Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45

Research in Veterinary Science 89 (2010) 223–229

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Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45 I. Buchenau a, F. Poumarat b, D. Le Grand c, H. Linkner d, R. Rosengarten d, M. Hewicker-Trautwein a,* a

Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany UMR Mycoplasmoses des Ruminants, Agence Française de Sécurité Sanitaire des Aliments, Site de Lyon, 31 Avenue Tony Garnier, F-69364 Cedex 07, Lyon, France UMR Mycoplasmoses des Ruminants, Pathologie du Bétail, Ecole Nationale Vétérinaire de Lyon, 1 Avenue Bourgelat, F-69280 Marcy-l0 Étoile, France d Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria b c

a r t i c l e

i n f o

Article history: Accepted 8 March 2010

Keywords: Mycoplasma bovis Calves Pneumonia Variable surface membrane proteins (Vsps) Immunohistochemistry

a b s t r a c t The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Mycoplasma (M.) bovis is one of the most pathogenic bovine mycoplasma species and is associated with a wide range of diseases including pneumonia, mastitis, arthritis, subcutaneous abscesses, keratoconjunctivitis, meningitis and infertility (Pfützner & Sachse, 1996; Nicholas & Ayling, 2003; Caswell & Archambault, 2008). A major characteristic of most mycoplasma infections, including those caused by M. bovis, is that they are usually chronic in nature. Pulmonary lesions in calves with natural or experimental M. bovis infection are mainly characterised by bronchointerstitial pneumonia, i.e. thickening of alveolar walls by infiltration with mononuclear inflammatory cells, suppurative bronchopneumonia, necrotising lesions of lung parenchyma and hyperplasia of peribronchial lymphoid tissue (Gourlay et al., 1976, 1989; Lopez et al., 1986; Thomas et al., 1986; Howard et al., 1987; Adegboye et al., 1995; Rodríguez et al., 1996; Khodakaram-Tafti & López, 2004; Gagea et al., 2006; Caswell & Archambault, 2008). The pathogenic mechanisms involved in the development of lung lesions after infection with M. bovis are largely unknown. In previous M. * Corresponding author. Tel.: +49 511 953 8630; fax: +49 511 953 8675. E-mail address: [email protected] (M. HewickerTrautwein). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.014

bovis infection experiments pathomorphological examination of lung lesions and antigen distribution was carried out between 14 and 27 days post infection (p.i.) (Thomas et al., 1986; Howard et al., 1987; Rodríguez et al., 1996). Except from one investigation describing histopathological findings in the lungs of few calves necropsied at seven days p.i. (Lopez et al., 1986), information about lung lesions and antigen distribution during early stages of respiratory M. bovis infections is not available. Studies on lungs from M. bovis infected calves have shown that, although chronically infected animals have M. bovis-specific antibodies circulating in the sera and develop a proliferation of bronchus-associated lymphoid tissue (BALT), the organism is able to persist in the lungs of the host for several weeks p.i. (Thomas et al., 1986; Rodríguez et al., 1996). The possible mechanisms for this chronicity of M. bovis in the host are still under investigation, but it has become apparent during the last years that the high variability of the organism’s membrane surface proteins could be involved (Behrens et al., 1994; Rosengarten et al., 1994; Le Grand et al., 1996). M. bovis possesses a large family of variable surface lipopropteins designated Vsps which are encoded by 13 different genes in the type strain PG45 (Behrens et al., 1994; Rosengarten et al., 1994; Lysnyansky et al., 1996, 1999, 2001; Sachse et al., 2000). Detailed in vitro analysis revealed that individual proteins of the Vsp system undergo high-frequency variation in expression

