cows. C. burnetii serostatus was determined in 719 dairy cows by individual serological testing. According to their serostatus, cows were randomly assigned to a control (n = 359) or vaccine (n = 360) group (inactivated phase I on Days 171–177 and 192–198 of gestation, Coxevac-Ceva Sante Animale). Using a χ²-test, vaccination had no effect on abortion before parturition, retention of placenta and stillbirth, either in seropositive as in seronegative cows. Cox's proportional hazards model revealed that cows in the vaccine group were 1.22 times more likely to conceive during the first 150 days in milk than cows in the control group. Moreover, the likelihood of pregnancy was lower in multiparous cows, cows with a retained placenta and cows undergoing first AI during the warm season compared to the remaining animals (by factors of 0.75, 0.69 and 0.69, respectively). In animals testing seronegative for C. burnetii, the likelihood of pregnancy was 1.25 times higher in vaccinated cows compared to non-vaccinated seronegative animals. No effect of vaccination on subsequent fertility was detected in seropositive animals. In conclusion, the results of this study indicate that phase I vaccination against C. burnetii during advanced pregnancy in dairy cows is safe and improves subsequent fertility of C. burnetii seronegative animals.     

Efficacy of vaccination strategies against intranasal challenge with Brucella abortus in BALB/c mice

Brucellosis is a zoonotic disease affecting 500,000 people worldwide annually. Inhalation of aerosol containing a pathogen is one of the major routes of disease transmission in humans. Currently there are no licensed human vaccines available. Brucella abortus strain RB51 is a USDA approved live attenuated vaccine against cattle brucellosis. In a mouse model, strain RB51 over-expressing superoxide dismutase (SOD) administered intraperitoneally (IP) has been shown to be more protective than strain RB51 against an IP challenge with B. abortus pathogenic strain 2308. However, there is lack of information on the ability of these vaccine strains to protect against intranasal challenge. With the long-term goal of developing a protective vaccine for animals and people against respiratory challenge of Brucella spp., we tested a number of different vaccination strategies against intranasal infection with strain 2308. We employed strains RB51 and RB51SOD to assess the efficacy of route, dose, and prime-boost strategies against strain 2308 challenge. Despite using multiple protocols to enhance mucosal and systemic protection, neither rough RB51 vaccine strains provided respiratory protection against intranasal pathogenic Brucella infection. However, intranasal (IN) administration of B. abortus vaccine strain 19 induced significant (p ≤ 0.05) pulmonary clearance of strain 2308 upon IN challenge infection compared to saline. Further studies are necessary to address host-pathogen interaction in the lung microenvironment and elucidate immune mechanisms to enhance protection against aerosol infection.     

Intradermal vaccination of pigs against FMD with 1/10 dose results in comparable vaccine efficacy as intramuscular vaccination with a full dose

The aim of this study was to investigate whether intradermal (ID) vaccination against foot-and-mouth disease (FMD) is suitable as an alternative for the usually used intramuscular (IM) route. We compared vaccine efficacy in groups of pigs in which vaccine administration differed with respect to antigen payload of the vaccine, administrated volume and administration route.