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Immunohistochemical Characterization of Lung Lesions Induced Experimentally by Mycoplasma agalactiae andMycoplasma bovis in Goats
J. Comp. Path. 2000, Vol. 123, 285–293 doi:10.1053/jcpa.2000.0418, available online at http://www.idealibrary.com on
Immunohistochemical Characterization of Lung Lesions Induced Experimentally by Mycoplasma agalactiae and Mycoplasma bovis in Goats F. Rodrı´guez, J. Sarradell, J. B. Poveda∗, H. J. Ball† and A. Ferna´ndez Department of Comparative Pathology and ∗Infectious Diseases, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontan˜a, 35416 Arucas, Gran Canaria, Spain and †Department of Agriculture for Northern Ireland, Veterinary Sciences Division, Stoney Road, Stormont, BT43SD Belfast, Northern Ireland, UK
Summary Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. Both Mycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3+ T lymphocyte, and the ratio of CD4+: CD8+ cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4+ T lymphocytes, plays a prominent role in the acute and subacute phase 2000 Harcourt Publishers Ltd of these infections.
Introduction Mycoplasma agalactiae is the main cause of contagious agalactia in sheep and goats, an economically important disease occurring primarily in the Mediterranean countries but also reported from many other areas of the world. The disease usually takes the form of mastitis and arthritis in the postpartum female (Hasso et al., 1994), but cases of pneumonia and pleurisy have also been reported (Cottew and Lloyd, 1965; DaMassa et al., 1992; Hasso et al., 1994). Mycoplasma bovis, an important cause of pneumonia, arthritis and mastitis, occurs in cattle in many areas of the world. The agent has also been isolated from the goat respiratory tract (Ojo and 0021–9975/00/080285+09 $35.00
Kede, 1976; DaMassa et al., 1992). Contagious agalactia has been reported in goats in the Canary Islands (Real et al., 1994), M. agalactiae being the predominant (90%) cause of the disease. A feature common to infections of the respiratory tract is the persistence of the causal organisms at the mucosal surface. It has been suggested 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 infection (Cassell et al., 1974; Fernald, 1979; Howard et al., 1987; Ross and Young, 1993). Phenotypic characterization of these cells in the lungs of infected mice, hamsters and cows has indicated that many are plasma cells, actively synthesizing immunoglobulins (Cassell et al., 1974; Gourlay and 2000 Harcourt Publishers Ltd
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Howard, 1982; Howard et al., 1987). Consequently, the local humoral immune response appears to play a major role in recovery from infection and subsequent immunity by promoting immunoglobulin-mediated host defences (Cassell et al., 1974; Howard et al., 1987). Studies in rats have indicated that a cell-mediated T-lymphocyte response produces resistance after infection, due to a non-specific mitogenic property of some mycoplasma strains (Fernald, 1979). In the present study, the immune response to M. agalactiae and M. bovis in the caprine respiratory system was investigated by determining immunohistochemically the distribution of CD3, CD4, CD8, MHC class II and / T lymphocytes and B cells in the bronchus-associated lymphoid tissue (BALT) of young experimentally infected goats. Materials and Methods Animals For the experiment, 12 goats aged 3 months were purchased shortly after birth from a local farm in which mycoplasma disease had never been detected. Before infection, nasal mucus, nasopharyngeal swabs and blood samples were obtained on three occasions from each animal for microbiological and serological examination for viruses, mycoplasmas and bacteria pathogenic for the caprine respiratory tract. The tests were carried out by monoclonal and polyclonal antibody-based ELISAs or by culture on routine and specific media for bacteria and mycoplasmas, respectively (Real et al., 1994; Ball et al., 1996; Rodrı´guez et al., 1996). Clinical examinations were also carried out on three occasions. Experimental Infection Ten goats were inoculated by the intranasal and intratracheal routes with recent mycoplasma isolates: five animals (nos 1–5) received M. agalactiae strain 123/97 and five (nos 6–10) M. bovis strain MC1750. Seed cultures of these organisms, consisting of mycoplasma broth culture or suspensions of mycoplasmas grown on solid medium, were stored at −70°C. When required, they were thawed, diluted 1 in 10 in fresh mycoplasma broth and incubated overnight to provide the inocula. Each animal was given 5 ml intranasally and 5 ml intratracheally, these doses being repeated 24 h later. The total number of mycoplasmas received by each goat was approximately 3 × 109 per day. Two control animals (nos 11 and 12) were similarly
treated with sterile mycoplasma broth. The goats were humanely killed 14 or 21 days after the second day of inoculation. The experiment was carried out in accordance with the Code of Practice (International Council for Laboratory Animal Science, Bethesda, 1985) for Housing and Care of Animals used in Scientific Procedures. Samples At necropsy, tracheal and lung tissue was taken for histopathological and immunohistochemical examination. Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax and sectioned (4 m). Additional samples were snapfrozen in OCT (Miles, Elkhart, IN, USA), immersed in 2-methylbutane (Merk, Darmstadt, Germany) and cooled in liquid nitrogen. Frozen samples were stored at −80°C; serial tissue sections (7 m) were then cut with a cryostat at −20°C and stored at −80°C until used. Histopathology Sections of formalin-fixed, paraffin wax-embedded tissue samples were stained with haematoxylin and eosin (HE). Microbiology Samples from trachea and lung were cultured in mycoplasma broth medium. Additional samples were cultured by routine methods for bacteria and fungi. Animals were examined for mycoplasmaemia at the time of killing by adding 10 ml of blood containing heparin 10 U/ml to 40 ml of mycoplasma broth. After incubation for 5 days, the broth cultures were examined for M. agalactiae and M. bovis by plating on mycoplasma solid medium. Serology Antibody to M. agalactiae or M. bovis in serum samples taken shortly before infection and at the time of killing was measured by an indirect ELISA with whole cell antigen preparations (De´niz et al., 1996). A fourfold or greater rise in titre was considered to indicate infection. Immunohistochemistry The avidin-biotin-peroxidase (ABC) method (Navarro et al., 1996), with some modifications, was used on sections of both formalin-fixed paraffin
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Experimental Mycoplasma Pneumonia in Goats Table 1 Primary antibodies used in the immunohistochemical technique Antibody 295 (pAb) Lysozyme (pAb) S-100 (pAb) MAC 387 (mAb) CD3 (pAb) GC50A1 (mAb) CACT80C (mAb) H42A (mAb) CACTB6A (mAb) Goat Igs (pAb)
Specificity
Source
Dilution 1 in
M. bovis/agalactiae Lysozyme S-100 Myeloid/histiocyte Ag CD3 CD4 CD8 MHC II / Goat Igs
VSD DAKO DAKO DAKO DAKO VMRD VMRD VMRD VMRD DAKO
1000 500 500 100 500 50 100 10 000 200 100
pAb, polyclonal antibody; mAb, monoclonal antibody; Igs, immunoglobulins; VSD, Veterinary Science Division, Department of Agriculture, Belfast; DAKO, Dako, Glostrup, Denmark; VMRD, VMRD Pullman Inc., Pullman, USA.
