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BACKGROUND: Mycoplasma (M.) hyopneumoniae is associated with respiratory disease in pigs and is the primary agent of enzootic pneumonia. Quantification of M. hyopneumoniae-related outcome parameters can be difficult, expensive, and time-consuming, in both research and field settings. In addition to well-established methods, technological tools are becoming available to monitor various aspects of relevant animal- and environment-related features, often in real-time. Therefore, this study aimed to assess whether certain parameters, such as animal movement and body temperature using microchips (IMT), correlate with established parameters and whether the currently used parameters can be rationalized. RESULTS: The percentage of movement was significantly reduced by M. hyopneumoniae infection in pigs (p < 0.05), where the M. hyopneumoniae-infected group showed a lower percentage of movement (1.9%) when compared to the negative control group (6.9%). On the other hand, macroscopic (MLCL) and microscopic (MLL) lung lesions, respiratory disease score (RDS), M. hyopneumoniae-DNA load, and anti-M. hyopneumoniae antibody levels increased significantly in the M. hyopneumoniae-infected group 28 days post-inoculation (p < 0.05). Moderate (r > 0.30) to very strong correlations (> 0.80) were observed between the abovementioned parameters (p < 0.05), except for IMT. A significant and moderate correlation was reported between IMT and rectal temperature (r = 0.49; p < 0.05). Last, the average daily weight gain and the percentage of air in the lung were not affected by M. hyopneumoniae infection (p > 0.05). CONCLUSIONS: M. hyopneumoniae infection significantly reduced the movement of piglets and increased lung lesions, M. hyopneumoniae-DNA load, and anti-M. hyopneumoniae antibody levels; and, good correlations were observed between most parameters, indicating a direct relationship between them. Thus, we suggest that changes in movement might be a reliable indicator of M. hyopneumoniae infection in pigs, and that a selected group of parameters-specifically RDS, MLCL, MLL, M. hyopneumoniae-DNA load, anti-M. hyopneumoniae antibody levels, and movement-are optimal to assess M. hyopneumoniae infection under experimental conditions.

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Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease, M. hyopneumoniae vaccination is commonly practiced. However, the persistence of M. hyopneumoniae vaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence of M. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection with M. hyopneumoniae was assumed in both farms. However, the studied pigs remained M. hyopneumoniae negative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms. Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitro M. hyopneumoniae stimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses after M. hyopneumoniae vaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-term M. hyopneumoniae vaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.

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Introduction: Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Methods: Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. Results: After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1ß was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. Discussion: In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

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In this paper, we describe a new quantitative method to evaluate and quantify in vitro growth inhibition of mastitis-related bacteria. Colony-forming units of Staphylococcus (S.) aureus (n = 10), Escherichia (E.) coli (n = 10), and Streptococcus (S.) uberis (n = 10) were quantified after their growth on top of layers of trypticase soy agar (TSA) containing six different concentrations (varying from 102 to 107 CFU/mL) of bovine non-aureus staphylococci (NAS), i.e., S. chromogenes (n = 3) and S. simulans (n = 3) isolates. Growth inhibition of the mastitis-related major bacterial pathogens, including E. coli, was confirmed by all NAS, an effect that varied highly among NAS isolates and was not evident from the semiquantitative method with which the new method was compared. By subsequent application of the new method on a larger set of 14 bovine NAS isolates, we observed that S. simulans and NAS originating from teat apices (especially S. epidermidis) required lower concentrations to inhibit both methicillin-sensitive (MSSA) (n = 5) and methicillin-resistant S. aureus (MRSA) isolates (n = 5) originating from milk. Therefore, the new assay is a promising tool to precisely quantify the intra- and inter-species differences in growth inhibition between NAS.

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Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

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Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. Although cell mediated immunity (CMI) may play a role in protection against M. hyopneumoniae, its transfer from sows to their offspring is poorly characterized. Therefore, maternally-derived CMI was studied in piglets from vaccinated and non-vaccinated sows. The potential influence of cross-fostering before colostrum ingestion on the transfer of CMI from dam to piglets was also investigated. Six M. hyopneumoniae vaccinated sows from an endemically infected herd and 47 of their piglets, of which 24 piglets were cross-fostered, were included, as well as three non-vaccinated control sows from an M. hyopneumoniae-free herd and 24 of their piglets. Vaccinated sows received a commercial bacterin intramuscularly at 6 and 3 weeks prior to farrowing. The TNF-α, IFN-γ and IL-17A production by different T-cell subsets in blood of sows, colostrum and blood of piglets was assessed using a recall assay. In blood of sows cytokine producing T-cells were increased upon M. hyopneumoniae vaccination. Similarly, M. hyopneumoniae-specific T-cells were detected in blood of 2-day-old piglets born from these vaccinated sows. In contrast, no M. hyopneumoniae-specific cytokine producing T-cells were found in blood of piglets from control sows. No difference was found in M. hyopneumoniae-specific CMI between cross-fostered and non-cross-fostered piglets. In conclusion, different M. hyopneumoniae-specific T-cell subsets are transferred from the sow to the offspring. Further studies are required to investigate the role of these transferred cells on immune responses in piglets and their potential protective effect against M. hyopneumoniae infections.

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