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and size, generating extensive surface diversification in a given M. bovis strain or isolate. Successful persistence of M. bovis organisms in the bovine host and its ability to evade the immune system might be attributed, at least partially, to its capability for rapid diversification of its cell surface antigens, as has been speculated for this and other pathogenic mycoplasma species (Razin et al., 1998; Wise et al., 1992; Caswell & Archambault, 2008). So far, published data about the occurrence of Vsp surface antigenic variation of M. bovis in vivo, in the respiratory tract of its host, are not available. This study investigates the pathomorphological findings and proliferation of BALT in lungs of calves after experimental infection with M. bovis during early postinfectious stages between 2 and 10 days p.i. and monitors the in vivo expression of M. bovis Vsps in lung tissue by using Vsp-specific monoclonal antibodies. 2. Materials and methods 2.1. Calves and experimental infection Eight calves aged two weeks were used for infection and one three-week old calf served as control. The animals originated from a herd without any recent history of respiratory disease, mastitis or arthritis and had been clinically healthy since birth. Before infection, calves were shown to be free of M. bovis respiratory infection by bacteriological examination of individual tracheobronchial lavage (TBL) fluid samples and by examination of serum samples for the presence of antibodies to M. bovis by an ELISA test (Vetoquinol, France) (Cho et al., 1976; Poumarat et al., 1987). This was retrospectively confirmed by Western blot analysis of M. bovis wholecell antigens using preimmune sera (Brank et al., 1999). TBL was performed by using two different catheters. First, a tube with an internal diameter of 0.5 cm was introduced into a nostril and moved until the bifurcation of the trachea. This tube protected a second tube (internal diameter 0.127 cm) from contamination. TBL was then collected via the second tube which was moved until the entry of one of the two main bronchi. Calves were housed in separate pens, fed with commercial milk substitute and examined clinically every day. Each calf, except the control animal, was inoculated endobronchially with approximately 50 ml of fresh culture containing 4  1010 colony forming units (CFU) of a clonal variant designated Vsp A67 (see also Brank et al., 1999) in the exponential phase of growth. Endobronchial inoculation was performed by placing the inoculum at the entry of one of the two main bronchi as described for collecting TBL fluid. The medium used was described elsewhere (Poumarat et al., 1991). This clonal variant, which expresses a 67 kDa size version of the variable surface protein VspA, was selected from a collection of isogenic variants generated from M. bovis type strain PG45 (Behrens et al., 1994). The control calf received approximately 50 ml of sterile broth alone. After inoculation, all calves were monitored daily for clinical signs of disease, and sampling of sera and TBL were performed daily until necropsy. TBL fluid samples were analysed for the presence of mycoplasmas and other bacteria by standard cultivation procedures (Poumarat et al., 1991), and serum samples were analysed for the presence of antibodies to M. bovis by ELISA (Le Grand et al., 2002) and Western blot (Brank et al., 1999). 2.2. Necropsy and collection of samples Two animals each were euthanased by intravenous injection of sodium pentobarbitone 2, 4, 6 and 10 days post inoculation (p.i.). The control calf was euthanased and necropsied at day 14 of the experiment. On each animal a complete necropsy was performed. From the lungs, after being examined for macroscopic lesions, tis-

sue samples were taken from six different regions, i.e. from the cranial parts of the apical lobes, from the medial part of the middle lobe, and from the caudal parts of the diaphragmatic lobes for microbiological, histopathological and immunohistochemical examinations. 2.3. Histopathology Lung tissue samples were fixed in 5% neutral-buffered formalin and embedded in paraffin wax. Paraffin sections from samples of all lung lobes were stained with haematoxylin and eosin (H&E) for histopathological examination. 2.4. Antibodies and immunohistochemistry For immunohistochemical detection of M. bovis, rabbit hyperimmune anti-M. bovis antiserum (pAb) D490 and monoclonal antibodies (mAbs) 1E5 and 1A1 specific for a number of Vsp antigens and mAb I2 specific for the Vsp-unrelated variable membrane surface protein antigen (pMB67) (for sources/references see Table 1) were tested by applying the avidin–biotin-peroxidase (ABC) technique. Mouse mAb 1E5 was raised against a clonal variant derived from M. bovis type strain PG45 and was shown to specifically react with M. bovis Vsp antigens A, B and C (Behrens et al., 1994; Rosengarten et al., 1994). Mouse mAb 1A1 was obtained by a similar procedure and was shown to react with Vsp A and Vsp C and other Vsps, but not with Vsp B (Le Grand et al., 1996). Both mAbs exclusively react with M. bovis but not with any other ruminant mycoplasma species. For immunolabelling of macrophages mouse mAb anti-human CD68 (clone EBM 11) was applied diluted 1:20 on sections after pre-treatment with 0.05% pronase E for 20 min at 37 °C. Paraffin sections were dewaxed, rehydrated and endogenous peroxidase activity was inhibited by incubation in 0.5% H2O2 in 70% ethanol for 30 min at room temperature (RT). For all M. bovis-specific antibodies, different treatments for antigen retrieval (AR) were tested to determine their effect on immunohistochemical staining. For AR with proteases, sections were pretreated with 0.25% trypsin from hog pancreas (Fluka, Chemie, Buchs, Switzerland) in phosphate-buffered saline (PBS) containing 0.02% CaCl2 (pH 7.6 for 60 min at 37 °C) or with 0.05% protease XIV (Sigma–Aldrich Chemie GmbH, Steinheim, Germany) in PBS (pH 7.4, 8 min at 37 °C). For AR by heating, a microwave (MW) pre-treatment step was included before the primary antibody was applied. The sections were placed in a container filled with 0.01 M citric acid buffer, pH 6.0, and irradiated for 10 min in a conventional MW oven at the highest power level setting. After heating, sections were allowed to cool and were consecutively rinsed for 5 min in fresh citric acid buffer followed by two rinses in PBS. Following enzymatic or non-enzymatic pre-treatment, sections were preincubated for 20 min at RT with normal goat or horse serum diluted 1:5 in PBS. Primary antibodies were diluted in PBS containing 1.0% bovine serum albumin (BSA) and were allowed to incubate for approximately 16–18 h at 4 °C. Thereafter, sections were incubated for 30 min at RT with biotin-conjugated antibodies to rabbit IgG, mouse IgG or mouse IgM (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 in PBS. Incubation with the ABC solution (Vectastain Elite ABC kit, Vector) was performed according to the manufacturer’s instructions. The chromogen used was 3,30 -diaminobenzidine-tetrahydrochloride (Fluka) with 0.03% H2O2 as substrate in 0.1 M Trisbuffered saline (Tris-hydroxymethyl-aminomethane, Merck, Darmstadt, Germany) pH 7.6. Tissue sections were counterstained with Mayer’s haematoxylin and mounted. For establishing the immunohistochemical staining reactions, paraffin sections from the lung of a calf from another experiment, which had been sacrificed after respiratory infection with M. bovis strain 1067 and from