wax-embedded tissues and frozen tissues. The former were de-waxed and rehydrated, and endogenous peroxidase activity was blocked by incubation of the sections with hydrogen peroxide 0·3% in methanol for 30 min at room temperature. Sections were then treated with pronase (Sigma, St Louis, MO, USA) 0·1% in Tris-buffered saline (TBS), pH 7·2, for 4 min at room temperature. When snap-frozen tissue samples were used, endogenous peroxidase activity was blocked by incubation with phenyl-hydrazine (Sigma) 0·05% in TBS, without subsequent pronase treatment. All tissue sections were incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. The primary antibodies (Table 1) were then applied overnight at 4°C (formalin-fixed tissue) or for 2 h at 37°C (frozen tissue). Preliminary experiments were carried out to determine the optimal dilution for the different antibodies. All monoclonal antibodies (mAbs) were raised against lymphocytes of bovine origin but had been shown previously to cross-react with caprine antigens (Navarro et al., 1996). Biotinylated goat or rabbit anti-mouse IgG (for mAbs) and anti-rabbit IgG (for polyclonal antibodies), both obtained from Vector Laboratories and diluted 1 in 200, were used as the secondary reagents. An ABC complex (Vector) diluted 1 in 50 was applied as the third reagent. The sections were then incubated for 1 min with 3,3′-diaminobenzidine tetrahydrochloride (Sigma) 0·035% in TBS containing hydrogen peroxide 0·1%. After rinsing in tap water, they were lightly counterstained with Harris’s haematoxylin, and mounted under DPX for microscopy. Sections in which the specific primary antibodies were replaced by
TBS, normal goat or rabbit serum or inappropriate antibodies were included as negative controls. Sections from the lymph nodes of one control animal (no. 12) were used as a positive control for all of the primary antibodies except those specific for M. agalactiae and M. bovis. Immunohistochemical labelling of mammary and lung lesions induced by these agents was carried out with M. bovis antiserum (pAb 295), which had been shown to cross-react with M. agalactiae (Rodrı´guez et al., 1996). Cell Counting and Statistical Analysis Positively labelled cells were counted in 20 selected BALT fields (×400 magnification) in each of two tissue sections of lung from each goat. The result was expressed as the mean of the percentage of positive cells per field ± standard deviation (SD). A comparative analysis of the number of CD4+, CD8+, MHCII+ and plasma cells containing immunoglobulins, in the control and infected animals, was made with the Pearson test of correlation by means of a Statgraphics 5·0 computer program (Statical Graphics Corporation, Rockville, Maryland, USA). P<0·05 was considered significant. Results Clinical, Post-mortem, Microbiological and Serological Findings Clinical examinations (body temperature, appetite, pulse and respiratory rate, and presence of nasal secretions) were carried out twice daily from the time of infection until slaughter. One day postinfection, two goats (nos 2 and 5) exhibited a moderate and transient increase in respiratory rate
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(50–60/min), coughing and inappetance, all of which returned to normal within 3 days after infection. All 10 infected goats showed transient pyrexia. At necropsy, no pulmonary consolidation was seen. Before infection, no viruses, pathogenic bacteria or mycoplasmas were detected in any animal. M. agalactiae or M. bovis was recovered post mortem from the trachea or lung (or both) of all but two (nos 8 and 10) infected animals. No septicaemia was detected in any animal at the time of killing, but high titres (600–800) of antibodies to M. agalactiae or M. bovis were invariably demonstrated. No bacterial respiratory pathogens were isolated from the lungs. Histopathology The main change in the goats killed 14 days (nos 1, 2, 6, 7) or 21 days (nos 3, 4, 5, 8, 9, 10) after infection consisted of peribronchiolar and perivascular lymphoid hyperplasia (Table 2). Moderate to dense accumulations of lymphocytes and smaller numbers of plasma cells were found in the walls of the airways and around adjacent blood vessels (Fig. 1). This caused narrowing of some bronchiolar lumina, and atelectasis of alveolar regions distal to occluded bronchioles. The alveolar septa appeared thickened due to the accumulation of macrophages, lymphocytes of variable size and plasma cells. The intra-alveolar exudate consisted predominantly of macrophages, but small numbers of lymphocytes, plasma cells and neutrophils were also present. These changes were more prominent in animals killed at 21 days post-infection (Table 2) than in those killed at 14 days.
Expression of S-100. Diffuse nuclear and cytoplasmic immunolabelling was detected in stellate cells with long cytoplasmic processes (dendritic cells) in the BALT. Some T lymphocytes, smooth muscle cells, nerve endings, chondrocytes and macrophages of the alveolar walls were also positive. Expression of myeloid-histiocyte antigen. Immunoreactivity with anti-human myeloid-histiocyte antigen mAb was observed in a few intravascular neutrophils and monocytes. No immunoreaction was noted within alveoli or in cells within the lumina of airways. Expression of CD3 antigen. With the CD3 polyclonal antibody, intense immunoreactivity was detected in the cytoplasm of lymphocytes located in the BALT, mainly in the perifollicular areas, as well as within infiltrating lymphoid cells in the alveolar walls (Fig. 3). Expression of CD4, CD8, /, and MHC class II antigens. With the CD4 monoclonal antibody, the CD4+ subpopulation was more numerous than the CD8+, resulting in a CD4:CD8 ratio of >1 (Table 2) in all infected goats. The CD4+ lymphocytes (Fig. 4) were mainly located in the perifollicular areas of the BALT, whereas the CD8+ lymphocytes (Fig. 5) were located under the epithelium and scattered in the follicles. The / T lymphocytes in the BALT were sparse and appeared closely associated with the bronchioles. MHC class II+ antigen (Fig. 6) was labelled both in lymphocytes and in cells with an irregular morphology and cytoplasmic prolongations, between the lymphocytes and under the epithelial basal membrane.
Immunohistochemistry The results obtained from the BALT are summarized in Table 2. Expression of M. agalactiae and M. bovis antigen. The polyclonal antibody pAb 295 invariably failed to detect mycoplasma antigens in lung tissue. Expression of lysozyme. Immunoreactivity with the anti-lysozyme polyclonal antibody was observed within the cytoplasm of some macrophages, mainly those located in the perifollicular areas of the BALT. Numerous immunoreactive macrophages were observed in alveolar spaces, and in the bronchiolar lumina (Fig. 2). Some intravascular neutrophils were also immunoreactive.
