A Morphologic and Immunohistochemical Study of ... - SAGE Journals

Vet Pathol 40:395–404 (2003)

A Morphologic and Immunohistochemical Study of the Bronchus-associated Lymphoid Tissue of Pigs Naturally Infected with Mycoplasma hyopneumoniae J. SARRADELL, M. ANDRADA, A. S. RAMI´REZ, A. FERNAŃDEZ, J. C. GO´MEZ-VILLAMANDOS, A. JOVER, H. LORENZO, P. HERRAÉZ, AND F. RODRI´GUEZ Department of General Pathology, School of Veterinary Medicine, National University of Rosario, Casilda, Santa Fe, Argentina (JS, MA); Section of Epidemiology and Preventive Medicine, Veterinary Faculty, University of Las Palmas de Gran Canaria, Gran Canaria, Spain (ASR); Department of Comparative Pathology, Veterinary Faculty, University of Las Palmas de Gran Canaria, Gran Canaria, Spain (AF, HL, PH, FR); and Department of Pathological Anatomy, Veterinary Faculty, University of Co´rdoba, Spain (JCGV, AJ) Abstract. Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchusassociated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD31, CD41, and CD81), IgG1 or IgA1 lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG1 and IgA1 plasma cells. CD41 cells predominated over CD81 cells. Local humoral immunity appears to play an important role in the infection. Key words: Bronchus-associated lymphoid tissue; immune response; immunohistochemistry; Mycoplasma hyopneumoniae; pigs.

Porcine enzootic pneumonia (PEN), primarily caused by Mycoplasma hyopneumoniae (Mh),18 is a contagious disease found in most swine herds. The disease is characterized by high morbidity and low mortality rates. The intensive evolution of the pork production industry during the past few decades has been a primary factor associated with pneumonia being the most economically important disease in finishing pigs.30 A common feature in infections of the respiratory tract is persistence of the causal organisms at the mucosal surface. It has been reported that this contributes to the formation of the lymphoid accumulations seen in the lungs of mice, rats, hamsters, cattle, and pigs as a result of mycoplasma infections.6,9,13,25 Several mycoplasmal products and mycoplasma membrane proteins, which are immunogenic during Mh infection, have been described so far as being responsible for the massive lymphoid hyperplasia around air-

ways.11,17,19,22,26,29 Histologic lesions in the chronic severe stages of PEN are characterized by lymphoid hyperplasia of the bronchus-associated lymphoid tissue (BALT) causing obliteration of the lumens of bronchioles and atelectasis of surrounding alveoli.3,8,11,16,17 Mh colonizes the luminal surface of bronchial and bronchiolar epithelial cells without penetration into the lung parenchyma,5,7,15 inducing reduction in ciliary activity, loss of cilia, microcolony formation, and accumulation of Mh organisms in relation to exfoliated epithelial cells.14 Infections with Mh may result in suppression of the humoral immune response due to a decrease in antibody production by B lymphocytes. Cellular immunity also may be suppressed by the inhibition of macrophage-mediated phagocytosis. These effects are most pronounced in the early stages of the infection, although they may remain for several weeks after the initial infection.17

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Sarradell, Andrada, Ramı´rez, Fernańdez, Go´mez-Villamandos, Jover, Lorenzo, Herraéz, and Rodrı´guez

Table 1.

Vet Pathol 40:4, 2003

Methods of analysis used to evaluate naturally infected and control pigs in this study (n 5 60).*

Pig Nos.

Histology

Microbiology

Transmission electron microscopy

1–25‡ 26–29§ 30–60\

1 1 1

1 1 1

1 1 2

Immunohistochemistry Infectious agents†

Cellular immunophenotyping

1 1 1

1 1 2

* All laboratory procedures were used on three lung samples taken from each pig. † Immunohistological staining included the antigen detection of Mycoplasma hyopneumoniae, Porcine Respiratory and Reproductive Syndrome virus, and Aujeszky’s Disease virus. ‡ Porcine enzootic pneumonia-infected pigs. § Negative control pigs. \ Pigs excluded from the ultrastructural and immunohistochemical studies.