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I. Buchenau et al. / Research in Veterinary Science 89 (2010) 223–229 Table 1 M. bovis-specific antibodies.

a b

Antibody designation

Type or isotype

Specificity

References/source

D 490 mAb 1E5

Rabbit hyperimmune serum Mouse IgM

M. bovis M. bovis VspaA, B and C

mAb 1A1 mAb I2

Mouse IgG1 Mouse IgG1

M. bovis VspA, C and other Vsps M. bovis pMB67b

Rosengarten et al., 1994 Rosengarten et al., 1994 Behrens et al., 1994 Le Grand et al.,1996 Poumarat et al., 1994 Behrens et al., 1996

Variable membrane surface proteins belonging to the Vsp protein family of M. bovis. Variable membrane surface protein of M. bovis not belonging to the Vsp protein family.

which M. bovis organisms had been reisolated were used. Paraffin sections from this calf served as positive control. Negative control sections, in which the primary antibody was replaced by normal Balb/c serum (BioLogo, Kronshagen, Germany) were included in all staining runs. Additionally, formalin-fixed paraffin sections of lung tissue obtained from two calves aged 3.5 months were used as negative controls. These two gnotobiotic calves had been vaccinated and challenged with bovine respiratory syncytial virus and, since the age of two months, kept under strict specific pathogen free (SPF) conditions until necropsy. They were microbiologically and serologically negative for M. bovis and did not show any macroscopical or histological lung lesions. 3. Results 3.1. Clinical, serological and microbiological findings In serum samples taken before inoculation, antibodies to M. bovis were not present. M. bovis or other bacterial species were not isolated from any of the TBL fluid samples obtained before experimental infection except from calves Nos. 2 and 4, from which Pasteurella multocida was isolated before inoculation. As we have previously reported (Brank et al., 1999), antibodies to VspA were detected in serum samples of inoculated animals as early as 6 days after infection. The sera also contained antibodies that reacted with VspC and VspB. After infection with M. bovis, clinical signs of respiratory disease were only found in two calves from which Mannheimia haemolytica (calf No. 3) and P. multocida (calf No. 4) were isolated from lung tissue samples at necropsy (Table 2). Signs of lameness were not seen in any animal. The results of the microbiological findings in lung tissue samples obtained at necropsy are given in Table 2. M. bovis was recovered from TBL (data not shown) of two calves (Nos. 5 and 6) and lung samples of four infected calves (Nos. 2, 3, 5 and 6; Table 2). Furthermore, other mycoplasma species were isolated from the lungs of three infected calves (Nos. 3, 4 and 6; Table

2). From the control calf, neither M. bovis nor other mycoplasma or other bacterial species were isolated at necropsy. 3.2. Macroscopical and histopathological findings in lungs Macroscopic lung lesions were only found in three animals. Lungs of calf No. 3, from which M. haemolytica was isolated, had atelectasis and multiple necrotic foci were present in both apical lobes and in the middle lobe. The calf from which P. multocida was isolated (calf No. 4) had macroscopically visible areas of consolidation with suppurative bronchitis in the apical lobes and in the middle lung lobe. In these two animals, the respiratory lymph nodes were enlarged. In calf No. 6 macroscopically visible areas of consolidation with abscesses in the left and right apical lobes were present. Microscopically, apical and middle lung lobes from all inoculated calves showed moderate infiltration of interalveolar septa with numerous macrophages and lower numbers of neutrophilic granulocytes and lymphocytes whilst in the diaphragmatic lobes only slight infiltrations of interalveolar septa by such inflammatory cells were found (Fig. 1). The majority of CD68-positive macrophages were located in the interalveolar septa often being present within small blood vessels whilst only few CD68-positive alveolar macrophages were found (Fig. 2). Calf No. 3 had multifocal to coalescing, variably sized foci with a central area of coagulative necrosis. Within the necrotic areas remnants of alveoli and bronchiole were visible. Necrotic foci were surrounded by numerous neutrophilic granulocytes, so-called oat cells, macrophages and a peripheral wall of fibrous tissue. In samples from consolidated lung lobes of calf No. 4 marked suppurative bronchopneumonia was present. With the exception of calf No. 2, interstitial pneumonia was associated with slight (calves Nos. 5–8) or slight to moderate (calves Nos. 1, 3, 4) proliferation of peribronchial lymphoid tissue, which was more prominent in the apical and middle lobes than in the caudal lung lobes. In lung samples from the non-inoculated calf no significant microscopic lesions were seen, and there were only few CD68-positive macrophages.

