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 [1
] 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 [23
] 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.
Obliterative bronchiolitis was seen in three inoculated calves of this investigation. The occurrence of bronchiolitis obliterans in animals naturally infected with M. bovis
has been described by other investigators [2
]. Furthermore, bronchiolitis obliterans has been reported in calves with chronic pneumonic lesions associated with spontaneous and experimental infections with other bacteria, e.g. Mannheimia haemolytica, P. multocida, Histophilus somni
and Mycoplasma dispar
, and with bovine respiratory syncytial virus [25
]. The obliterative changes seen in M. bovis
-infected calves resemble lesions classified as "bronchiolitis obliterans with intraluminal polyps" in man [30
], which occur in cases of organizing pneumonia in humans and are known as "bronchiolitis obliterans organizing pneumonia" (BOOP). Organizing lesions in the alveolus, i.e. alveolar fibrosis, as described for BOOP in man [31
] were not present in the calves with obliterative bronchiolitis of this study, but were described in calves spontaneously infected with M. bovis
]. Therefore, and also because re-epithelization of fibrous tissue within affected bronchioli was not present, the obliterative changes found in the bronchioli of the three calves of the present study might represent an early stage of organization. Recent studies on the lung tissue from these three calves demonstrated increased production of inducible nitric oxide and nitrotyrosine suggesting that nitric oxide and peroxynitrite are potentially involved in the development of obliterative bronchiolitis [23
In a previous study, we demonstrated in vivo
expression of M. bovis
Vsps in lung tissue of calves infected with a clonal variant of M. bovis
type strain PG45 during the first ten days p.i. [9
]. The present investigation revealed that there is long-term persistence of M. bovis
in chronic bronchopneumonic lesions as demonstrated by bacteriology and IHC for antigens, i.e. Vsp antigens and pMB67.
The distribution of Vsp and non-Vsp antigens of M. bovis
found in this study closely resembles the pattern described by other investigators who used different poly- and/or monoclonal antibodies to M. bovis
]. With mAb 1A1, apart from the positive macrophages, positive reactions for Vsp antigens and also for the antigen pMB67 occurred less frequently than with the mAb pool. A possible reason for this is that the mAb pool detects both variable and non-variable antigens. A constant finding in lungs of all inoculated calves in this study was the presence of M. bovis
Vsp and non-Vsp antigens in the cytoplasm of macrophages. This suggests that M. bovis
is taken up by phagocytosis following opsonisation and that residual antigen, possibly after killing of the organism, persists detected by IHC. Another possibility would be that whole organisms of M. bovis
, after being phagocytosed, survive within the phagosome of macrophages.
studies have shown that, except from variable surface antigens recognized as potential virulence factor of persistence in the host, M. bovis
is able to generate a biofilm [32
]. Further studies to determine if biofilms also occur in vivo
, i.e. on the surfaces of the respiratory tract in M. bovis
infected calves, and if or how they contribute to the persistence of the agent in the host, are necessary.
In all M. bovis
infected calves, hyperplasia of BALT was characterized by strong MHC class II expression of lymphoid cells within the BALT. This finding indicates ongoing stimulation of the local pulmonary immune system in response to persisting M. bovis
antigen. Only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II, further supporting the hypothesis that, although M. bovis
antigen is still present in necrotizing lesions, the antigen-presenting mechanisms are down-regulated at chronic stages of the disease. Nitric oxide is known to play a role as a modulator of immune responses. Therefore, the low expression of MHC class II by macrophages in perinecrotic areas of M. bovis
infected calves reported by other investigators [33
] and also seen in this study, possibly represents down-regulation of MHC class II-mediated antigen presentation as a result of the production of inducible nitric oxide and nitric oxide by activated macrophages. Beside macrophages, pulmonary dendritic cells play an important role in antigen presentation and induction of T cell-mediated immune responses in the lung [34
]. A previous study, in which quantification of MHC class II expressing dendritic cells in calves examined in this study was carried out, showed that statistically significantly increased numbers of MHC class II-expressing dendritic cells were present in the mucosa of bronchi and bronchioli of M. bovis
infected animals [35
]. In this study, examination of lung sections revealed that, in caseonecrotic foci and obliterated bronchioli, in contrast to the respiratory mucosa, only few MHC class II expressing dendritic cells were present, possibly indicating down-regulation of antigen presentation in these areas.
Reduced numbers of MHC class II expressing dendritic cells could be the result of the production of inducible nitric oxide and nitric oxide by activated macrophages. Otherwise, lesser expression of MHC class II could be a non-specific consequence of chronic immunostimulation reflecting lower amounts of MHC class II-inducing cytokines, e.g. IL-1 and IFN-γ, at the chronic stage of the disease.
