The trial was developed as a longitudinal study in which weaned piglets were included for 6 consecutive weeks. The reason for choosing this protocol, instead of a protocol in which different treatment groups were mixed in the same week, was to eliminate the effect of the interaction of treatments within the same week. Additionally, the protocol used in this trial has allowed us to avoid the possible interaction between the different treatment groups if they were to have temporarily cohabited. As seen in other trials, when cohabiting, the control group may benefit from the decrease in infection pressure, fostered by the presence of vaccinated groups; alternatively, cohabiting may mask the lesser effect of a vaccine with reduced protection by mixing with other animals vaccinated with another more effective vaccine [21]. This protocol also facilitated the slaughterhouse assessment. This type of sample design may indeed have other drawbacks, which we will discuss later on.
1. Growth study discussion:
First, it should be noted that, despite the great care that was taken at the time of designing the trial and selecting the animals to make all the groups as homogeneous as possible, differences between them were detected at the time of the first weighing (WEIGHT 1). Obviously, at this moment there is no point in assuming any possible vaccination effect against M. hyo since the piglets had only been vaccinated for 3 days. It would be possible that this kind of trial, to assess the performance of different M. hyo vaccines, which begins only once the animals enter the fattening unit, could have its final result affected by such an undetected variable. Therefore, throughout the statistical study carried out in the different stages of animal growth (comparisons at the time of WEIGHT 2 and WEIGHT 3), the effect of the initial weight has been considered when assessing possible statistically significant differences between treatments.
In the case of the data obtained in WEIGHT 2, both WEIGHT 1 and SOW CYCLE affected the weight obtained at that time, around 57 days of age. This shows the great influence that weaning weight has on the weight of piglets just 5 weeks later, before moving on to the fattening units [22,23]. Likewise, the cycle of the sow from which they were farrowed has an influence, being only in this case restricted to piglets born to primiparous sows, which are significantly larger than piglets born to sows of cycles 2 to 6. However, once the highly significant effect of WEIGHT 1 has been corrected, the influence of the interaction SOW CYCLE*TREATMENT or the TREATMENT GROUP is no longer detected, which seems to indicate that the vaccine used, at this time of the trial, acts in the same way in all cycles and that the influence of the cycles on the mean weight is similar in all treatment groups. This could lead to the hypothesis that, until this stage in development, M. hyo infection is not important in the population.
Finally, as for the growth study, there is one last weighing (WEIGHT 3), carried out around 161-162 days of age for all the animals that kept their individual identification. The analysis of the weights recorded leads us to assertain that the effect of weight at weaning (WEIGHT 1) does not influence WEIGHT 3, additionally, WEIGHT 1 does not affect the weight just before leaving for the fattening units (WEIGHT 2), which in turn, affects growth at the time of WEIGHT 3, perhaps because there may have been little time between the two weighings. Once the highly significant effect of WEIGHT 2 has been corrected, the effect of the interaction SOW CYCLE*TREATMENT GROUP is not detected, but an important influence is maintained separately from both the SOW CYCLE and, for the first time in the trial, from the TREATMENT GROUP. Regarding the SOW CYCLE, we may conclude that the piglets born to primiparous sows, show a mean weight significantly lower than that of cycles 7 to 9. Therefore, we may confirm the worse performance during the fattening stage was for the piglets born to primiparous sows. When analysing the TREATMENT GROUP, we have already mentioned that WEIGHT 3 is the first time in the trial where there is evidence of a significant effect of the vaccine against M. hyo with which the piglets had been immunised pre-weaning. Statistically, once the estimation of the marginal means has been performed, it may be affirmed that the group vaccinated against M. hyo with the intradermal vaccine (group I) has a final mean weight (WEIGHT 3) that is significantly higher than the piglet group vaccinated with the other intramuscular vaccine (group H). However, no other significant differences are detected, although there are numerical differences with the N group (control group- not vaccinated against M. hyo). At this point, it is also important to note that the latter weighing (WEIGHT 3) occurs just before the pigs are sent to the slaughterhouse to preserve as many animals as possible out of those originally identified. However, many of the animals remained for several weeks more in the fattening units until they are sent to the slaughterhouse, and if they were affected at that time by M. hyo, the difference in growth between the groups could be greater if one more weighing were to be performed on these animals just before they are sent to the slaughterhouse. Therefore, we could consider likely that, if this last weighing were to be performed a few weeks later, the differences in mean weights could have increased between the different groups due to the effect of the vaccine compared to non-vaccination. Finally, the fact that the differences with the unvaccinated group are only numerical could be based on the mean age of the pigs that belong to that group compared to the other two. We must remember that group N, with 162.3 ± 1.52 (SD) average days of age, is 0.9 days older than group I, and 1.4 days older than group H. At such ages, according to some studies, the growth rate of pigs can approach or even exceed 1Kg per day [23,24].