Table 2 Bacteriological findings obtained for lung tissue samples from calves experimentally infected with a 67 kDa Vsp A-expressing clonal variant of M. bovis type strain PG45.

a b c d e

Calf No.

Euthanasia (days p.i.)

Recovery of M. bovis

Isolation of other bacteria

Isolation of other mycoplasma speciesa

1 2 3 4 5 6 7 8 NCe

2 2 4 4 6 6 10 10 14

Negative 6/6b (103–105)c 1/6 (10) Negative 1/6 (10) 2/6 (10–102) Negative Negative Negative

Negative Negative 2/6 (Mannheimia haemolytica) 6/6 (Pasteurella multocida) Negative 2/6 (Bacillus sp.)d Negative Negative Negative

Negative Negative 2/6 1/6 Negative 3/6 Negative Negative Negative

M. bovirhinis, M. bovigenitalium, M. arginini, and/or Acholeplasma sp. Number of positive samples of six examined lung tissue samples. CFU/ml of M. bovis organisms. Only few organisms. Negative (uninfected) control calf.

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Fig. 1. Inflammatory cell response in the lung of a calf experimentally infected with a 67 kDa Vsp A-expressing clonal variant of M. bovis (4 days p.i.). Infiltration with numerous macrophages and fewer neutrophilic granulocytes. H&E. 400.

numerous macrophages being located in different lung compartments (Fig. 3) (Table 3). Additionally, positive immunolabelling with mAb 1A1 was found in 4 calves in the lumina and on the surfaces of larger airways and/or alveoli. Within the inflammatory exudate of bronchi and bronchioli fine granular reaction product was present extracellularly and in the cytoplasm of neutrophils and macrophages including large foamy macrophages and few multinucleate cells. In 3 calves (Nos. 1, 3, 4), mAb 1A1 showed a cytoplasmic reaction of intravascular mononuclear cells. With mAb 1E5, only in two calves (Nos. 2 and 3) positive immunohistochemical staining reactions occurred (see Table 3). M. bovis antigens detected with the antibodies used was mainly found in tissue samples obtained from the apical and from the middle lung lobes whilst in tissue samples from the diaphragmatic lobes antigen was seldom seen. Necrotic foci of calf No. 3 did not contain M. bovis antigen. In lung tissue samples from the uninoculated control calf and also from the two gnotobiotic control calves M. bovis antigen was not present (Table 3). In sections of all inoculated calves and also of the control calves stained with pAb D490 and with mAb 1A1 epithelial cells of bronchi and bronchioli showed granular or diffuse cytoplasmic staining. In bronchi, such granular staining was mainly located in Goblet cells. Furthermore, in all inoculated calves and also in the control animals granular staining of epithelial cells of peribronchial glands occurred with pAb D490 and mAb 1A1 (Table 3).

4. Discussion

Fig. 2. Increased numbers of CD68-immunopositive macrophages in interalveolar septa from a calf experimentally infected with a 67 kDa Vsp A-expressing clonal variant of M. bovis (6 days p.i.). ABC method. 630.

3.3. Evaluation of antigen retrieval techniques for immunohistochemical staining In sections of the positive control calf pAb D490 gave a strong specific immunohistochemical staining reaction both after enzymatic and MW pre-treatment. With mAb 1E5, specific staining was only seen in sections pretreated with trypsin. In sections stained with pAb D490 and mAb 1E5 slight to moderate background staining of interlobular connective tissue and of vessel walls occurred. With mAb 1A1, stronger specific staining was achieved on sections pretreated with trypsin than on those pretreated with protease XIV or by MW heating. Initially, for each pre-treatment method, the optimal working dilution for the different primary antibodies was established. The optimal dilutions were 1:1800 for pAb D490 and 1:200 for mAb 1A1 whilst mAb 1E5 had to be used undiluted. With mAb I2, no staining of M. bovis antigen was achieved. Because of these results, immunohistochemical staining in lungs of calves was carried out on sections pretreated with trypsin. 3.4. Immunohistochemical labelling of M. bovis antigens The distribution of M. bovis antigen detected with the different antibodies used is summarised in Table 3. With pAb D490, M. bovis antigen was detected in all 8 inoculated calves. In 6 of these 7 animals, mAb 1A1 gave positive cytoplasmic immunolabelling of