Phenotypic characterization of the cells present in the lungs of mycoplasma-infected mice, hamsters, and cows has indicated that many are plasma cells actively synthesizing immunoglobulins.6,11,13 There was an increase in the number of cells producing immunoglobulins in the lungs of pigs experimentally infected with Mh.19 Locally secreted IgA prevented the adhesion of mycoplasmas to ciliated epithelium, and IgG participated in opsonization and phagocytosis by alveolar macrophages,26,27,31 but the exact role of antibody in the development of the infection or protection from the disease has not been elucidated. The determination of histologic changes in the CD41, CD81, macrophages, dendritic cells, and IgG1 and IgA1 cells present in the BALT may help identify the local immune response in PEN-affected animals. The objectives of this research were to describe changes in the histologic relationships and distribution of different cell types in the BALT of pigs naturally infected with Mh by using histologic and immunohistochemical techniques. Materials and Methods Animals Lung specimens from 25 (Nos. 1–25) pigs were selected from 56 pigs submitted to the slaughterhouse of Las Palmas (Spain) that had gross lesions indicative of PEN. The three selection criteria that were required to be fulfilled by the pigs included in the study were: 1. Animals with areas of pulmonary consolidation affecting 20–40% of the lung parenchyma, ranging from dark red through grayish pink, and with the presence of pale nodules indicative of lymphoid hyperplasia. 2. Lungs with histologic lesions indicative of PEN without the presence of exudative bronchopneumonia, suggesting the participation of other bacteria. These lesions consisted of different gradations of peribronchial and peribronchiolar lymphocytic hyperplasia, infiltration of mononuclear cells in alveolar septa and lamina propria of bronchioles, and slight or moderate number of inflammatory cells in airways and alveoli. 3. Immunohistochemical and microbiologic detection of

Mh without the presence of other mycoplasmas, bacteria, or viral pathogens for the porcine respiratory tract. Animals containing more than 103 color-changing units of Mh per gram of lung were included as positive controls. Four control lungs (Nos. 26–29) were included in the study as negative controls. Table 1 summarizes the samples and the procedures carried out in the study. Three samples from the cranio-ventral region of the lungs of all 60 pigs (56 natural cases and 4 negative controls) were taken for the microbiologic, histologic, ultrastructural, and immunohistochemical analyses. Each sample was divided into four parts: one for the microbiologic study, one for formalin fixation, one to be cooled in liquid nitrogen, and the last one for glutaraldehyde fixation. All tissues embedded in paraffin were tested by using immunohistochemistry to rule out infection with porcine respiratory and reproductive syndrome virus (PRRSV) and Aujeszky’s disease virus. After the 25 Mh-infected pigs were selected on the basis of the criteria described above, immunohistochemistry for cellular antigens and ultrastructural examination were carried out in 29 animals (25 selected Mhinfected and 4 control pigs). Tissue processing Lung tissues were processed for histopathologic and immunohistochemical examination. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin and sectioned at 4 mm. Additional tissues were snap-frozen in Optimal Cutting Temperature (O.C.T.y, Tissue-Tekt, Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands), immersed in 2-methylbutane (Merck, Darmstadt, Germany), and cooled in liquid nitrogen. Frozen tissues were stored at 270 C. Serial tissue sections (7 mm) were then cut with a cryostat at 220 C and stored at 270 C until use. These snapfrozen tissues were used for the detection of the CD4 and CD8 antigens using the immunohistochemical technique. Histopathology Sections of formalin-fixed, paraffin-embedded, and frozen lung samples were stained with hematoxylin and eosin (HE). Classification of the histologic lesions followed the semiquantitative criteria of Livingston et al. (1972):16 2 indicates negative;

BALT in Porcine Enzootic Pneumonia


Table 2. Antibodies

397

Primary antibodies used in the immunohistochemical studies.