Experimental infections of calves have shown that M. bovis
has both stimulating and suppressing effects on the bovine immune response such as stimulating the production of nitric oxide and TNF-α by macrophages, inducing apoptosis of lymphocytes, producing a lympho-inhibitory peptide, impairing lymphocyte responses to mitogens and suppressing the neutrophil oxidative burst [36
Aside from a few studies [11
], only limited information is available on the local immune response in lung tissue of calves infected with M. bovis
The present study revealed a considerable increase of IgG1- and IgG2-positive plasma cells at 21 days p.i. among which IgG1-containing plasma cells clearly predominated. This finding is consistent with the results previously obtained by Howard et al.
]. In one study [12
], increased IgG1 antibodies were found in the sera of experimentally infected cattle, but only small amounts of IgG2 antibodies. The authors concluded that the immune response mounted against M. bovis
infection was skewed toward a T helper 2 immune response. It has been speculated by others [12
] that, because IgG2, in comparison to IgG1, is the superior opsonin, the low IgG2 response may contribute to the chronicity of M. bovis
infection. In vitro
studies with bovine alveolar macrophages and bovine polymorphonuclear leukocytes indicate that opsonisation, i.e. specific sera, promote phagocytosis and killing of M. bovis
by phagocytes [40
The immunohistochemical finding of many antigen-positive macrophages suggests that phagocytosis, possibly opsonophagocytosis, does occur in vivo. However, because in necrotic foci high amounts of extracellular antigen are found adjacent to phagocytes, the process of phagocytosis could be modified during the course of infection by yet unknown mechanism. Differentiation of B cells into antibody secreting plasma cells usually is due to cytokine secretion by helper function of CD4+ T lymphocytes. Statistical evaluation of the numbers of CD4+ and CD8+ T cells in this study, however, did not reveal statistically significant differences between inoculated and control calves.
In this study, M. bovis
was isolated from the lungs of 7 of 8 experimentally infected calves at necropsy. In the lungs of all calves inoculated with M. bovis
, suppurative inflammatory changes of bronchi and bronchiole, often associated with suppurative bronchopneumonia, were found. Since pyogenic bacteria were isolated from the majority of these calves, they are possibly responsible for the development of the suppurative lung lesions. These results agree with the findings of other investigators that M. bovis
is a predisposing factor in bovine respiratory disease allowing colonization of the lower respiratory tract by commensal pathogenic bacteria [3
]. Although caseonecrotic pneumonia is considered to be a distinctive lesion caused by M. bovis
], the present findings support the hypothesis of other investigators that severe caseonecrotic lesions mainly occur when other bacteria are present [42
]. Co-infection of calves after spontaneous or experimental infection with M. bovis
has also been described by other investigators [1
]. One report, in which the same M. bovis
field strain as in this study was used, describes co-occurring P. multocida
infection in 10 of 16 conventionally reared experimentally infected calves [43
]. The polymicrobial infection being present at 21 days p.i. in the calves of this study is complicating the interpretation of the role of M. bovis
in the development of the local immune response in the lungs of infected calves. It cannot be excluded that the other bacteria isolated at the end of the experiment together with M. bovis
participated in the generation of the immune response in these animals.
In lung tissue of the control calves, which were microbiologically negative for M. bovis, minimal or mild inflammatory changes including mild suppurative bronchitis and bronchiolitis, being associated with the presence of S. aureus, were seen. Therefore, it cannot be excluded that during the repeated manipulations necessary for collecting TBLF samples bacteria from the upper respiratory tract were flushed into the lungs of control calves animals and possibly also of M. bovis infected animals.
The three calves with caseonecrotic pneumonia and/or obliterative bronchiolitis had signs of clinical disease, i.e. increased body temperature and respiratory rate, nasal discharge, coughing and reduced appetite. In the other five calves, however, in spite of having lung lesions such as suppurative inflammatory changes of larger airways and/or suppurative bronchopneumonia, no clinical signs, i.e. increased body temperature, respiratory rate or pulse rate or abnormal findings by auscultating heart and lung, were recorded. These findings indicate that, at least under the experimental conditions of this study, respiratory M. bovis
infections of calves can cause lung lesions, which, by applying conventional methods of clinical examination, are not associated with detectable signs of respiratory disease. The clinical signs and their presence in experimentally infected animals reported in the literature vary concerning type of signs and number of animals showing such signs per experiment. In one report, the occurrence of subclinical pneumonia, i.e. the absence of clinical signs of respiratory disease in spite of lung lesions was recorded in nine of ten calves 14 days after inoculation with M. bovis
]. These findings are similar to our observations in five of eight of the inoculated animals. Clinical signs associated with bovine respiratory disease vary and signs may be minimal or absent in cases with minor and/or chronic lung lesions [44
]. Other methods such as radiology, ultrasonography and lung function testing are considered as useful techniques for diagnosing clinically silent pneumonic lesions and for correlating clinical signs with pathological findings [44