This study demonstrated that in lungs of calves after experimental respiratory infection with a clonal variant of M. bovis type strain PG45 an acute inflammatory response develops within 48 h p.i. and that variation in expression of M. bovis Vsp antigens occurs in vivo. In the lungs of all inoculated calves of this study numerous macrophages and fewer numbers of neutrophilic granulocytes were found. In cattle, both alveolar macrophages (AMs) and pulmonary intravascular macrophages (PIMs) are recognised by the CD68 antibody used in the present study (Singh et al., 2004). Lung macrophages are known to produce several pro-inflammatory cytokines, i.e. TNF-a, IL-8 and IL-1b (Delclaux & Azoulay, 2003). TNF-a is thought to be a key mediator in eliciting the recruitment of neutrophils to the lung in man and cattle as well (Zhang et al., 2000; Singh et al., 2004). In vitro studies showed that bovine AMs produce TNF-a after infection with M. bovis strain PG45 (Jungi et al., 1996). So far, information about the possible production of specific mediators and cytokines in lung tissue of M. bovis infected calves are not available. It may be speculated, however, that induction of pro-inflammatory cytokines responsible for attraction of neutrophils also occurs in vivo. Specific virulence factors of M. bovis which could be involved in the induction of lung lesions are still unknown. In former studies, a polysaccharide toxin was isolated from M. bovis, but direct toxic effects of this toxin on bovine cells were not observed (Geary et al., 1981). Recruitment of neutrophils into lungs is a characteristic feature found in several pulmonary bacterial infections in man and animals and neutrophils are thought to induce lung lesions by releasing toxic oxygen radicals and proteolytic enzymes (Khair et al., 1996). M. bovis, beside immune reactive effects (Razin et al., 1998), has been reported to have also immunosuppressive characteristics inhibiting neutrophil degranulation, oxidative burst and mitogen-induced proliferation of bovine lymphocytes (Finch & Howard, 1990; Thomas et al., 1991; Vanden Bush & Rosenbusch, 2004). Studies in naturally and experimentally infected calves suggest that M. bovis is an important predisposing factor in bovine respira-

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I. Buchenau et al. / Research in Veterinary Science 89 (2010) 223–229 Table 3 Immunohistochemical labelling results for M. bovis antigens in lungs of experimentally infected calves and of control calves. Calf No.

Euthanasia (days p.i.)

Macrophagesa

AMb

Interalveolar septa

Exudate in lumina of bronchi/bronchioli

Surface of larger airway and/ or alveolar epithelium

Epithelium (larger airways, peribronchial glands)

1 2 3

2 2 4

D490 D490, 1E5, 1A1 D490, 1E5, 1A1

D490 D490, 1A1 D490, 1A1

D490c D490d No immunolabelling

D490, 1A1 D490, 1E5 D490, 1A1

D490, 1A1 D490, 1A1 D490, 1A1

4

4

D490

D490, 1A1

D490d, 1A1d

No immunolabelling

D490, 1A1

5 6 7

6 6 10

D490, 1A1 D490 D490, 1A1

D490 D490, 1A1 D490

No immunolabelling D490d, 1A1d No immunolabelling

No immunolabelling No immunolabelling D490

D490, 1A1 D490, 1A! D490, 1A1

8 NCe

10 14

No immunolabelling No immunolabelling

D490, 1A1 D490, 1A1

–g

D490 No immunolabelling No immunolabelling

No immunolabelling No immunolabelling

GNCf

D490 No immunolabelling No immunolabelling

D490 D490 No immunolabelling No immunolabelling D490 D490 No immunolabelling D490 No immunolabelling No immunolabelling

No immunolabelling

No immunolabelling

D490, 1A1

a

Macrophages located in interalveolar septa. Alveolar macrophages. c Extracellularly located antigen. d Positive immunolabelling of macrophages. D490, positive immunolabelling with pAb D490. 1E5, positive immunolabelling with mAb 1E5. 1A1, positive immunolabelling with mAb 1A1. e Negative (M. bovis uninfected) control calf. f Two negative (M. bovis uninfected) gnotobiotic control calves (see acknowledgements). g Euthanased after vaccination with bovine respiratory syncytial virus. b

Fig. 3. Immunolabelling of M. bovis Vsp antigens with mAb 1A1 in pulmonary macrophages of an experimentally infected calf (6 days p.i.). ABC method. 630.

tory disease leading to invasion of other highly pathogenic bacteria, i.e. P. multocida and M. haemolytica (Gourlay & Houghton, 1985; Rodríguez et al., 1996). The findings that in two calves of our study a secondary infection with these two bacterial species occurred supports this view of other investigators, even though it is not unlikely that the TBL procedure itself was the cause of the secondary infection. In chronic cases of natural and experimental M. bovis infection often necrotising lung lesions appearing as necrotic foci or marked caseonecrotic bronchopneumonia occur (Caswell & Archambault, 2008). Caseonecrotic bronchopneumonia is a distinctive lesion and there is strong evidence that M. bovis plays a causal role, but pulmonary coagulative necrosis is not pathognomonic for any infection (Caswell & Archambault, 2008; Thomas et al., 1986). In one calf of this study, which had necrotic lung lesions at 4 days p.i. both M. bovis and M. haemolytica were isolated from lung tissue sampled at necropsy. Caseous necrosis in experimental M. bovis infection was not seen in calves examined less than 2 weeks p.i. (Thomas et al., 1986; Rodríguez et al., 1996). Therefore, and because so-called oat cells, which are a characteristic feature of M. haemolytica infection (Tatum et al., 1998), were present, the necrotic changes found in one calf of our study were most likely induced by secondary infection with M. haemolytica. It cannot be ex-