Expression of immunoglobulins. With the polyclonal antibody against caprine immunoglobulins, immunoreactivity was detected in the cytoplasm of a moderate number of plasma cells and on the surface of some lymphocytes, located mainly in the perifollicular and subepithelial tissues. Plasma cells and lymphocytes in the alveolar walls were also labelled. Discussion A common feature of mycoplasma pneumonias is the increase of mononuclear cells in the lung, as for example with M. pulmonis in mice, M. bovis in cattle, M. gallisepticum in chickens and M. hyopneumoniae in pigs (Fernald, 1979; Howard et al., 1987). This leads to peribronchiolar and perivascular accumulations of these cells, many of
– 0·63±0·23 3·30±1·68 0·23±0·14
41·21±5·56 31·21±4·67 14·28±3·55 2·18 0·18±0·10 36·12±7·98 7·81±2·77
52·15±3·21 37·52±4·12 12·95±2·17 2·89 0·34±0·11 37·02±3·67 6·97±1·97
2† (++)
– 0·71±0·20 3·3±0·43 0·31±0·18
1† (++)
44·22±5·65 30·55±3·34 12·79±2·89 2·38 1·26±0·65 45·39±6·84 5·91±1·63
– 1·23±0·78 3·61±1·89 0·20±0·11
3‡ (+++)
38·81±4·88 35·65±5·33 15·83±4·21 2·25 0·76±0·32 41·50±7·43 7·11±2·04
– 0·44±0·75 2·25±0·54 0·43±0·42
4‡ (+++)
6† (+)
59·10±6·23 37·05±5·44 11·05±2·64 3·35 0·97±0·22 48·51±4·29 6·38±1·87
40·32±6·33 31·14±3·45 12·17±2·58 2·55 1·16±0·34 49·30±7·49 3·72±1·12
– – 0·32±0·12 1·30±0·42 3·14±0·44 2·94±1·65 0·37±0·21 0·42±0· 12
5‡ (++)
40·31±7·47 28·25±4·87 14·10±3·75 2·00 0·58±0·21 46·01±5·31 7·32 ±1·68
– 0·74±0·24 3·23±1·68 0·41±0·22
7† (++)
59·12±6·88 42·70±5·56 20·16±4·87 2·11 0·35±0·14 44·50±5·56 8·90±2·32
– 1·15±0·66 2·37±0·75 0·34±0·11
8‡ (+++) – 0·83±0·36 4·16±1·68 0·30±0·12
9‡ (+++)
57·84±6·31 31·90±5·66 15·37±4·21 2·07 0·89±0·21 44·69±4·76 9·48±2·66
Mycoplasma bovis
52·13±5·46 34·23±7·43 16·19±2·64 2·11 0·78±0·31 45·75±5·56 8·54±1·67
– 1·91±0·35 2·98±0·68 0·39±0·14
10‡ (+++)
34·23±3·56 16·33±2·74 17·72±3·56 0·92 0·84±0·21 25·14±3·54 1·22±0·35
– 0·97±0·18 2·37±0·91 0·46±0·32
11† (−)
38·35±5·33 19·15±3·54 21·23±4·36 0·90 0·91±0·24 23·53±5·67 1·40±0·14
– 1·22±0·24 3·88±1·44 0·36±0·75
12‡ (−)
sterile broth controls
∗Means ± SD of the percentages of positive cells in the BALT. †Killed 14 days (‡21 days) after inoculation. Lymphoid hyperplasia: (−), none; (+), slight; (++), moderate; (+++), severe. The Pearson test revealed a significant difference (P<0·05) for CD4+, CD8+, MHCII+ and Igs+ cells between the control and infected goats; no significant difference was obtained with the other antibodies.
pAb 295 Lysozyme S-100 Myeloid/ histiocyte antigen CD3 CD4 CD8 CD4/CD8 / MHC II Immunoglobulins
Antibodies
Mycoplasma agalactiae
Results∗ in goats 1–12, inoculated with
Table 2 Histological and immunohistochemical results
Experimental Mycoplasma Pneumonia in Goats
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Fig. 1. Lung of goat no. 7, showing peribronchiolar and perivascular lymphocytic hyperplasia. HE. ×200.
Fig. 2. Goat no. 2. Immunoreaction to lysozyme in the cytoplasm of macrophages within alveoli. ABC.×200.
Fig. 3. Goat no. 1. CD3+ lymphocytes located in the BALT and alveolar walls. ABC.×400.
Fig. 4. Goat no. 9. Numerous CD4+ lymphocytes, mainly located in the perifollicular areas of the BALT. ABC.×400.
Fig. 5. Goat no. 10. Occasional CD8+ lymphocytes among mononuclear cells in the BALT. ABC.×400.
Fig. 6. Goat no. 8. Immunoreaction with MHC class II mAb in the cytoplasm of mononuclear cells, some of them showing cytoplasmic processes (dendritic cells). ABC.×400.
Experimental Mycoplasma Pneumonia in Goats
which are likely to be synthesizing antibody (Cassell et al., 1974; Gourlay and Howard, 1982; Howard et al., 1987) as part of the immune response to the infection. In the present study, goats inoculated with M. agalactiae or M. bovis via the respiratory tract showed similar accumulations of leucocytes. Although significant increases in immunoglobulinproducing cells in the BALT and humoral antibody were detected, the main cellular type in the lymphoreticular hyperplasia was the CD3+ T lymphocyte, which indicated the significant role of cellular immunity. Alveolitis, the presence of inflammatory cells in alveoli, and peribronchiolar accumulations of mononuclear cells are typical of the cuffing interstitial pneumonia (Gourlay and Howard, 1982) that characterizes various mycoplasma respiratory infections. The persistence of organisms and antigen at the mucosal surface may be responsible for the formation of lymphoid accumulations around airways (Howard et al., 1987). The severity of the lesions produced under experimental conditions may be influenced by the degree of colonization or virulence (or both) of the mycoplasma strain used. Under the conditions used in the present experiment, neither M. agalactiae nor M. bovis alone induced respiratory disease severe enough to cause death. The polyclonal antiserum pAb 295 used in this study failed to demonstrate M. agalactiae or M. bovis antigens in lung tissue proved by microbiological methods to be infected, despite the fact that polyclonal and monoclonal antibodies against mycoplasma species are regarded as a potential diagnostic tool (Howard et al., 1987; Rodrı´guez et al., 1996). Immune defence mechanisms in the respiratory tract include humoral immunity, mediated by B lymphocytes. This has three major effects, namely, influencing those biological activities mediated by specific antibody, increasing phagocytic defence mechanisms, and initiating the inflammatory response (Busse, 1991). In the present study, plasma cells containing goat immunoglobulins were observed in the BALT of all infected animals, and specific antibodies to M. agalactiae or M. bovis were demonstrated in serum. This would suggest a significant role for the humoral immune response in clearing mycoplasmas from the respiratory tract. In addition, cell-mediated immunity, effected by T lymphocytes, has two major effects, namely, increasing the phagocytic and cytotoxic activities of macrophages, and initiating the chronic inflammatory response. The predominant phagocytic
