388
E. A. FREUNDT ET AL.
specificity of the test was, however, considerably improved when formalinised rather than fresh red blood cells were used.
Because of the rather laborious technique and the difficulties involved
in standardising the IHA method, this test is of only limited practical value
for classification and identification purposes. The high sensitivity of the
test does, on the other hand, make it particularly useful for sero-epidemiological studies.
(f) Complement Jixation. The complement fixation (CF) test has been
used widely in the classification of mycoplasmas (Edward, 1950; Edward
and Fitzgerald, 1951 ; Huijsmans-Evers and Ruys, 1956; Card, 1959;
Taylor-Robinson et al., 1963, 19651; Lemcke, 1964; Clyde, 1964; Fox
et al., 1969), although it is currently less popular for routine identification
than, for example, GI and M I tests. Nevertheless, the fact that the C F
test is used extensively in the serological diagnosis of mycoplasma infections
of man and animals and in sero-epidemiological surveys should encourage
the continued use of this test in the classification of mycoplasmas.
Although considerable interspecies cross-reactivity has been reported
with this test, quantitative differences between homologous and heterologous titres are usually sufficient to allow clear taxonomic distinctions to be
made. The high levels of cross-reactivity noted in some reports may in
some cases be attributed to contamination of both test and immunising
antigens with antigenic material from the medium, in particular adsorbed
serum proteins, when the immunising antigen is grown in the presence of
serum foreign to the animal being immunised. Apart from this, the use of
antigens at dilutions that tend to be anticomplementary and the practice of
comparing antigens at a fixed unitage or at a standard opacity have probably
magnified the cross-reactions between species. Heterologous reactions can
be much reduced or even eliminated by testing each antigen at a previously
determined optimal concentration (Section V . F, 2 (h)).
Nevertheless, when all technical precautions have been taken to avoid
non-specific cross-reactions and thus to increase the specificity of the test,
low-level cross-reactions (at 2% or less of the homologous titre) still occur
between some species. These may be due to the presence of common or
related antigens. I n C F reactions it appears that cytoplasmic antigens as
well as membrane antigens are involved (Hollingdale and Lemcke, 1969;
Pollack et al., 1970). I n this respect, C F differs from tests such as direct
agglutination, GI, MI or immunofluorescence. Cross-reactions revealed
by C F but not by GI, M I or immunofluorescence tests may therefore be
due to cytoplasmic antigens, especially in species which share a common
metabolic pathway and in which serologically related enzymes have been
demonstrated (Thirkill and Kenny, 1974). I n this sense, C F tests can be
E. A. FREUNDT ET AL.
specificity of the test was, however, considerably improved when formalinised rather than fresh red blood cells were used.
Because of the rather laborious technique and the difficulties involved
in standardising the IHA method, this test is of only limited practical value
for classification and identification purposes. The high sensitivity of the
test does, on the other hand, make it particularly useful for sero-epidemiological studies.
(f) Complement Jixation. The complement fixation (CF) test has been
used widely in the classification of mycoplasmas (Edward, 1950; Edward
and Fitzgerald, 1951 ; Huijsmans-Evers and Ruys, 1956; Card, 1959;
Taylor-Robinson et al., 1963, 19651; Lemcke, 1964; Clyde, 1964; Fox
et al., 1969), although it is currently less popular for routine identification
than, for example, GI and M I tests. Nevertheless, the fact that the C F
test is used extensively in the serological diagnosis of mycoplasma infections
of man and animals and in sero-epidemiological surveys should encourage
the continued use of this test in the classification of mycoplasmas.
Although considerable interspecies cross-reactivity has been reported
with this test, quantitative differences between homologous and heterologous titres are usually sufficient to allow clear taxonomic distinctions to be
made. The high levels of cross-reactivity noted in some reports may in
some cases be attributed to contamination of both test and immunising
antigens with antigenic material from the medium, in particular adsorbed
serum proteins, when the immunising antigen is grown in the presence of
serum foreign to the animal being immunised. Apart from this, the use of
antigens at dilutions that tend to be anticomplementary and the practice of
comparing antigens at a fixed unitage or at a standard opacity have probably
magnified the cross-reactions between species. Heterologous reactions can
be much reduced or even eliminated by testing each antigen at a previously
determined optimal concentration (Section V . F, 2 (h)).
Nevertheless, when all technical precautions have been taken to avoid
non-specific cross-reactions and thus to increase the specificity of the test,
low-level cross-reactions (at 2% or less of the homologous titre) still occur
between some species. These may be due to the presence of common or
related antigens. I n C F reactions it appears that cytoplasmic antigens as
well as membrane antigens are involved (Hollingdale and Lemcke, 1969;
Pollack et al., 1970). I n this respect, C F differs from tests such as direct
agglutination, GI, MI or immunofluorescence. Cross-reactions revealed
by C F but not by GI, M I or immunofluorescence tests may therefore be
due to cytoplasmic antigens, especially in species which share a common
metabolic pathway and in which serologically related enzymes have been
demonstrated (Thirkill and Kenny, 1974). I n this sense, C F tests can be