cluded that this secondary infection was the result of TBL procedure by which bacteria from the upper airways were flushed into the lung. Following respiratory infection with M. bovis, a specific immune response resulting in production of antibodies to M. bovis Vsp antigens occurs (Brank et al., 1999). Furthermore, a local pulmonary immune response develops in inflamed lungs which is characterised by hyperplasia of BALT also known as ‘‘cuffing pneumonia” (Nicholas & Ayling, 2003). In this study, hyperplasia of BALT was found in 7 of 8 infected calves examined between 2 and 10 days p.i. Because of histological findings in lungs of clinically healthy calves and cattle proliferation of BALT is thought to be induced by various infectious stimuli occurring after birth (Anderson et al., 1986). The finding that in the present study proliferation of BALT was present in the two calves necropsied at day 2 p.i. may indicate that M. bovis stimulates a local immune response in the lungs of the host already during these early postinfectious stages. Histological investigations showed that BALT is missing in neonatal calves but is present in four-month old animals (Anderson et al., 1986). At necropsy, the inoculated calves of this study were between three and four weeks old. Histological descriptions of the presence or absence of BALT in healthy calves of this age range are not available. Therefore, although in the control calf of this study BALT was not present and although the animals used in this study had been clinically healthy since birth, it cannot be completely excluded that BALT hyperplasia seen in infected animals could, at least partially, represent a reaction to infectious agents other than M. bovis. Furthermore, we cannot exclude that in those two animals, from which other pathogenic bacteria, i.e.P. multocida and M. haemolytica, either alone or together with M. bovis were isolated at day 4 p.i., BALT proliferation could also, at least in part, be due to these secondary bacterial infections. In this study, positive immunohistochemical staining reactions for Vsp antigens by applying AR techniques was achieved for two of the three mAbs (1A1, 1E5) tested. Negative immunohistochemical staining reaction with mAb I2 with specificity for the non-Vsp membrane surface antigen pMB67 of M. bovis is presumably due to destruction of antigenic epitopes recognised by this antibody because of formalin fixation and processing techniques used for embedding of lung tissue samples.

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Immunohistochemical staining seen in epithelial cells of bronchi, bronchioli and peribronchial glands with pAb D490 and mAb 1A1 used in this study most likely is non-specific because a similar reaction pattern was also found in the control animal. In future studies, application of a recently developed in situ hybridisation method (Jacobsen et al., 2010) by using DNA probes on tissue sections from the calves of this study may be useful to analyse in which types of cells and/or other lung tissue locations M. bovis DNA is present and to compare the results with immunohistochemical staining reactions for protein antigens. The results of this study indicate that M. bovis Vsps undergo phase variation in the respiratory tract of infected calves in vivo. Vsp antigens were widely distributed within the lungs of the infected calves of this study and were already present at day two p.i. within the cytoplasma of numerous macrophages and in other compartments of the lung. The two Vsp-specific mAbs used in this study, besides with Vsp A, also react with Vsp B (mAb 1E5), Vsp C (mAb 1E5 and mAb 1A1), and other Vsps (mAb 1A1), respectively. By immunohistochemical examination with these two mAbs, positive staining reactions were seen in 7 of 8 calves. However, as the immunohistochemical labelling pattern obtained with mAb 1A1 in the majority of lung tissue samples examined did not correspond with the immunohistochemical staining pattern obtained with mAb 1E5 and vice versa (see Table 3), it can be concluded that in these samples Vsps other than Vsp A or Vsp C were expressed. Calf No. 2, for instance, revealed positive immunohistochemical staining reaction of the surface of larger airways with mAb 1E5, not however with mAb 1A1, indicating the expression of Vsp B. In contrast, the same locations in lung tissue samples from calves No. 1 and 3 showed positive staining reactions with mAb 1A1 but not with mAb 1E5, assuming the expression of antigens of the Vsp protein family with a Vsp specificity different from that of Vsp A, Vsp B and Vsp C. Likewise, the failure to immunostain with mAb 1A1 and mAb 1E5 those tissue areas that positively reacted with pAb D490 (e.g., the surface of larger airways of calf No. 7) indicates the OFF expression state of at least Vsp A, Vsp B and Vsp C in these tissue areas. The results therefore suggest that after inoculation of the host with a Vsp A-expressing clonal variant of M. bovis, clonal subpopulations develop, which also express other Vsps, i.e. Vsp B, or Vsps other than Vsp A, Vsp B and Vsp C. Furthermore, it was demonstrated by Western blot analysis that M. bovis recovered from the lung of one of the infected animals (No. 5) expressed a Vsp phenotype different from the Vsp A phenotype of the inoculum (Poumarat et al., 1998). Interestingly, these additional studies have also indicated that the Vsps of M. bovis appear not only to vary in expression but also in size during in vivo infection, as in the TBL samples from two infected animals (Nos. 5 and 6) the size of the Vsp A product expressed was found to decrease over time (Poumarat et al., 1998). Overall the results obtained in this study demonstrate that the bovine host is obviously unable to eliminate M. bovis during infection, at least during the time period examined shortly after inoculation (between 2 and 10 days p.i.). The results presented also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease in M. bovis infected calves. Conflict of interest statement None declared. Acknowledgements The authors are grateful to Dr L.H. Thomas, Institute of Animal Health, Compton, Newbury, Berkshire, UK, for donating paraffinembedded tissue blocks from two calves used as additional negative controls for the immunohistochemical staining reactions.