Specificity*

Source†

Dilution

Fixation

Polyclonal antibodies Aujeszky Mycoplasma hyopneumoniae Lysozyme S-100 SDOW17 CD3 IgG GASw/IgA(Fc)

Aujeszky M. hyopneumoniae Lysozyme S-100 PRRS CD3 IgG IgA

UCO DVL

1 : 800 1 : 1000

Formalin Formalin

DAKO DAKO DVS DAKO SIGMA NORDIC

1 : 500 1 : 500 1 : 1000 1 : 500 1 : 400 1 : 1000

Formalin Formalin Formalin Formalin Formalin Formalin

Monoclonal antibodies 1AC7 MAC 387 74-12-4 MCA 1223 MCA 1335

PRRS L1 CD4 CD8 SLA-II

INGENASA DAKO VMRD SEROTEC SEROTEC

1 : 250 1 : 100 1 : 300 1 : 100 1 : 100

Formalin Formalin Snap-frozen Snap-frozen Formalin

* Porcine Respiratory and Reproductive Syndrome. † UCO 5 University of Co´rdoba, Spain; DVL 5 Danish Veterinary Laboratory, Copenhagen V, Denmark; DAKO 5 Dako, Glostrup, Denmark; DVS 5 Department of Veterinary Science, South Dakota State University, USA; SIGMA 5 Sigma Chemical Company, St. Louis, MO, USA; NORDIC 5 Nordic Immunological Laboratories, Tilburg, The Netherlands; INGENASA 5 Ingenierıá y Gene´tica Aplicada, Madrid, Spain; VMRD 5 Veterinary Medical Research and Development, Pullman Inc., Pullman, WA, USA; SEROTEC 5 Serotec Ltd., Oxford, UK.

1 indicates one or more lymphoid nodules involving the muscularis mucosae of bronchi and bronchioles; 11 indicates lymphoid nodules affecting the muscularis mucosae of bronchi and bronchioles as well as the presence of inflammatory cells in the septal wall, bronchus, and alveolar lumen; 111 indicates perivascular and peribronchiolar hyperplasia of the lymphoid tissue with inflammatory cells in the alveolar septa and neutrophils in the bronchial and alveolar lumens; and 1111 indicates massive perivascular and peribronchiolar lymphoid hyperplasia in extended areas of the lung parenchyma. Ultrastructural study Lung tissues were fixed by immersion in 2.5% glutaraldehyde buffered to pH 7.4 with 0.1 M phosphate buffer. For transmission electron microscopy, tissue blocks of 1–2 mm3 were postfixed in 1% osmium tetroxide and stained with 0.5% uranyl acetate. Tissues dehydrated in graded alcohols and propylene oxide were embedded in epoxy resin (Epon 812, Fluka, Switzerland). Sections 50–70 nm thick were cut on an LKB Ultratome Nova ultramicrotome (LKB-Produkter AB, Bromma, Sweden) using a diamond knife (Diatome LTD, Bienne, Switzerland), counterstained with uranyl acetate and lead citrate, and examined under a Philips CM-10 (Philips, Eindhoven, The Netherlands) electron microscope. From each block, 0.5-mm-thick sections were cut with glass knives, stained with toluidine blue, and scanned under a light microscope to ensure that the bronchial or bronchiolar epithelia, and BALT or parenchymal tissue were present.

Microbiology A modified SP4 medium (SP4-II)23 was used for mycoplasma isolation. Lung samples were taken using sterile swabs, which were then immersed in a tube containing 2 ml SP4-II liquid medium and incubated with agitation at 37 C. Additional samples were cultured by routine methods for bacteria and fungi. Subcultures on solid SP4-II medium were incubated at 37 C in a humidified ambient atmosphere and read on the basis of the Mh colony morphology.10 Mh was differentiated from M. hyorhinis by using routine biochemical tests.23 Complementarily, an indirect immunoperoxidase test on nitro-cellulose filter papers was used to differentiate Mh from M. flocculare and M. hyorhinis.29 Three primary antibodies raised against Mh were used, one polyclonal (Danish Veterinary Laboratory, DK-1790 Copenhagen V, Denmark) and two monoclonal (Veterinary Science Division, DANI, Belfast, UK). The J strain of Mh (American Type Culture Collection) was included as a positive control. The results were considered positive when positively immunolabeled microcolonies were observed. Immunohistochemistry The avidin–biotin–peroxidase (ABC) method,21 with some modifications, was used on sections of both formalin-fixed, paraffin-embedded tissues and frozen tissues. The former were dewaxed and rehydrated, and endogenous peroxidase activity was blocked by incubation of the sections with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature. Sections were then treated with pronase (Sigma, St. Louis, MO, USA) 0.1% in Tris-buffered saline (TBS),

