Evaluation of colonization, variable lipoprotein-based serological response, and cellular immune response of Mycoplasma hyorhinis in experimentally infected swine.

Mycoplasma hyorhinis (Mhr) is a commensal of the upper respiratory tract that can be shed by nasal secretions and transmitted by direct contact in neonatal and nursery pigs. Lesions associated with Mhr infection include polyserositis and arthritis; however, systemic Mhr disease pathogenesis is not well characterized. This study aimed to investigate the immunopathogenesis and bacterial dissemination pattern of Mhr using single and multiple inoculation approaches in a caesarian-derived colostrum-deprived (CDCD) pig model. Animals in three treatment groups were inoculated once (Mhr 1; n = 12) or four (Mhr 2; n = 8) times with Mhr or sham-inoculated (NC group; n = 3) nasally and by tonsillar painting. Inoculum consisted of a triple cloned Mhr field isolate (4.5 × 107 CFU/mL) in Friis medium. Clinical signs were evaluated daily during the study. Serum and oral fluid antibody (IgA and IgG) response and cellular immune response were assessed using a recombinant chimeric VlpA-G-based indirect ELISA and by ELISpot, respectively. The presence of Mhr in oral fluids, nasal and oropharyngeal swabs were evaluated by qPCR. At 6 wpi, pigs were euthanized and evaluated for gross lesions consistent with Mhr and bacterial colonization in tonsils by qPCR. No clinical signs or gross lesions consistent with Mhr-associated disease were observed throughout the study. For Mhr 2 group, the presence of IgA and IgG in serum and oral fluids were detected at 2 and 4 weeks post-inoculation (wpi), respectively, while in Mhr 1, only IgA was detected in oral fluids at 6 wpi. The proportion of animals shedding Mhr in nasal secretions varied from 20 to 40 % in the Mhr 1 and 62.5-100% in the Mhr 2 group. However, the proportion of animals shedding Mhr in oropharyngeal swabs was consistent through the study (60 %) in Mhr 1 and fluctuated from 20 % to 87.5 % in Mhr 2 group. The lack of clinical signs and the presence of Mhr specific humoral response and bacterial colonization indicates that the multiple inoculation experimental model may mimic subclinical natural infection in the field. In addition, the humoral and transient cellular response did not result in bacterial clearance. Based on these results, animals would have to be exposed multiple times to mount a detectable immune response.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Pasteurella multocida and its role in porcine pneumonia.

Pasteurella multocida has been recognized as a contributor to debilitating and fatal porcine pneumonia for at least 120 years and there continues to be sustained, unabated high prevalence of the organism in cases submitted for diagnostic work up. Understanding of its role in disease has been limited, in part because of difficulty in reproducing the disease experimentally with capsular type A strains of P. multocida, the predominant type associated with porcine pneumonia. This limitation has stymied the development of improved methods for disease control. In this review, the reports of efforts to reproduce the disease are compared. Reports have indicated induction of pneumonia in combined infections with agents such as hog cholera virus, pseudorabies virus and Mycoplasma hyopneumoniae. Pneumonia has been induced with intratracheal or endobronchial inoculation of anesthetized swine using capsular type A strains. Substantial recent progress in understanding the putative virulence attributes and molecular genetics of P. multocida will likely lead to better understanding of the host-parasite and parasite-parasite interactions in porcine pneumonia associated with this organism. In particular, it seems important to consider the role of biofilm formation in the pathogenesis of this disease. Ultimately, this understanding should provide a foundation for better methods for induction of the experimental disease, development of improved diagnostics, development of better therapeutic/prophylactic pharmaceutical approaches and development of immunoprophylactic products.

Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells.

We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 +/- 3 nM (n = 217 cells). The [Ca2+]i increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 microg/ml), and this increase lasted approximately 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 microg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 microM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 microM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.