Date Published: February 4, 2012
Publisher: BioMed Central
Author(s): Kathrin Hermeyer, Inka Buchenau, Anne Thomasmeyer, Berit Baum, Joachim Spergser, Renate Rosengarten, Marion Hewicker-Trautwein.
Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.
Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4+ and CD8+ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.
Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4+ and CD8+ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.
The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.
Mycoplasma bovis is an important cause of chronic pneumonia in feedlot cattle and dairy calves. Both in spontaneous and experimentally infected animals, different patterns of inflammatory lung lesions occur, among which caseonecrotic pneumonia is considered distinctive . Findings in spontaneously occurring M. bovis infections suggest that necrotic lesions originate from bronchioles or small bronchi . The chronicity of lung lesions and the persistence of M. bovis implies that the immune response is insufficient in eliminating the pathogen [2,3]. However, the mechanisms leading to tissue damage and how M. bovis evades the host immune response are incompletely understood [1,4]. The factors of M. bovis potentially associated with virulence are the variable surface membrane proteins (Vsps) . In addition, other surface proteins, unrelated to the Vsps, e.g. pMB67, have been described [6-8]. Variable expression of these proteins may be a major mechanism by which M. bovis evades the immune response . In a previous report the in vivo expression of Vsp antigens in lung tissue of calves inoculated with a clonal variant of M. bovis type strain PG45 by using immunohistochemistry (IHC) and different monoclonal Vsp-specific antibodies during early postinfectious stages, i.e. between 2 and 10 days post inoculation (p.i.) was described . So far, it is not known if Vsp antigens are still present during the chronic stages of pneumonic lesions induced by M. bovis.
The results of this study show that inoculation with M. bovis strain 1067 causes caseonecrotic pneumonic lesions that originate from small bronchi and bronchioli. The possible mechanisms, however, by which M. bovis induces these lesions, are not clear. Whether M. bovis infects airway epithelial cells is controversial  and recent findings in experimentally infected animals suggest that positive immunohistochemical staining with antibodies to M. bovis seen in airway epithelial cells is non-specific. Therefore, beside direct effects of M. bovis, certain factors released by the host’s lung tissue could be involved in the development of necrotizing lesions in large airways. A recent study of lung sections of the calves examined in this investigation revealed the co-localization of M. bovis antigen and of strongly expressed inducible nitric oxide and nitrotyrosine by macrophages in perinecrotic tissue areas  indicating that the production of nitric oxide and peroxynitrite is potentially involved in the development of necrotizing lung lesions. Increasing concentrations of peroxynitrite lead to the generation of reactive oxygen and nitrogene species (ROS and RNS) which both have cytotoxic capacities. Therefore, both ROS and RNS are potentially involved in the development of severe necrotizing lung lesions seen in the animals of this study.
Our findings show that infection of calves with M. bovis strain 1067 results in various lung lesions including caseonecrotic pneumonia. IHC for wide spectrum cytokeratin in two calves with caseonecrotic foci demonstrated that these lesions originated from bronchioli and bronchi. Our results show that there is long-term persistence of M. bovis as demonstrated by bacteriology and IHC for M. bovis antigens, i.e. VSp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from (i) local downregulation of antigen presenting mechanisms and (ii) an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.
The authors declare that they have no competing interests.
KH and MH-T have conducted the histopathology and drafted the manuscript. IB and BB have performed the IHC and evaluation of M. bovis Vsp antigens, CD4+ and CD8+ T cells and immunoglobulins in plasma cells and performed the statistical analysis. AT performed the IHC and evaluation of MHC class II. JS and RR have designed the bacteriological part of the study and analysis and interpretation of bacteriological results. All authors have revised the manuscript and approved the final manuscript.