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Fig. 1. Lung; pig No. 7. Peribronchiolar and perivascular lymphocytic hyperplasia and some macrophages and neutrophils within the lumen of an airway (arrow). HE. Bar 5 95 mm. Fig. 2. Lung; pig No. 9. Mh observed as granular brown labeling lining the ciliated epithelium of a bronchus. ABC method, Harris’s hematoxylin counterstain. Bar 5 65 mm. Fig. 3. Lung; pig No. 18. Immunolabeling with the anti-lysozyme pAb in numerous macrophages located in perifollicular areas of the BALT. ABC method, Harris’s hematoxylin counterstain. Bar 5 65 mm.



pH 7.2, for 4 minutes at room temperature. When snap-frozen tissue samples were used, endogenous peroxidase activity was blocked by incubation with 0.05% phenyl-hydrazine (Sigma) in TBS. All tissue sections were incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. The primary antibodies (Table 2) were then applied overnight at 4 C (formalin-fixed tissues) or for 2 hours at 37 C (frozen tissues). Preliminary experiments were carried out to determine the optimal dilution for the different antibodies. A biotinylated goat or rabbit anti-mouse IgG (Vector) for monoclonal antibodies or anti-rabbit IgG (Vector) for polyclonal antibodies, both diluted 1 : 200, were applied as secondary reagents. An ABC complex (Vector) diluted 1 : 50 was applied as the third reagent. The sections were then incubated for 1 minute with 3,39-diaminobenzidine tetrahydrochloride (Sigma) 0.035% in TBS containing hydrogen peroxide 0.1%. After rinsing in tap water, slides were lightly counterstained with Harris’s hematoxylin and mounted under DPX mountant (BDH Laboratory Supplies, Poole, UK) for microscopy. Sections in TBS treated with normal goat or rabbit serum, or inappropriate antibodies that replaced the specific primary antibodies, were included as negative controls. Sections from the lymph nodes of one control animal (No. 26) were used as a positive control for primary antibodies, except those specific for Mh, Aujeszky’s disease virus and PRRSV, in which experimentally infected tissue sections were processed alongside the test slides. Cell counting and statistical analysis Positively labeled cells were counted in 20 selected BALT fields (4003 magnification) from each animal. The result was expressed as the mean of the percentage of positive cells per field 6 SD. A comparative analysis of the number of immunolabeled cells in the control and infected animals was made using the nonparametric test W of Wilcoxon by means of the SPSSt 10.0 computer program (SPSS Inc. Headquarters, S. Wacker Drive, Chicago, IL, USA). P , 0.05 was considered statistically significant.

399

Results Histology

Lung tissues infected with Mh showed a catarrhal bronchointerstitial pneumonia, with development of prominent peribronchial, peribronchiolar, and perivascular accumulations of lymphoid cells and formation of lymphoid follicles (Fig. 1). In severe cases, the lymphoid nodules caused narrowing of the lumina of airways. The epithelium over prominent nodules of all animals was degenerated and exfoliated with different severities. Diffuse loss of cilia from many bronchial surfaces was detected when PEN-infected animals were compared with similar areas of controls. There was hyperplasia of goblet cells in bronchi and large bronchioles. Accumulations of small and large lymphocytes, plasma cells, and neutrophils thickened the alveolar septa. There was moderate hyperplasia of type II pneumocytes of inflamed alveoli. In accordance with the semiquantitative criteria of Livingston et al. (1972),16 there were 4 lungs without lesions (controls), 811, 5111, and 121111 (PENaffected animals). Microbiology

Mycoplasmal and bacterial isolation results are listed in Table 3. Mh was isolated and immunolabeled in all the 25 infected animals included as positive controls in the study. From the lung tissue of the infected animals discharged from subsequent studies (pig Nos. 30–60), Mh was the most frequent infectious agent detected (17 animals), followed by M. hyorhinis (8 animals). Immunohistochemistry