2. Discussion of the tracheobronchial swab test (TBS)
The analysis of the results of tracheobronchial swabs suggests the hypothesis that the group vaccinated with the intradermal vaccine (group I), enjoys greater protection from M. hyo, which would be in line with the results of the weight study. It seems that, according to one study [12], intradermal vaccination offers levels of local immunity (at the level of the lung mucosa), and systemic immunity which is greater compared to other intramuscularly administered bacterin against the same agent (specific IgA antibody levels of M. hyo and IL-10 expression levels, responsible for controlling inflammation locally). Continuing with the second part of this study of lung samples, we may confirm, given the results, that not only do the animals vaccinated intradermally show fewer statistically significant positive samples, but also that the value of Ct is greater, which indicates a lower pathogen load, contributing even more to the above hypothesis.
3. Discussion of the assessment at the slaughterhouse:
Some studies have suggested that the greater the lung area affected by lesions the lower the growth rate in the animal [25]. In view of the results of the slaughterhouse visits to assess the lung lesions compatible with both EP and pleuritis, it may be inferred that all groups were infected by M. hyo. The least affected group, in relation to the previous results of the tracheobronchial swab tests and growth studies, is group I (Intradermal vaccination). Group I presented data on the prevalence of lesions compatible with EP and pleuritis that are significantly lower than the other two groups, as well as a disease index (DI) and an APPI index numerically lower than those of the other groups, thus offering new evidence of better control of lung involvement, in line with the results obtained in the tracheobronchial swab test as well as in the growth of the animals. No significant differences were detected between the groups in terms of a maximum lesion, perhaps because the number of animals with a maximum lesion is very low in all groups and this affects the power of the statistical test.
It is worth highlighting the role of pleuritis lesions on the C-reactive protein (CRP testing is a blood test marker for inflammation), and this case reveals, once again, a certain association between pleuritis lesions that may have been possibly caused by App and the results of the assessments of EP-compatible lesions at the slaughterhouse [26]. It is clear that the prevalence of pleuritis in group I (intradermal vaccination), with a lower prevalence of EP, is significantly lower than that of the unvaccinated group.
In the lung assessment study, the act of withdrawing the first and last batches from the barns, has enabled us to rule out the potential positive effect of the leader pigs in each batch (normally those that have shown better and more weight growth) and the potential negative effect of the end pigs in the fattening barn from that same batch (on some occasions, the last pigs in a batch at the fattening units stay longer due to marketing and company logistics rather than for their health status).
4. Discussion of the serological study:
The serology and PCR data against the PRRS virus indicates that the virus recirculated in the animals from approximately 6 weeks of age. The negative PCR results for PRRS at 3 weeks of age indicate that the breeders were probably stable [27]. Therefore, at the time of vaccinations against both PCV2 and M. hyo, the animals were healthy concerning PRRS, and there was no interference in vaccination due to the immunosuppressive effect of the PRRS virus [28]. However, we can verify that around 6 weeks of age, several of the pools are positive, and as soon as they enter the fattening unit, in the third sampling, all the pools are positive. With this information, we may suggest that the influence of the PRRS virus may have been the same in all the study groups.