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cell infiltrating the lung after M. agalactiae or M. bovis infection in the goats in this study was the macrophage. This observation, which was confirmed by the use of the anti-lysozyme pAb, is consistent with the view that this cell type is a major effector mechanism operating against mycoplasma infections (Howard et al., 1987; Rodrı´guez et al., 1996). CD4+ T lymphocytes represented the predominant subset responsible for the observed BALT hyperplasia. These T cells are known to be capable of activating macrophages, resulting in efficient intracellular killing of mycoplasmas (Nash et al., 1992). It is likely that CD4+ cells, through the production of T helper (Th)1 cytokines such as interferon (IFN), play an important role in the clearance of mycoplasmas from the lung. Synthesis of interleukin (IL)1 and tumour necrosis factor (TNF) by activated macrophages may also result in profound effects on the immune response, as these two cytokines are important mediators of the inflammatory response, inducing secretion of acute phase proteins, stimulating release of prostaglandins and proteolytic enzymes, and enhancing cellular infiltration through chemotaxis and mitogenicity. IL-1 also plays an important role in specific antigenic immune responses, acting as an early signal in T-cell activation, and participating in the cytokine network that regulates Bcell differentiation (Mossmann and Coffman, 1989; Nash et al., 1992). Cytokines play a key role in the regulation of macrophage functional capacity during antigen-specific and non-specific immune responses, firstly as molecules that directly influence the functional status of macrophages, and secondly as the effector molecules of activated macrophages (Cao et al., 1989). Cytokines can also “upregulate” MHC antigen expression on macrophages (Nash et al., 1992) and, since T-cell receptors recognize antigen in the context of MHC class I or class II molecules, this activation of the receptors of macrophages has important implications for the initiation and development of the immune response. In this study, the number of mononuclear cells expressing MHC class II antigen showed a significant increase in the infected goats. Such activation might participate in the signal regulation of antigen-presenting cells, through both humoral and cellular immunity. In the current study, sparse (2–3%) / T lymphocytes were observed in paracortical areas and medullary cords of lymph nodes of the uninfected goat (no. 12) used as a positive control (data not shown), while they were under 1% in the lungs of infected and control animals. The results closely
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resembled those obtained by Caro et al. (1998), with the same mAb, in peripheral blood and lymph nodes from goats aged 3 months. The number of T cells of this subset decreases with age in goats (Caro et al., 1998) and other young ruminants (Wyatt et al., 1994). Some authors have related such decreases to the functional capacity of this Tcell population to confer early cellular immunity on young animals that do not yet possess a mature system of / T cells (Hein et al., 1991). In conclusion, infection of young goats with M. agalactiae or M. bovis by the respiratory route induced a moderate bronchointerstitial pneumonia, characterized by BALT hyperplasia and infiltration of mononuclear cells into the alveolar walls. CD3+ T lymphocytes, cells expressing MHC class II+, and plasma cells containing caprine immunoglobulins constituted the main cellular types in the lymphoreticular hyperplasia. Acknowledgments Support for J. Sarradell during his stay in the Veterinary Faculty of Las Palmas de GC was provided by a grant (FOMEC 70-609-B) from the National University of Rosario, Argentina. This work was supported by grants PB96-0140 and PI1999-148 from the “Direccio´ n General de Ensen˜ anza Superior e Investigacio´ n Cientı´fica” and “Consejerı´a de Educacio´ n, Cultura y Deportes del Gobierno de Canarias”, respectively. References Ball, H. J., Finlay, D., Rodrı´guez, F. and Mackie, D. (1996). Monoclonal antibody detection of mycoplasma antigen as a diagnostic tool. In: Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, J. Frey and K. Sarris, Eds, European Cooperation on Scientific and Technical Research, Luxembourg, pp. 78–80. Busse, W. W. (1991). Pathogenesis and sequelae of respiratory infections. Review of Infectious Diseases, 13, 477–485. Cao, H., Wolf, R. G., Mreltzer, M. S. and Crawford, R. M. (1989). Differential regulation of Class II MHC determinants on macrophages by IFN- and IL-4. Journal of Immunology, 143, 3524–3531. Caro, M. R., Gallego, M. C., Buendia, A. J., Navarro, E. and Navarro, J. A. (1998). Postnatal evolution of lymphocyte subpopulations in peripheral blood and lymphoid organs in the goat. Research in Veterinary Science, 65, 145–148. Cassell, G. H., Lindsey, J. R. and Baker, H. J. (1974). Immune response of pathogen-free mice inoculated intranasally with Mycoplasma pulmonis. Journal of Immunology, 112, 124–136.
Cottew, G. S. and Lloyd, L. S. (1965). An outbreak of pleurisy and pneumonia in goats in Australia attributed to a mycoplasma species. Journal of Comparative Pathology, 75, 363–374. DaMassa, A. J., Wakenell, S. and Brooks, D. L. (1992). Mycoplasmas of goats and sheep. Journal of Veterinary Diagnostic Investigation, 4, 101–113. De´ niz, S., Real, F., Poveda, J. B., Acosta, B., Ramı´rez, A. S. and Ferna´ ndez, A. (1996). Epidemiological study of M. agalactiae and Mycoplasma mycoides subsp. mycoides in the Canary Islands. In: Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, J. Frey and K. Sarris, Eds, European Cooperation on Scientific and Technical Research, Luxembourg, pp. 128–130. Fernald, G. W. (1979). Humoral and cellular immune responses to mycoplasmas. In: The Mycoplasmas, Vol. 2, J. G. Tully and R. F. Whitcomb, Eds, Academic Press, New York, pp. 399–423. Gourlay, R. N. and Howard, C. J. (1982). Respiratory mycoplasmosis. Advances in Veterinary Science and Comparative Medicine, 26, 289–332. Hasso, S. A., Al-Darraji, A. M. and Al-Aubaidi, J. M. (1994). Pathology of experimentally induced contagious agalactia in goats. Small Ruminant Research, 13, 79–84. Hein, W. R., Dudler, L., Beya, M. F. and Mackay, C. R. (1991). Epitopes of T19 lymphocytes surface antigen are extensively conserved in ruminants. Veterinary Immunology and Immunopathology, 27, 173–181. Howard, C. J., Thomas, L. H. and Parsons, K. R. (1987). Immune response of cattle to respiratory mycoplasmas. Veterinary Immunology and Immunopathology, 17, 401–412. Mossmann, T. R. and Coffman, R. L. (1989). Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, 145–173. Nash, A. D., Barcham, G. J., Andrews, A. E and Brandon, M. R. (1992). Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production. Veterinary Immunology and Immunopathology, 31, 77–94. Navarro, J. A., Caro, M. R., Seva, J., Rosillo, M. C., Go´ mez, M. A. and Gallego, M. C. (1996). Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes. Veterinary Immunology and Immunopathology, 51, 147–156. Ojo, M. O. and Kede, B. O. (1976). Pathology of Mycoplasma agalactiae subsp. bovis in goat mammary gland. Veterinary Microbiology, 1, 19–22. Real, F., De´ niz, S., Acosta, B., Ferrer, O. and Poveda, J. B. (1994). Caprine contagious agalactia caused by Mycoplasma agalactiae in the Canary Islands. Veterinary Record, 135, 15–16. Rodrı´guez, F., Bryson, D. G., Ball, H. J. and Forster, F. (1996). Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves. Journal of Comparative Pathology, 115, 151–162.
