Histological and immunohistochemical findings in thoracic lymph nodes of cattle with contagious bovine pleuropneumonia (CBPP).

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS IN THORACIC LYMPH NODES OF CATTLE WITH CONTAGIOUS BOVINE PLEUROPNEUMONIA (CBPP) E. Scanziani, S. Paltrinieri*, M. Boldini**, G. Mandelli, V. Grieco, C. Monaci** Istituto di Anatomia Patologica Veterinaria e Patologia Aviare - Milano; * Istituto di Patologia Generale Veterinaria - Milano; ** Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia - Brescia Introduction Since the 1550 the contagious bovine pleuropneumonia (CBPP) spred all of the world. The prophilaxis and the increasing knowledges of the aetiology and the pathogenesis of the disease led to a decrease of the prevalence of the CBPP with a complete eradication from many countries: it was eradicated from the USA in 1982 and from Australia in 1967. In this century the CBPP was confined in some endemic african countries. In Europe some outbreaks of this disease occured in 1958 in Portugal (Ferronha et al. 1990) and between 1980 and 1984 in Spain, Portugal and France. At the end of 1990 the CBPP has been diagnosed in Lombardy, Northern Italy (Guadagnini et al., 1991). The clinico-pathological findings of CBPP are not specific: similar patterns of fibrinous pleuropneumonia are detectable in diseases such as the IBR, non specific mycoplasmosis etc... (Mandelli, 1989). The isolation and identification of Mycoplasma mycoides subsp. mycoides from infected animals is the only one reliable diagnostic test, but have many methodological problems (Al Aubaidi and Fabricant, 1970 Cottral 1978). Serological diagnosis is hampered either by false positive cattles due to cross-reactivity between Mycoplasma mycoides subsp. mycoides and other mycoplasmas or false negative animals in early and late phases of the disease. Recently Mycoplasma mycoides subsp. mycoides antigen has been detected in the tissues by immunofluorescence (Ferronha et al., 1988) and immunohistochemistry (Ferronha et al. 1990; Scanziani et al. 1991a; Scanziani et al., 1991b): the localisation and the pattern of the positivity was variable with the evolutive step of the disease: in the acute phase antigens were detectable in alveolar macrophages as well as in the interlobular septa where a diffuse and weak positivity was found; with chronicity a strong positivity was found in the macrophages of fibrotic septa and in the follicular center of the broncho-alveolar associated lymphoid tissue (BALT). Necrotic debris in sequestra as well as the macrophages on their walls were strongly positive (Scanziani et al., 1991). The lymph nodes only in the acute cases show gross pathological lesions: lymphoid hyperplasia and ectasia of the sinuses with a large number of macrophages were histologically detectable in the acute cases, while follicular hyperplasia with a large germinal center and fibrosis were present with chronicity. Large necrosis are rarely detectable: most frequently histology showed necrotic debris in the sinuses. In preliminary studies we detected Mycoplasma mycoides subsp. mycoides antigens in the lymph nodes (Scanziani et al. 1991a; Scanziani et al., 1991b) The present work was assesed to study histological lesions and to detect the presence and localization of Mycoplasma mycoides subsp. mycoides antigens in thoracic lymph nodes of cattle affected by CBPP and to compare the results with bacteriological and serological positivity. Matherial and methods Animals For the present study XX adult Holstein-Freisian cattle at slaughtering were selected. They came from 5 dairy herds on wich CBPP were present. All the animals were affected by CBPP: the diagnosis was based on the presence of tipical lung lesions and on the isolation of M.mycoides from the lungs. For each animal macroscopic lesions of the lungs and thoracic lymph nodes were recorded at slaughtering. Furthermore the lungs and the thoracic lymph nodes were sampled for bacteriological and histological analysis. On the basis of macroscopic and histological lesions of the lungs each case was classified as acute, subacute or chronic. The thoracic lymph nodes of five cattle that had no pulmnary lesions, that were bacteriologically negative for M.mycoides and belonged to herds in which CBPP was not present were also sampled as controls. Serology Bacteriology Coltural examination for mycoplasma was carried ut by plating on Difco PPLO agar (Difco----) medium supplemented with horse serum (xx per cent), yest extract (yy per cent), glucose, tallium acetate and penicillin. In addition the samples were homogeneized (10 per cent w/v) in Difco PPLO broth supplemented as for the solid medium. Three serial 10-fold dilutions were then made in the medium and subcultured on the solid medium after two days (?). The isolated mycoplasma were identified by biochamical tests, growth inhibition test and direct immunofluorescence. Histology and immunohistochemistry The samples of the lungs and of the thoracic lymhp nodes were fixed in 10% formalin and routinely embedded in paraffin. 5mm thick sections were then stained with haematoxilin and eosin and withthe avidin-biotin-peroxidase complex (ABC) procedure (Hsu et al. 1981), using commercial immunoperoxidase kits (Vectastain Elite; Vector Laboratories): sections were deparafiined, treated with 0,5 per cent hydrogen peroxide in methanol for 20 minutes, and rehydrated. They were then incubated at 4°C for 18 hours with a 1:20000 dilution of a policlonal rabbit antiserum against M. mycoides subsp. mycoides (kindly provided by Prof. M.C.D. De Lage of the Laboratorio Nazionale della Ricerca Veterinaria of Lisbona). After washing, the secions were covered with a secondary antibody biotinylated goat anti-rabbit immunoglobulin, and left at room temperature for 30 minutes. The peroxidase-conjugate ABC was allowed to react at room temperature for 30 minutes. Sectins were incubated with diaminobenzidine-hydrogen peroxide for seven minutes, washed in tap water and counterstained with Mayer’s haematoxilin. For each sample a negative control was made by replacing the primary antibody with normal rabbit serum. Known negative and positive control sections were included in each assay. Results Pathological findings All the animals showed typical macrscopic lesions of CBPP. 13 animals had acute lung lesions with fibrinous interstitial pleuropneumonia with marmorization, mainly in the basal lobe, and lymph nodes were frequently affected, with enlarged (up than 10 times the normal size) and edematous lymph nodes and with a yellow coloured perilymphonodal fluid; 12 cases showed subacute lung lesions with a more evident marmorization of the acute lesions: thoracic lymph nodes tributaries of hte affected areas wererenlarged and slightly edematous; 28 animals had chronic lung lesions with sequestra and fibrosis: only some of this cases showed chronic lesions in the lymph nodesthat were moderately enlarged. Lymph node histology In the acute cases the marginal sinuses were ectasic and filled by lymph macrphages and granulocytes; macrophages were also detectable in the medullary sinuses. In the subacute cases macrophages were presnt in the sinuses and mainly in the medullary sinuses: mild hyperplasia of the follicular germinal centers was also present in some cases The germinal centers of the follicles was ex