Distribution and location of Mycoplasma ovipneumoniae in the lungs of pneumonic sheep

Abstract

The localisation and distribution of Mycoplasma ovipneumoniae was demonstrated using both immunofluorescent and. immunoperoxidase techniques.Paraffin-wax sections were prepared by the "Sainte-Marie" (1962) method and cut to a thickness of 6 u. An indirect fluorescent antibody technique (F.A.T.) was employed using rabbit anti- M. ovipneumoniae antiserum and swine anti-rabbit antiserum labelled with fluorescein isothiocyanate (SWAR/FITC). A triple step "sandwich" immunoperoxidase technique was used by treating sections with rabbit anti-M. ovipneumoniae antiserum, sheep anti-rabbit IgG and rabbit anti-sheep conjugated to horseradish peroxidase (HRP). Destruction of endogenous peroxidase was by sodium borohydride in disodium hydrogen phosphate, and SPF sheep serum was applied to sections to reduce non-specific staining. An indirect F.A.T. was used to demonstrate Immunoglobulin G (igG) and Immunoglobulin A (IgA) in sheep lungs. Rabbit anti-sheep IgG (IgA) and then SViAR/FITC were applied to sections of normal and pneumonic lungs.

Positively stained mycoplasma were demonstrated lining ciliated bronchial epithelium. Bright fluorescence was seen slightly away from the cell surface, and particulate peroxidase stain in the tips of cilia. No staining was seen in non-ciliated bronchi or alveolar lining. Particulate stain was seen in the bronchial and alveolar exudate.

F.A.T. revealed coating of a proportion of alveolar macrophages with a layer of fluorescence. Peibronchial and perivascular cuffs fluoresced brightly when treated with rabbit anti-sheep IgG, as did the intra-alveolar exudate and some alveolar macrophages. Cells stained for IgA. were found largely in the interstitium of the lungs, and fluorescence of exudate and alveolar macrophages appeared as for IgG.

Thus, M. ovipneumoniae was demonstrated lining bronchi in close association with cilia, and possibly within the intralaminar exudate. There was an increase in both IgG and IgA. in pneumonic lungs compared to normal lungs, but relative importance of each immunoglobulin was not determined.