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Received, January 21st, 2000 Accepted, May 15th, 2000
Immunohistochemical Characterization of Lung Lesions Induced Experimentally by Mycoplasma agalactiae and Mycoplasma bovis in Goats F. Rodrı´guez, J. Sarradell, J. B. Poveda∗, H. J. Ball† and A. Ferna´ndez Department of Comparative Pathology and ∗Infectious Diseases, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontan˜a, 35416 Arucas, Gran Canaria, Spain and †Department of Agriculture for Northern Ireland, Veterinary Sciences Division, Stoney Road, Stormont, BT43SD Belfast, Northern Ireland, UK
Summary Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. Both Mycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3+ T lymphocyte, and the ratio of CD4+: CD8+ cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4+ T lymphocytes, plays a prominent role in the acute and subacute phase 2000 Harcourt Publishers Ltd of these infections.
Introduction Mycoplasma agalactiae is the main cause of contagious agalactia in sheep and goats, an economically important disease occurring primarily in the Mediterranean countries but also reported from many other areas of the world. The disease usually takes the form of mastitis and arthritis in the postpartum female (Hasso et al., 1994), but cases of pneumonia and pleurisy have also been reported (Cottew and Lloyd, 1965; DaMassa et al., 1992; Hasso et al., 1994). Mycoplasma bovis, an important cause of pneumonia, arthritis and mastitis, occurs in cattle in many areas of the world. The agent has also been isolated from the goat respiratory tract (Ojo and 0021–9975/00/080285+09 $35.00
Kede, 1976; DaMassa et al., 1992). Contagious agalactia has been reported in goats in the Canary Islands (Real et al., 1994), M. agalactiae being the predominant (90%) cause of the disease. A feature common to infections of the respiratory tract is the persistence of the causal organisms at the mucosal surface. It has been suggested 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 infection (Cassell et al., 1974; Fernald, 1979; Howard et al., 1987; Ross and Young, 1993). Phenotypic characterization of these cells in the lungs of infected mice, hamsters and cows has indicated that many are plasma cells, actively synthesizing immunoglobulins (Cassell et al., 1974; Gourlay and 2000 Harcourt Publishers Ltd
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Howard, 1982; Howard et al., 1987). Consequently, the local humoral immune response appears to play a major role in recovery from infection and subsequent immunity by promoting immunoglobulin-mediated host defences (Cassell et al., 1974; Howard et al., 1987). Studies in rats have indicated that a cell-mediated T-lymphocyte response produces resistance after infection, due to a non-specific mitogenic property of some mycoplasma strains (Fernald, 1979). In the present study, the immune response to M. agalactiae and M. bovis in the caprine respiratory system was investigated by determining immunohistochemically the distribution of CD3, CD4, CD8, MHC class II and / T lymphocytes and B cells in the bronchus-associated lymphoid tissue (BALT) of young experimentally infected goats. Materials and Methods Animals For the experiment, 12 goats aged 3 months were purchased shortly after birth from a local farm in which mycoplasma disease had never been detected. Before infection, nasal mucus, nasopharyngeal swabs and blood samples were obtained on three occasions from each animal for microbiological and serological examination for viruses, mycoplasmas and bacteria pathogenic for the caprine respiratory tract. The tests were carried out by monoclonal and polyclonal antibody-based ELISAs or by culture on routine and specific media for bacteria and mycoplasmas, respectively (Real et al., 1994; Ball et al., 1996; Rodrı´guez et al., 1996). Clinical examinations were also carried out on three occasions. Experimental Infection Ten goats were inoculated by the intranasal and intratracheal routes with recent mycoplasma isolates: five animals (nos 1–5) received M. agalactiae strain 123/97 and five (nos 6–10) M. bovis strain MC1750. Seed cultures of these organisms, consisting of mycoplasma broth culture or suspensions of mycoplasmas grown on solid medium, were stored at −70°C. When required, they were thawed, diluted 1 in 10 in fresh mycoplasma broth and incubated overnight to provide the inocula. Each animal was given 5 ml intranasally and 5 ml intratracheally, these doses being repeated 24 h later. The total number of mycoplasmas received by each goat was approximately 3 × 109 per day. Two control animals (nos 11 and 12) were similarly
treated with sterile mycoplasma broth. The goats were humanely killed 14 or 21 days after the second day of inoculation. The experiment was carried out in accordance with the Code of Practice (International Council for Laboratory Animal Science, Bethesda, 1985) for Housing and Care of Animals used in Scientific Procedures. Samples At necropsy, tracheal and lung tissue was taken for histopathological and immunohistochemical examination. Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax and sectioned (4 m). Additional samples were snapfrozen in OCT (Miles, Elkhart, IN, USA), immersed in 2-methylbutane (Merk, Darmstadt, Germany) and cooled in liquid nitrogen. Frozen samples were stored at −80°C; serial tissue sections (7 m) were then cut with a cryostat at −20°C and stored at −80°C until used. Histopathology Sections of formalin-fixed, paraffin wax-embedded tissue samples were stained with haematoxylin and eosin (HE). Microbiology Samples from trachea and lung were cultured in mycoplasma broth medium. Additional samples were cultured by routine methods for bacteria and fungi. Animals were examined for mycoplasmaemia at the time of killing by adding 10 ml of blood containing heparin 10 U/ml to 40 ml of mycoplasma broth. After incubation for 5 days, the broth cultures were examined for M. agalactiae and M. bovis by plating on mycoplasma solid medium. Serology Antibody to M. agalactiae or M. bovis in serum samples taken shortly before infection and at the time of killing was measured by an indirect ELISA with whole cell antigen preparations (De´niz et al., 1996). A fourfold or greater rise in titre was considered to indicate infection. Immunohistochemistry The avidin-biotin-peroxidase (ABC) method (Navarro et al., 1996), with some modifications, was used on sections of both formalin-fixed paraffin
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Experimental Mycoplasma Pneumonia in Goats Table 1 Primary antibodies used in the immunohistochemical technique Antibody 295 (pAb) Lysozyme (pAb) S-100 (pAb) MAC 387 (mAb) CD3 (pAb) GC50A1 (mAb) CACT80C (mAb) H42A (mAb) CACTB6A (mAb) Goat Igs (pAb)
Specificity
Source
Dilution 1 in
M. bovis/agalactiae Lysozyme S-100 Myeloid/histiocyte Ag CD3 CD4 CD8 MHC II / Goat Igs
VSD DAKO DAKO DAKO DAKO VMRD VMRD VMRD VMRD DAKO
1000 500 500 100 500 50 100 10 000 200 100
pAb, polyclonal antibody; mAb, monoclonal antibody; Igs, immunoglobulins; VSD, Veterinary Science Division, Department of Agriculture, Belfast; DAKO, Dako, Glostrup, Denmark; VMRD, VMRD Pullman Inc., Pullman, USA.