References Adegboye, D.S., Halbur, P.G., Cavanaugh, D.L., Werdin, R.E., Chase, C.C.L., Miskimins, D.W., Rosenbusch, R.F., 1995. Immunohistochemical and pathological study of Mycoplasma bovis-associated lung abscesses in calves. Journal of Veterinary Diagnostic Investigation 7, 333–337. Anderson, M.L., Moore, P.F., Hyde, D.M., Dungworth, D.L., 1986. Bronchus-associated lymphoid tissue in the lungs of cattle: relationship to age. Research in Veterinary Science 41, 211–220. Behrens, A., Heller, M., Kirchhoff, H., Yogev, D., Rosengarten, R., 1994. A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis. Infection and Immunity 62, 5075–5084. Behrens, A., Poumarat, F., Le Grand, D., Heller, M., Rosengarten, R., 1996. A newly identified immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic variation. Microbiology 142, 2463–2470. Brank, M., Le Grand, D., Poumarat, F., Bezille, P., Rosengarten, R., Citti, C., 1999. Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle. Clinical and Diagnostic Laboratory Immunology 6, 861–867. Caswell, J.L., Archambault, M., 2008. Mycoplasma bovis pneumonia in cattle. Animal Health Research Reviews 8, 161–186. Cho, H.J., Ruhnke, H.L., Langford, E.V., 1976. The indirect hemagglutination test for the detection of antibodies in cattle naturally infected with mycoplasmas. Canadian Journal of Comparative Medicine 40, 20–29. Delclaux, C., Azoulay, E., 2003. Inflammatory response to infectious pulmonary injury. European Respiratory Journal (Suppl. 42), 10s–14s. Finch, J.M., Howard, C.J., 1990. Inhibitory effect of Mycoplasma dispar and Mycoplasma bovis on bovine immune responses in vitro. Zentralblatt für Bakteriologie (Suppl. 20), 563–569. Gagea, M.I., Bateman, K.G., van Dreumel, T., McEwen, B.J., Carman, S., Archambault, M., et al., 2006. Diseases and pathogens associated with mortality in Ontario beef feedlots. Journal of Veterinary Diagnostic Investigation 18, 18–28. Geary, S.J., Tourtelotte, M.E., Cameron, J.A., 1981. Inflammatory toxin from Mycoplasma bovis: isolation and characterisation. Science 212, 1032–1033. Gourlay, R.N., Thomas, L.H., Howard, C., 1976. Pneumonia and arthritis in gnotobiotic calves following inoculation with Mycoplasma agalactiae subsp bovis. The Veterinary Record 98, 506–507. Gourlay, R.N., Houghton, S.B., 1985. Experimental pneumonia in conventionally reared and gnotobiotic calves by dual infection with Mycoplasma bovis and Pasteurella haemolytica. Research in Veterinary Science 38, 377–382. Gourlay, R.N., Thomas, L.H., Wyld, S.G., 1989. Increased severity of calf pneumonia associated with the appearance of Mycoplasma bovis in a rearing herd. The Veterinary Record 124, 420–422. Howard, C.J., Thomas, L.H., Parsons, K.R., 1987. Immune response of cattle to respiratory mycoplasmas. Veterinary Immunology and Immunopathology 17, 401–412. Jacobsen, B., Hermeyer, K., Jechlinger, W., Zimmermann, M., Spergser, J., Rosengarten, R., Hewicker-Trautwein, M., 2010. In situ hybridization for the detection of Mycoplasma bovis in paraffin-embedded lung tissue from experimentally infacted calves. Journals of Veterinary Diagnostic Investigation 22, 90–93. Jungi, T.W., Krampe, M., Sileghem, M., Griot, Ch., Nicolet, J., 1996. Differential and strain-specific triggering of bovine alveolar macrophage effector functions by mycoplasmas. Microbial Pathogenesis 21, 487–498. Khair, O.A., Davies, R.J., Devalia, J.L., 1996. Bacterial-induced release of inflammatory mediators by bronchial epithelial cells. European Respiratory Journal 9, 1913–1922. Khodakaram-Tafti, A., López, A., 2004. Immunohistopathological findings in the lungs of calves naturally infected with Mycoplasma bovis. Journal of Veterinary Medicine A 51, 10–14. Le Grand, D., Solsona, M., Rosengarten, R., Poumarat, F., 1996. Adaptive surface antigen variation in Mycoplasma bovis to the host immune response. FEMS Microbiology Letters 144, 267–275. Le Grand, D., Calavas, D., Brank, M., Citti, C., Rosengarten, R., Bezille, P., et al., 2002. Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France. Veterinary Record 150, 268–273. Lopez, A., Maxie, G., Ruhnke, L., Savan, M., Thomson, R.G., 1986. Cellular inflammatory response in the lungs of calves exposed to bovine viral diarrhoea virus, Mycoplasma bovis and Pasteurella multocida. American Journal of Veterinary Research 47, 1283–1286. Lysnyansky, I., Rosengarten, R., Yogev, D., 1996. Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. Journal of Bacteriology 178, 5395–5401. Lysnyansky, I., Sachse, K., Rosenbusch, R., Levisohn, S., Yogev, D., 1999. The vsp locus of Mycoplasma bovis: gene organisation and structural features. Journal of Bacteriology 181, 5734–5741. Lysnyansky, I., Ron, Y., Yogev, D., 2001. Juxtaposition of an active promoter to vsp genes via site-specific DNA inversions generates antigenic variation in Mycoplasma bovis. Journal of Bacteriology 183, 5698–5708. Nicholas, R.A., Ayling, R.D., 2003. Mycoplasma bovis: Disease, diagnosis, and control. Research in Veterinary Science 74, 105–112. Pfützner, H., Sachse, K., 1996. Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Revue Scientifique et Technique – Office International des Épizooties 15, 1477–1494. Poumarat, F., Perrin, M., Belli, P., Martel, J.L., 1987. Recherche des anticorps antiMycoplasma bovis dans le serum des bovins à l’aide de la réaction