Secondly, and referring to another of the agents that play a key role in respiratory pathology, a specific ELISA was performed for the Actinobacillus pleuropneumoniae (App) Apx IV toxin to determine only its presence on the farm. Until now, we had only been guided by the slaughterhouse assessments for diagnosis, as well as by lesions found at the level of field necropsies. Given the results, in which almost 100% of the samples were positive (in each of groups H and I, only 1 of 16 samples was a suspect) we may affirm that App could also be involved in the respiratory pathology on this farm and that it could have affected all the study groups equally.
From the serum analysis to detect antibodies against PCV2, we can confirm that, in the first sampling, between 95 and 100% of the animals remain positive in all the treatment groups, due to the colostral immunity received from their mothers. Additionally, this result was seen in the second sampling as a result of the response to vaccination (Porcilis® PCV, MSD Animal Health) [29]. By performing additional quantitative PCR tests in a 4-sample pool, it is clear that in all treatment groups there is no presence of PCV2 viraemia until the last sampling, at a minimum of 21 weeks of age. The viraemia levels found are subclinical in all groups and not related to clinical symptoms of porcine circovirosis [30], which together with having vaccinated all animals, leads us to believe that although the type 2 porcine circovirus was present at the fattening unit, its possible impact has been negligible, and in any case similar in all groups.
Finally, regarding the detection of antibodies against M. hyo, we see that group N, unvaccinated, does not show antibodies present against M. hyo until the last sampling at the end of the fattening period. We must remember that, at that time, 96.7% of tracheobronchial swab samples were positive for this agent, as such the animals were facing a recent infection, and hence this fact supports the idea that if we had performed another weighing some weeks after WEIGHT 3, we might have found greater differences than those observed in this trial. On the other hand, the intramuscular treatment group, (group H) seems to be presenting a seroconversion to vaccination with a decrease in antibody levels at around 13-14 weeks of age and a subsequent increase at 17 and 21 weeks of age (when 95% of the animals were ELISA-positive for M. hyo). This fact could confirm that, in group H, the recirculation of M. hyo occurred earlier than in group N (unvaccinated), and thus perhaps does not show significant differences in growth (although there are numerical differences). Additionally, at 21 weeks of age, the time of the swab, 100% of the samples of group H were PCR-positive for M. hyo. Finally, treatment group I, vaccinated intradermally, shows a slight seroconversion to vaccination and it is not until 21 weeks of age, just like group N, when the presence of M.hyo is detected again in the serum samples. In addition, and in contrast to groups N and H, only 36.7% of tracheobronchial swab samples are positive, and this fact might be related to greater local protection offered by the vaccine. Moreover, it must be remembered that within the samples of positive lung swabs, the intensity of the positivity or Ct value was significantly lower in the intradermally vaccinated group.
Perhaps the greatest limitation of this trial is its longitudinal character, that is, the development of the trial over 6 weeks, since according to some publications [31] the variability of positive piglets to M. hyo at the time of weaning is very high between batches, and such positivity to M. hyo at the time of weaning may be a valid predictor for detecting a higher incidence of M. hyo problems during the growth phase of the animals. However, other studies make it clear that, although this fact is important, many other factors are also key to the onset of problems related to EP (management, density, concomitant pathologies) [11,26,32]. In Fano's study [31], the importance of primiparous sows as the main responsible agent for the existence of M. hyo on farms, due to the horizontal sow-piglet transmission during the lactation phase, is discussed. In this current study, it was taken into account that all batches of animals created week by week had the same number of primiparous sows; therefore, if these sows were the M. hyo transmitters to their progeny, the probability would be the same in all treatment groups in terms of the number of possible shedding animals. Future trials may need to include a correction factor by introducing the assessment of tracheobronchial swabs, or nasal swabs to determine the prevalence of M. hyo at the time of weaning and thus evaluate the effect of this fact on the results at the end of the trial.