Expression of Mh antigen. Mh organisms were immunolabeled as a granular brown reaction at the luminal surface of bronchial and bronchiolar epithelial

← Fig. 4. Lung; pig No. 20. Immunoreaction to S-100 in mononuclear cells with a stellate morphology (dendritic cells) (arrows) located in the germinal centers of the BALT. ABC method, Harris’s hematoxylin counterstain. Bar 5 75 mm. Fig. 5. Lung; pig No. 20. Immunoreaction with the anti-human myeloid-histiocyte antigen in neutrophils and macrophages located within blood vessels (arrow), between epithelial cells, and in the lumen of an airway. ABC method, Harris’s hematoxylin counterstain. Bar 5 50 mm. Fig. 6. Lung; pig No. 17. CD31 immunolabeled lymphocytes prominently located in the perifollicular areas of the BALT and in the lamina propria of a bronchiole (arrow). ABC method, Harris’s hematoxylin counterstain. Bar 5 65 mm. Fig. 7. Lung; pig No. 19. Numerous aggregates of CD41 lymphocytes among mononuclear cells in the BALT. ABC method, Harris’s hematoxylin counterstain. Bar 5 65 mm. Fig. 8. Lung; pig No. 8. Immunoreaction with SLA-II mAb in the cytoplasm of monunuclear cells scattered in a germinal center and more pronounced in perifollicular areas. ABC method, Harris’s hematoxylin counterstain. Bar 5 65 mm. Fig. 9. Lung; pig No. 12. IgA1 B lymphocytes around bronchial submucosal glands. ABC method, Harris’s hematoxylin counterstain. Bar 5 55 mm.

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Table 3.


Summary of microbiologic and histologic finding for pig groups I–III.* Group I (1–25)†

Group II (26–29)†

Group III (30–60)†

Infectious agent Mycoplasma hyopneumoniae Mycoplasma hyorhinis Mycoplasma flocculare Pasteurella multocida Bordetella bronchiseptica Haemophilus spp. Actinomyces pyogenes Escherichia coli Streptococcus suis PRRS‡ Aujeszky’s disease virus

25/25 0/25 0/25 0/25 0/25 0/25 0/25 0/25 0/25 0/25 0/25

0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4

17/31 8/31 4/31 2/31 3/31 2/31 1/31 3/31 2/31 1/31 0/31

Predominant histological change Catarrhal bronchointerstitial pneumonia Fibrinous bronchopneumonia Suppurative bronchopneumonia

25/25 0/25 0/25

0/4 0/4 0/4

18/31 7/31 6/31

* Microbiology included mycoplasma, bacteria, and viruses isolated or immunolabeled in naturally infected and control lungs from pigs in the study (n 5 60). † Number of positive lungs per number of lungs evaluated in each group. ‡ Porcine Respiratory and Reproductive Syndrome.

cells of all PEN-infected animals (Fig. 2). Control tissues did not have immunostaining with this antibody. Expression of lysozyme. The immunoreactivity with the anti-lysozyme polyclonal antibody (Fig. 3) was granular to diffuse in the cytoplasm of a moderate number of macrophages, mainly in the perifollicular areas of the BALT and scattered in the germinal centers. Numerous immunoreactive cells were observed in alveolar spaces and in the bronchiolar lumina of PEN-infected animals. Expression of S-100. Diffuse nuclear and cytoplasmic immunolabeling was detected in numerous (Table 4) stellate cells with long cytoplasmic processes (dendritic cells) in the BALT (Fig. 4). Some T lymphoTable 4.

