IX. IDENTIFICATION OF MYCOPLASMAS
403
serum proteins. These in turn will react with serum proteins adsorbed to the
test antigen, if the latter has been grown in the presence of the same serum.
For growth inhibition and metabolism inhibition tests this is unimportant,
since antibodies to medium components in the immunising antigen do not
affect the growth of the test organisms. For immunofluorescence, complement fixation, double immunodiffusion or direct agglutination, it will
result in spurious cross-reactions between species.
The most effective method of preventing such reactions is to grow the
immunising antigen in medium containing serum from the animal species
to be immunised. Some workers recommend that this should apply to all
the medium constituents and that, for example, rabbits should be immunised with organisms grown in a rabbit meat infusion broth supplemented with rabbit serum. However, it is only components of serum which
have been shown unequivocally to adsorb to mycoplasmas during growth
(Bradbury and Jordan, 1972), and many species grow very poorly in rabbit
infusion medium. An alternative method suggested by Kenny (1969) is to
grow the immunising antigen in medium supplemented with agamma calf
serum and the test organism in medium containing agamma horse serum.
Reference antisera for a total of 29 Mycoplasma or Acholeplasma species
or subspecies, as well as seed reference reagents of the corresponding type
strains, are available from the Research Resources Branch, Collaborative
Research, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20014, U.S.A., and from the
FAO/WHO Collaborating Centre for Animal Mycoplasmas, Institute of
Medical Microbiology, Bartholin Building, University of Aarhus, DK-8000
Aarhus C, Denmark. These antisera were produced by immunisation of
horses or mules with antigens prepared from organisms normally grown in
PPLO Broth (Difco) with 10% added autologous preimmune horse or
mule serum and 2% yeast extract dialysate (Freundt et al., 1973b; Cunningham, 1978-1980). Antiserum and seed reference reagents for the vast
majority of the remaining currently recognised Mycoplasma and
Acholeplasma series are available, in limited quantities, from the
FAO/WHO Collaborating Centre for Animal Mycoplasmas. These lastmentioned antisera are produced in rabbits using antigens prepared from
organisms grown, whenever possible, in rabbit meat infusion broth
supplemented with rabbit serum (or PPLO Serum Fraction, Difco) and
yeast extract.
B. Growth inhibition
1. Disc method
(a) Equipment and reagents. Sterile 6 mm filter paper discs are impregnated
403
serum proteins. These in turn will react with serum proteins adsorbed to the
test antigen, if the latter has been grown in the presence of the same serum.
For growth inhibition and metabolism inhibition tests this is unimportant,
since antibodies to medium components in the immunising antigen do not
affect the growth of the test organisms. For immunofluorescence, complement fixation, double immunodiffusion or direct agglutination, it will
result in spurious cross-reactions between species.
The most effective method of preventing such reactions is to grow the
immunising antigen in medium containing serum from the animal species
to be immunised. Some workers recommend that this should apply to all
the medium constituents and that, for example, rabbits should be immunised with organisms grown in a rabbit meat infusion broth supplemented with rabbit serum. However, it is only components of serum which
have been shown unequivocally to adsorb to mycoplasmas during growth
(Bradbury and Jordan, 1972), and many species grow very poorly in rabbit
infusion medium. An alternative method suggested by Kenny (1969) is to
grow the immunising antigen in medium supplemented with agamma calf
serum and the test organism in medium containing agamma horse serum.
Reference antisera for a total of 29 Mycoplasma or Acholeplasma species
or subspecies, as well as seed reference reagents of the corresponding type
strains, are available from the Research Resources Branch, Collaborative
Research, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20014, U.S.A., and from the
FAO/WHO Collaborating Centre for Animal Mycoplasmas, Institute of
Medical Microbiology, Bartholin Building, University of Aarhus, DK-8000
Aarhus C, Denmark. These antisera were produced by immunisation of
horses or mules with antigens prepared from organisms normally grown in
PPLO Broth (Difco) with 10% added autologous preimmune horse or
mule serum and 2% yeast extract dialysate (Freundt et al., 1973b; Cunningham, 1978-1980). Antiserum and seed reference reagents for the vast
majority of the remaining currently recognised Mycoplasma and
Acholeplasma series are available, in limited quantities, from the
FAO/WHO Collaborating Centre for Animal Mycoplasmas. These lastmentioned antisera are produced in rabbits using antigens prepared from
organisms grown, whenever possible, in rabbit meat infusion broth
supplemented with rabbit serum (or PPLO Serum Fraction, Difco) and
yeast extract.
B. Growth inhibition
1. Disc method
(a) Equipment and reagents. Sterile 6 mm filter paper discs are impregnated