wax-embedded tissues and frozen tissues. The former were de-waxed and rehydrated, and endogenous peroxidase activity was blocked by incubation of the sections with hydrogen peroxide 0·3% in methanol for 30 min at room temperature. Sections were then treated with pronase (Sigma, St Louis, MO, USA) 0·1% in Tris-buffered saline (TBS), pH 7·2, for 4 min at room temperature. When snap-frozen tissue samples were used, endogenous peroxidase activity was blocked by incubation with phenyl-hydrazine (Sigma) 0·05% in TBS, without subsequent pronase treatment. All tissue sections were incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. The primary antibodies (Table 1) were then applied overnight at 4°C (formalin-fixed tissue) or for 2 h at 37°C (frozen tissue). Preliminary experiments were carried out to determine the optimal dilution for the different antibodies. All monoclonal antibodies (mAbs) were raised against lymphocytes of bovine origin but had been shown previously to cross-react with caprine antigens (Navarro et al., 1996). Biotinylated goat or rabbit anti-mouse IgG (for mAbs) and anti-rabbit IgG (for polyclonal antibodies), both obtained from Vector Laboratories and diluted 1 in 200, were used as the secondary reagents. An ABC complex (Vector) diluted 1 in 50 was applied as the third reagent. The sections were then incubated for 1 min with 3,3′-diaminobenzidine tetrahydrochloride (Sigma) 0·035% in TBS containing hydrogen peroxide 0·1%. After rinsing in tap water, they were lightly counterstained with Harris’s haematoxylin, and mounted under DPX for microscopy. Sections in which the specific primary antibodies were replaced by
TBS, normal goat or rabbit serum or inappropriate antibodies were included as negative controls. Sections from the lymph nodes of one control animal (no. 12) were used as a positive control for all of the primary antibodies except those specific for M. agalactiae and M. bovis. Immunohistochemical labelling of mammary and lung lesions induced by these agents was carried out with M. bovis antiserum (pAb 295), which had been shown to cross-react with M. agalactiae (Rodrı´guez et al., 1996). Cell Counting and Statistical Analysis Positively labelled cells were counted in 20 selected BALT fields (×400 magnification) in each of two tissue sections of lung from each goat. The result was expressed as the mean of the percentage of positive cells per field ± standard deviation (SD). A comparative analysis of the number of CD4+, CD8+, MHCII+ and plasma cells containing immunoglobulins, in the control and infected animals, was made with the Pearson test of correlation by means of a Statgraphics 5·0 computer program (Statical Graphics Corporation, Rockville, Maryland, USA). P<0·05 was considered significant. Results Clinical, Post-mortem, Microbiological and Serological Findings Clinical examinations (body temperature, appetite, pulse and respiratory rate, and presence of nasal secretions) were carried out twice daily from the time of infection until slaughter. One day postinfection, two goats (nos 2 and 5) exhibited a moderate and transient increase in respiratory rate
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(50–60/min), coughing and inappetance, all of which returned to normal within 3 days after infection. All 10 infected goats showed transient pyrexia. At necropsy, no pulmonary consolidation was seen. Before infection, no viruses, pathogenic bacteria or mycoplasmas were detected in any animal. M. agalactiae or M. bovis was recovered post mortem from the trachea or lung (or both) of all but two (nos 8 and 10) infected animals. No septicaemia was detected in any animal at the time of killing, but high titres (600–800) of antibodies to M. agalactiae or M. bovis were invariably demonstrated. No bacterial respiratory pathogens were isolated from the lungs. Histopathology The main change in the goats killed 14 days (nos 1, 2, 6, 7) or 21 days (nos 3, 4, 5, 8, 9, 10) after infection consisted of peribronchiolar and perivascular lymphoid hyperplasia (Table 2). Moderate to dense accumulations of lymphocytes and smaller numbers of plasma cells were found in the walls of the airways and around adjacent blood vessels (Fig. 1). This caused narrowing of some bronchiolar lumina, and atelectasis of alveolar regions distal to occluded bronchioles. The alveolar septa appeared thickened due to the accumulation of macrophages, lymphocytes of variable size and plasma cells. The intra-alveolar exudate consisted predominantly of macrophages, but small numbers of lymphocytes, plasma cells and neutrophils were also present. These changes were more prominent in animals killed at 21 days post-infection (Table 2) than in those killed at 14 days.
Expression of S-100. Diffuse nuclear and cytoplasmic immunolabelling was detected in stellate cells with long cytoplasmic processes (dendritic cells) in the BALT. Some T lymphocytes, smooth muscle cells, nerve endings, chondrocytes and macrophages of the alveolar walls were also positive. Expression of myeloid-histiocyte antigen. Immunoreactivity with anti-human myeloid-histiocyte antigen mAb was observed in a few intravascular neutrophils and monocytes. No immunoreaction was noted within alveoli or in cells within the lumina of airways. Expression of CD3 antigen. With the CD3 polyclonal antibody, intense immunoreactivity was detected in the cytoplasm of lymphocytes located in the BALT, mainly in the perifollicular areas, as well as within infiltrating lymphoid cells in the alveolar walls (Fig. 3). Expression of CD4, CD8, /, and MHC class II antigens. With the CD4 monoclonal antibody, the CD4+ subpopulation was more numerous than the CD8+, resulting in a CD4:CD8 ratio of >1 (Table 2) in all infected goats. The CD4+ lymphocytes (Fig. 4) were mainly located in the perifollicular areas of the BALT, whereas the CD8+ lymphocytes (Fig. 5) were located under the epithelium and scattered in the follicles. The / T lymphocytes in the BALT were sparse and appeared closely associated with the bronchioles. MHC class II+ antigen (Fig. 6) was labelled both in lymphocytes and in cells with an irregular morphology and cytoplasmic prolongations, between the lymphocytes and under the epithelial basal membrane.
Immunohistochemistry The results obtained from the BALT are summarized in Table 2. Expression of M. agalactiae and M. bovis antigen. The polyclonal antibody pAb 295 invariably failed to detect mycoplasma antigens in lung tissue. Expression of lysozyme. Immunoreactivity with the anti-lysozyme polyclonal antibody was observed within the cytoplasm of some macrophages, mainly those located in the perifollicular areas of the BALT. Numerous immunoreactive macrophages were observed in alveolar spaces, and in the bronchiolar lumina (Fig. 2). Some intravascular neutrophils were also immunoreactive.