Summary of immunohistochemical findings.*

Antibody

PEN-affected animals (n 5 25)

Controls (n 5 4)

Lysozyme S-100 L1 CD3 CD4† CD8† CD4/CD8† SLA-II IgG† IgA†

0.81 6 0.28 10.69 6 1.03 2.65 6 0.13 25.73 6 3.25 19.61 6 1.05 4.12 6 0.42 4.55 22.88 6 2.72 1.92 6 1.69 1.91 6 0.89

0.76 6 0.16 11.40 6 1.56 1.19 6 0.28 30.17 6 2.75 10.20 6 2.41 20.85 6 1.37 0.46 27.09 6 3.11 1.03 6 0.27 0.99 6 0.11

* Mean 6 SD of the percentage of positive cells in the bronchusassociated lymphoid tissue for each antibody. † Antigens with significant differences (P , 0.05) between infected and control pigs.

cytes, smooth muscle cells, nerve endings, chondrocytes, and macrophages of the alveolar walls also were positive. Expression of L1 antigen. Immunoreactivity with anti-human myeloid-histiocyte antigen (MAC 387) (Table 2) was observed in the cytoplasm of intravascular neutrophils and monocytes present in a small number in all animals as well as in neutrophils and macrophages between the epithelium of airways and within bronchi and bronchioles (Fig. 5). Expression of CD3 antigen. With the CD3 polyclonal antibody (Table 2), intense immunoreactivity was detected on the surface and in cytoplasm of mononuclear cells. CD31 lymphocytes were found mainly in perifollicular or interfollicular areas (primarily in cases with 1111 severity) (Fig. 6) as well as within infiltrating lymphoid cells in the alveolar walls. Numerous positive cells were observed in the lamina propria of bronchi and bronchioles and within the airway epithelium. Expression of CD4 and CD8 antigen. The CD41 subpopulation was more numerous than the CD81 in all infected lungs, resulting in a median CD4 : CD8 of 4.55 : 1 (Table 4). The CD41 lymphocytes (Fig. 7) were located mainly in the perifollicular areas of the BALT, whereas the CD81 lymphocytes were observed under the bronchial and bronchiolar epithelium, between epithelial cells, and scattered in the follicles. The same localization, but with opposite proportions (median ratio is 0.46 : 1), was observed in control lungs (Table 4), where the percentage of CD41 lym-



401

phocytes decreased to a median of 10.2, and the percentage of CD81 lymphocytes reached a median of 20.85. The CD4 : CD8 ratio was significantly higher in the PEN cases than in the controls (P , 0.0001) (Table 4). Expression of SLA II antigen. There was a positive immunoreaction for SLAII antigen in lymphocytes and in cells with an irregular morphology and cytoplasmic prolongations (dendritic cells) present in the central areas of the BALT. They were located between the lymphocytes of the BALT and under the lamina propria of bronchi and bronchioles. The immunoreaction was especially intense at the perifollicular areas of the BALT (Fig. 8). Expression of IgG. With the polyclonal antibody against porcine IgG (Table 2), immunoreactivity was detected in the cytoplasm and on the surface of a moderate number (Table 4) of plasma cells as well as on the surface of some lymphocytes, located primarily in the germinal centers of the BALT and in the lamina propria of bronchi and bronchioles. Plasma cells and lymphocytes in the alveolar walls and serum within some vessels also were labeled. There was a statistically significant difference (P , 0.001) between median values of 1.92% positive cells in test cases and 1.03% in controls (Table 4). Expression of IgA. When the polyclonal antibody against porcine IgA was used, immunoreaction was detected in the cytoplasm of a moderate (Table 4) number of plasma cells and on the surface of some lymphocytes located in the germinal areas of the BALT as well as in the submucosa of bronchi and bronchioles. Numerous positive cells were observed around bronchial submucosal glands (Fig. 9). There was a statistically significant difference (P , 0.001) between median values of 1.91% of positive cells in test cases and 0.99% in controls. Results obtained from the immunohistochemical analysis are shown in Table 4. Although the relative proportion of lysozyme, S-100, L1, CD3, and SLA-II positive cells was not significantly different between control and PEN-affected animals, the number of all these cell types increased in infected animals with BALT hyperplasia. Ultrastructural study

Numerous mycoplasma organisms were attached to cilia of epithelial cells in bronchi and bronchioles. In some cases, structures compatible with pili of adhesion were seen linking mycoplasma organisms to cilia (Fig. 10). Loss of cilia and vacuolization of the cytoplasm of epithelial cells (Fig. 11) were commonly detected. Intraepithelial lymphocytes (Fig. 12) and plasma cells in the submucosa confirmed the immunohistochemical

Fig. 10. Lung; pig No. 9. Mycoplasma organisms with a structure compatible with pilus of adhesion. Uranyl acetate and lead citrate. Bar 5 0.5 mm. Fig. 11. Lung; pig No. 17. Vacuolization and loss of cilia of bronchial epithelial cells. Uranyl acetate and lead citrate. Bar 5 3.75 mm.