I. Buchenau et al. / Research in Veterinary Science 89 (2010) 223–229 d’hemagglutination passive: valeur et limites de la réaction. Revue de Médicine Vétérinaire 138, 981–989. Poumarat, F., Perrin, B., Longchambon, D., 1991. Identification of ruminant mycoplasmas by dot immunobinding on membrane filtration (MF dot). Veterinary Microbiology 29, 329–338. Poumarat, F., Solsona, M., Boldini, M., 1994. Genotype, protein and antigen variability of Mycoplasma bovis. Veterinary Microbiology 40, 305–321. Poumarat, F., Le Grand, D., Citti, C., & Rosengarten, R. 1998. A new quest: following Mycoplasma bovis variable surface proteins in the host. In Proceedings of the 12th conference of the international organisation for mycoplasmology, sydney, Australia (p. 127). Razin, S., Yogev, D., Naot, Y., 1998. Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews 62, 1094–1156. Rodríguez, F., Bryson, D.G., Ball, H.J., Forster, F., 1996. Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves. Journal of Comparative Pathology 115, 151–162. Rosengarten, R., Behrens, A., Stetefeld, A., Heller, M., Ahrens, M., Sachse, K., et al., 1994. Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins. Infection and Immunity 62, 5066–5074. Sachse, K., Helbig, J.H., Lysnyansky, I., Grajetzki, C., Muller, W., Jacobs, E., et al., 2000. Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infection and Immunity 68, 680–687.

229

Singh, B., Pearce, J.W., Gamage, L.N., Janardhan, K., Caldwell, S., 2004. Depletion of pulmonary intravascular macrophages inhibits acute lung inflammation. American Journal of Physiology – Lung Cellular and Molecular Physiology 286, L363–L372. Tatum, F.M., Briggs, R.E., Srinand, S., Zehr, E.S., Hsuan, S.L., Whitely, L.O., et al., 1998. Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterisation and virulence. Microbial Pathogenesis 24, 37–46. Thomas, L.H., Howard, C.J., Stott, E.J., Parsons, K.R., 1986. Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus. Veterinary Pathology 23, 571–578. Thomas, C.B., Van Enss, P., Wolfgram, L.J., Riebe, J., Sharp, P., Schultz, R.D., 1991. Adherence to bovine neutrophils and suppression of neutrophil chemiluminescence by Mycoplasma bovis. Veterinary Immunology and Immunopathology 27, 365–381. Vanden Bush, T.J., Rosenbusch, R.F., 2004. Characterisation of a lympho-inhibitory peptide produced by Mycoplasma bovis. Biochemical and Biophysical Research Communications 315, 336–341. Wise, K., Yogev, D., Rosengarten, R., 1992. Antigenic variation. In: Maniloff, J., McElhaney, R.N., Finch, L.R., Baseman, J.B. (Eds.), Mycoplasmas: Molecular biology and pathogenesis. American Society for Microbiology, Washington, DC, pp. 473–489. Zhang, P., Summer, W.R., Bagby, G.J., Nelson, S., 2000. Innate immunity and pulmonary defence. Immunological Reviews 173, 39–51.