Expression of immunoglobulins. With the polyclonal antibody against caprine immunoglobulins, immunoreactivity was detected in the cytoplasm of a moderate number of plasma cells and on the surface of some lymphocytes, located mainly in the perifollicular and subepithelial tissues. Plasma cells and lymphocytes in the alveolar walls were also labelled. Discussion A common feature of mycoplasma pneumonias is the increase of mononuclear cells in the lung, as for example with M. pulmonis in mice, M. bovis in cattle, M. gallisepticum in chickens and M. hyopneumoniae in pigs (Fernald, 1979; Howard et al., 1987). This leads to peribronchiolar and perivascular accumulations of these cells, many of
– 0·63±0·23 3·30±1·68 0·23±0·14
41·21±5·56 31·21±4·67 14·28±3·55 2·18 0·18±0·10 36·12±7·98 7·81±2·77
52·15±3·21 37·52±4·12 12·95±2·17 2·89 0·34±0·11 37·02±3·67 6·97±1·97
2† (++)
– 0·71±0·20 3·3±0·43 0·31±0·18
1† (++)
44·22±5·65 30·55±3·34 12·79±2·89 2·38 1·26±0·65 45·39±6·84 5·91±1·63
– 1·23±0·78 3·61±1·89 0·20±0·11
3‡ (+++)
38·81±4·88 35·65±5·33 15·83±4·21 2·25 0·76±0·32 41·50±7·43 7·11±2·04
– 0·44±0·75 2·25±0·54 0·43±0·42
4‡ (+++)
6† (+)
59·10±6·23 37·05±5·44 11·05±2·64 3·35 0·97±0·22 48·51±4·29 6·38±1·87
40·32±6·33 31·14±3·45 12·17±2·58 2·55 1·16±0·34 49·30±7·49 3·72±1·12
– – 0·32±0·12 1·30±0·42 3·14±0·44 2·94±1·65 0·37±0·21 0·42±0· 12
5‡ (++)
40·31±7·47 28·25±4·87 14·10±3·75 2·00 0·58±0·21 46·01±5·31 7·32 ±1·68
– 0·74±0·24 3·23±1·68 0·41±0·22
7† (++)
59·12±6·88 42·70±5·56 20·16±4·87 2·11 0·35±0·14 44·50±5·56 8·90±2·32
– 1·15±0·66 2·37±0·75 0·34±0·11
8‡ (+++) – 0·83±0·36 4·16±1·68 0·30±0·12
9‡ (+++)
57·84±6·31 31·90±5·66 15·37±4·21 2·07 0·89±0·21 44·69±4·76 9·48±2·66
Mycoplasma bovis
52·13±5·46 34·23±7·43 16·19±2·64 2·11 0·78±0·31 45·75±5·56 8·54±1·67
– 1·91±0·35 2·98±0·68 0·39±0·14
10‡ (+++)
34·23±3·56 16·33±2·74 17·72±3·56 0·92 0·84±0·21 25·14±3·54 1·22±0·35
– 0·97±0·18 2·37±0·91 0·46±0·32
11† (−)
38·35±5·33 19·15±3·54 21·23±4·36 0·90 0·91±0·24 23·53±5·67 1·40±0·14
– 1·22±0·24 3·88±1·44 0·36±0·75
12‡ (−)
sterile broth controls
∗Means ± SD of the percentages of positive cells in the BALT. †Killed 14 days (‡21 days) after inoculation. Lymphoid hyperplasia: (−), none; (+), slight; (++), moderate; (+++), severe. The Pearson test revealed a significant difference (P<0·05) for CD4+, CD8+, MHCII+ and Igs+ cells between the control and infected goats; no significant difference was obtained with the other antibodies.
pAb 295 Lysozyme S-100 Myeloid/ histiocyte antigen CD3 CD4 CD8 CD4/CD8 / MHC II Immunoglobulins
Antibodies
Mycoplasma agalactiae
Results∗ in goats 1–12, inoculated with
Table 2 Histological and immunohistochemical results
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Fig. 1. Lung of goat no. 7, showing peribronchiolar and perivascular lymphocytic hyperplasia. HE. ×200.
Fig. 2. Goat no. 2. Immunoreaction to lysozyme in the cytoplasm of macrophages within alveoli. ABC.×200.
Fig. 3. Goat no. 1. CD3+ lymphocytes located in the BALT and alveolar walls. ABC.×400.
Fig. 4. Goat no. 9. Numerous CD4+ lymphocytes, mainly located in the perifollicular areas of the BALT. ABC.×400.
Fig. 5. Goat no. 10. Occasional CD8+ lymphocytes among mononuclear cells in the BALT. ABC.×400.
Fig. 6. Goat no. 8. Immunoreaction with MHC class II mAb in the cytoplasm of mononuclear cells, some of them showing cytoplasmic processes (dendritic cells). ABC.×400.
Experimental Mycoplasma Pneumonia in Goats
which are likely to be synthesizing antibody (Cassell et al., 1974; Gourlay and Howard, 1982; Howard et al., 1987) as part of the immune response to the infection. In the present study, goats inoculated with M. agalactiae or M. bovis via the respiratory tract showed similar accumulations of leucocytes. Although significant increases in immunoglobulinproducing cells in the BALT and humoral antibody were detected, the main cellular type in the lymphoreticular hyperplasia was the CD3+ T lymphocyte, which indicated the significant role of cellular immunity. Alveolitis, the presence of inflammatory cells in alveoli, and peribronchiolar accumulations of mononuclear cells are typical of the cuffing interstitial pneumonia (Gourlay and Howard, 1982) that characterizes various mycoplasma respiratory infections. The persistence of organisms and antigen at the mucosal surface may be responsible for the formation of lymphoid accumulations around airways (Howard et al., 1987). The severity of the lesions produced under experimental conditions may be influenced by the degree of colonization or virulence (or both) of the mycoplasma strain used. Under the conditions used in the present experiment, neither M. agalactiae nor M. bovis alone induced respiratory disease severe enough to cause death. The polyclonal antiserum pAb 295 used in this study failed to demonstrate M. agalactiae or M. bovis antigens in lung tissue proved by microbiological methods to be infected, despite the fact that polyclonal and monoclonal antibodies against mycoplasma species are regarded as a potential diagnostic tool (Howard et al., 1987; Rodrı´guez et al., 1996). Immune defence mechanisms in the respiratory tract include humoral immunity, mediated by B lymphocytes. This has three major effects, namely, influencing those biological activities mediated by specific antibody, increasing phagocytic defence mechanisms, and initiating the inflammatory response (Busse, 1991). In the present study, plasma cells containing goat immunoglobulins were observed in the BALT of all infected animals, and specific antibodies to M. agalactiae or M. bovis were demonstrated in serum. This would suggest a significant role for the humoral immune response in clearing mycoplasmas from the respiratory tract. In addition, cell-mediated immunity, effected by T lymphocytes, has two major effects, namely, increasing the phagocytic and cytotoxic activities of macrophages, and initiating the chronic inflammatory response. The predominant phagocytic