402



findings. The BALT areas showed changes indicative of lymphoid hyperplasia such as mitotic figures and apoptotic bodies. Dendritic cells had an irregular nucleus (Fig. 13) and condensed chromatin in a narrow zone in the inner nuclear membrane. Cytoplasmic prolongations between dendritic cells and lymphocytes (Fig. 14) and desmosomal junctions connecting dendritic cell extensions were frequently observed. Discussion

Fig. 12. Lung; pig No. 19. Intraepithelial lymphocyte showing a round nucleus with a pattern of peripheral heterochromatin and a narrow rim of cytoplasm. Uranyl acetate and lead citrate. Bar 5 2 mm. Fig. 13. Lung; pig No. 6. A cell compatible with dendritic cells in the germinal center of the BALT showing stellate nucleus with condensed chromatin in the inner nuclear membrane and cytoplasmic prolongations. Uranyl acetate and lead citrate. Bar 5 1 mm. Fig. 14. Lung; pig No. 12. Dendritic processes extending between the germinal center lymphocytes. Uranyl acetate and lead citrate. Bar 5 1 mm.

In this study, pigs infected with Mh had a cranioventral pattern of catarrhal bronchointerstitial pneumonia; prominent peribronchial, peribronchiolar, and perivascular accumulations of lymphoid tissue; formation of lymphoid follicles; and thickening of the alveolar septa. These findings closely resembled those previously described by others in the chronic stages of the infection.3,8,28 The obliteration of the lumens of bronchioli producing atelectasis of surrounding alveoli has been described previously1,8,16,17 and might be explained by the conjunction of four mechanisms: accumulation of mucus and inflammatory exudates due to loss of mucociliary function, increased activity of mucus-secreting cells and altered glycoprotein production in globet cells, bronchoconstriction by chemical mediators released by alveolar macrophages and inflammatory cells, and pressure from the aggregates of lymphoid tissue. A granular immunoreaction was shown by Mh located on the luminal surface of bronchial and bronchiolar epithelial cells of all PEN-affected animals. This localization was observed previously by Doster et al. (1988) using an indirect immunoperoxidase method and by Kwon and Chae (1999) using in situ hybridization. Mycoplasma organisms were not detected within epithelial cells, BALT, or alveolar macrophages, confirming the results of other authors15 using in situ hybridization. However, those workers detected Mh DNA in alveolar and interstitial macrophages of naturally infected pigs. This difference could be due to the higher sensitivity of the technique compared with immunocytochemistry or with the persistence of mycoplasmal DNA devoid of proteins within phagocytic cells. Previous workers3,16 also noted that Mh are rarely present in alveoli. It is suggested from these observations that the alveolar lesions may not be due to the direct pathogenic action of the mycoplasma but may occur secondary to the lesions (loss of cilia, exfoliation of epithelial cells, presence of viscous exudate in the airways, and accumulation of inflammatory cells) and functional impairments of the respiratory tract (reduction of mucociliary activity and suppression of the humoral immune response). Additionally, proinflammatory cytokines such as tumor necrosis factor alpha, interleukin 1 (IL-1), IL-6, IL-8, and prostaglandin E2, produced during the experimental infec-