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cell infiltrating the lung after M. agalactiae or M. bovis infection in the goats in this study was the macrophage. This observation, which was confirmed by the use of the anti-lysozyme pAb, is consistent with the view that this cell type is a major effector mechanism operating against mycoplasma infections (Howard et al., 1987; Rodrı´guez et al., 1996). CD4+ T lymphocytes represented the predominant subset responsible for the observed BALT hyperplasia. These T cells are known to be capable of activating macrophages, resulting in efficient intracellular killing of mycoplasmas (Nash et al., 1992). It is likely that CD4+ cells, through the production of T helper (Th)1 cytokines such as interferon (IFN), play an important role in the clearance of mycoplasmas from the lung. Synthesis of interleukin (IL)1 and tumour necrosis factor (TNF) by activated macrophages may also result in profound effects on the immune response, as these two cytokines are important mediators of the inflammatory response, inducing secretion of acute phase proteins, stimulating release of prostaglandins and proteolytic enzymes, and enhancing cellular infiltration through chemotaxis and mitogenicity. IL-1 also plays an important role in specific antigenic immune responses, acting as an early signal in T-cell activation, and participating in the cytokine network that regulates Bcell differentiation (Mossmann and Coffman, 1989; Nash et al., 1992). Cytokines play a key role in the regulation of macrophage functional capacity during antigen-specific and non-specific immune responses, firstly as molecules that directly influence the functional status of macrophages, and secondly as the effector molecules of activated macrophages (Cao et al., 1989). Cytokines can also “upregulate” MHC antigen expression on macrophages (Nash et al., 1992) and, since T-cell receptors recognize antigen in the context of MHC class I or class II molecules, this activation of the receptors of macrophages has important implications for the initiation and development of the immune response. In this study, the number of mononuclear cells expressing MHC class II antigen showed a significant increase in the infected goats. Such activation might participate in the signal regulation of antigen-presenting cells, through both humoral and cellular immunity. In the current study, sparse (2–3%) / T lymphocytes were observed in paracortical areas and medullary cords of lymph nodes of the uninfected goat (no. 12) used as a positive control (data not shown), while they were under 1% in the lungs of infected and control animals. The results closely
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resembled those obtained by Caro et al. (1998), with the same mAb, in peripheral blood and lymph nodes from goats aged 3 months. The number of T cells of this subset decreases with age in goats (Caro et al., 1998) and other young ruminants (Wyatt et al., 1994). Some authors have related such decreases to the functional capacity of this Tcell population to confer early cellular immunity on young animals that do not yet possess a mature system of / T cells (Hein et al., 1991). In conclusion, infection of young goats with M. agalactiae or M. bovis by the respiratory route induced a moderate bronchointerstitial pneumonia, characterized by BALT hyperplasia and infiltration of mononuclear cells into the alveolar walls. CD3+ T lymphocytes, cells expressing MHC class II+, and plasma cells containing caprine immunoglobulins constituted the main cellular types in the lymphoreticular hyperplasia. Acknowledgments Support for J. Sarradell during his stay in the Veterinary Faculty of Las Palmas de GC was provided by a grant (FOMEC 70-609-B) from the National University of Rosario, Argentina. This work was supported by grants PB96-0140 and PI1999-148 from the “Direccio´ n General de Ensen˜ anza Superior e Investigacio´ n Cientı´fica” and “Consejerı´a de Educacio´ n, Cultura y Deportes del Gobierno de Canarias”, respectively. References Ball, H. J., Finlay, D., Rodrı´guez, F. and Mackie, D. (1996). Monoclonal antibody detection of mycoplasma antigen as a diagnostic tool. In: Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, J. Frey and K. Sarris, Eds, European Cooperation on Scientific and Technical Research, Luxembourg, pp. 78–80. Busse, W. W. (1991). Pathogenesis and sequelae of respiratory infections. Review of Infectious Diseases, 13, 477–485. Cao, H., Wolf, R. G., Mreltzer, M. S. and Crawford, R. M. (1989). Differential regulation of Class II MHC determinants on macrophages by IFN- and IL-4. Journal of Immunology, 143, 3524–3531. Caro, M. R., Gallego, M. C., Buendia, A. J., Navarro, E. and Navarro, J. A. (1998). Postnatal evolution of lymphocyte subpopulations in peripheral blood and lymphoid organs in the goat. Research in Veterinary Science, 65, 145–148. Cassell, G. H., Lindsey, J. R. and Baker, H. J. (1974). Immune response of pathogen-free mice inoculated intranasally with Mycoplasma pulmonis. Journal of Immunology, 112, 124–136.
Cottew, G. S. and Lloyd, L. S. (1965). An outbreak of pleurisy and pneumonia in goats in Australia attributed to a mycoplasma species. Journal of Comparative Pathology, 75, 363–374. DaMassa, A. J., Wakenell, S. and Brooks, D. L. (1992). Mycoplasmas of goats and sheep. Journal of Veterinary Diagnostic Investigation, 4, 101–113. De´ niz, S., Real, F., Poveda, J. B., Acosta, B., Ramı´rez, A. S. and Ferna´ ndez, A. (1996). Epidemiological study of M. agalactiae and Mycoplasma mycoides subsp. mycoides in the Canary Islands. In: Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics, J. Frey and K. Sarris, Eds, European Cooperation on Scientific and Technical Research, Luxembourg, pp. 128–130. Fernald, G. W. (1979). Humoral and cellular immune responses to mycoplasmas. In: The Mycoplasmas, Vol. 2, J. G. Tully and R. F. Whitcomb, Eds, Academic Press, New York, pp. 399–423. Gourlay, R. N. and Howard, C. J. (1982). Respiratory mycoplasmosis. Advances in Veterinary Science and Comparative Medicine, 26, 289–332. Hasso, S. A., Al-Darraji, A. M. and Al-Aubaidi, J. M. (1994). Pathology of experimentally induced contagious agalactia in goats. Small Ruminant Research, 13, 79–84. Hein, W. R., Dudler, L., Beya, M. F. and Mackay, C. R. (1991). Epitopes of T19 lymphocytes surface antigen are extensively conserved in ruminants. Veterinary Immunology and Immunopathology, 27, 173–181. Howard, C. J., Thomas, L. H. and Parsons, K. R. (1987). Immune response of cattle to respiratory mycoplasmas. Veterinary Immunology and Immunopathology, 17, 401–412. Mossmann, T. R. and Coffman, R. L. (1989). Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, 145–173. Nash, A. D., Barcham, G. J., Andrews, A. E and Brandon, M. R. (1992). Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production. Veterinary Immunology and Immunopathology, 31, 77–94. Navarro, J. A., Caro, M. R., Seva, J., Rosillo, M. C., Go´ mez, M. A. and Gallego, M. C. (1996). Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes. Veterinary Immunology and Immunopathology, 51, 147–156. Ojo, M. O. and Kede, B. O. (1976). Pathology of Mycoplasma agalactiae subsp. bovis in goat mammary gland. Veterinary Microbiology, 1, 19–22. Real, F., De´ niz, S., Acosta, B., Ferrer, O. and Poveda, J. B. (1994). Caprine contagious agalactia caused by Mycoplasma agalactiae in the Canary Islands. Veterinary Record, 135, 15–16. Rodrı´guez, F., Bryson, D. G., Ball, H. J. and Forster, F. (1996). Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves. Journal of Comparative Pathology, 115, 151–162.
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Received, January 21st, 2000 Accepted, May 15th, 2000