tion, stimulate the accumulation and activation of inflammatory cells in alveolar lumen and septa, have a direct cytotoxic effect on endothelial cells, cause tissue injury, and can regulate the response of numerous cell types, especially neutrophils, macrophages, and B and T lymphocytes.1,17,22 The ultrastructural study confirmed the presence of mycoplasma organisms in the spaces between cilia and showed piluslike structures of adhesion between mycoplasma and cilia. This confirmed the observations of Blanchard et al. (1992),5 who described the presence of fine fibrils radiating from the mycoplasma to the cilia. We found epithelial cells with vacuolization of the cytoplasm and loss of cilia. These findings confirm that Mh adheres to cilia using fine fibrils, and this adherence is associated with degenerative changes in the ciliated epithelial cells. Cell-mediated immune mechanisms are important in the development of pneumonic lesions during Mh infection because of two major effects: increasing the phagocytic and cytotoxic activities of macrophages and initiating the chronic inflammatory response.1,9,11,16,17,19 Mh organisms, after colonizing the respiratory tract, stimulate alveolar macrophages to secrete cytokines that induce a persistent, immune and inflammatory response.1 These cytokines, nonspecific mitogenic activity of Mh on lymphocytes,19 or a number of immunogenic mycoplasmal proteins28 have been incriminated in the intense lymphocyte accumulation around bronchi and bronchioles, infiltration of inflammatory cells into the airways, and lymphoid hyperplasia characteristic of Mh-infected pigs. The predominant phagocytic cell infiltrating the lung in PEN-affected animals in this study was the macrophage. This was confirmed by the use of the anti-lysozyme polyclonal antibody (pAb) and is consistent with the view that this cell type is an effector mechanism operating against mycoplasmal infections.13,24 Antigen-presenting cells phagocytose, process, and present antigen.2,12 In the current study, stellate cells with long cytoplasmic processes (dendritic cells) were detected in germinal centers of the BALT in the ultrastructural study and by using the anti–SLA-II and S100 antibodies. This observation revealed that the BALT possesses a high structural organization that may facilitate presentation of antigen to CD41 helper T lymphocytes. CD41 T lymphocytes represented the predominant subset responsible for the observed BALT hyperplasia, with statistically significant differences between PEN-affected and control animals. These CD41 T cells are capable of activating macrophages, resulting in efficient intracellular killing of mycoplasmas.20 CD81 lymphocytes were observed between epithelial cells in both the immunohistochemical and the ultrastructural studies. This finding suggests

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some cytotoxic and regulatory activity of the immune system at this level.4 The local humoral immune response appears to play a role in recovery from mycoplasma infection and subsequent immunity.6,13 Phenotypic characterization of the cells present in the lungs of pigs experimentally infected with Mh revealed an increased number of cells producing immunoglobulins.19 There were significantly more IgG1 and IgA1 cells in PEN cases compared with controls in the current study. Numerous IgA1 cells were observed around bronchial submucosal glands. Immunoreaction to porcine IgG and IgA was detected as well on some lymphocytes, located mainly in the perifollicular and subepithelial tissues. Local IgA secretion prevents the adhesion of mycoplasmas to the ciliated epithelium, and IgG enhances their phagocytosis by alveolar macrophages.26,27,31 Consequently, the local humoral immune response appears to play a major role in the pathogenesis of the infection and subsequent immunity.6,13,19 The information obtained about the changes in the cellular populations in the BALT of naturally infected animals could help to identify strategies for vaccination for Mh. In conclusion, the BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were especially located in perifollicular areas of the BALT, in the lamina propria and within the airway epithelium. Plasma cells containing IgG or IgA were present at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG1 and IgA1 plasma cells. CD41 cells predominated over CD81 cells. Local humoral immunity, as determined by the increase of IgG1 and IgA1 lymphocytes and plasma cells in the BALT, bronchial and bronchiolar walls, and alveolar septa, appears to play an important role in the infection. Acknowledgements We thank P. Castro and A. Afonso for their excellent technical assistance. Support for J. Sarradell during his stay in the Veterinary Faculty of Las Palmas was provided by a grant (FOMEC 70-609-B) from the National University of Rosario, Argentina. This work was supported financially by grants from the Consejerıá de Educacioń y Ciencia, Gobierno de Canarias (PI2001-127) and from the Ministerio de Educacioń y Ciencia (AGL2001-3842).

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Request reprints from Dr. F. Rodrı´guez, Department of Comparative Pathology, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontanã, 35416 Arucas, Gran Canaria (Spain). E-mail: [email protected].