VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

DIAGNOSIS

Specimens

• Mycoplasmas are fragile and specimens must be kept refrigerated and delivered to laboratory within 24-48 hrs of collection.
• The samples may include mucousal scrapings, tracheal exudates, aspirates, and pneumonic tissue from the edge of the lesion, cavity or joint fluids and mastitic milk.
• Swabs should be submitted in transport medium.

Diagnosis

• Based on direct microscopy
  • FAT is most reliable than simple staining.
• By isolation and identification
  • The inoculation technique will vary according to the nature of the specimen.
    • Fluid materials such as foetal fluids and exudates can be inoculated directly into broth and spread over the surface of the agar medium.
    • Specimens such as semen, joint fluids and tissues may contain inhibitors for mycoplasmas.
    • Make ten fold dilutions in mycoplasma broth and then culture.
    • Tissues, such as pieces of lung can be moved across the surface of an agar plate for inoculation.
    • Alternatively the tissue can be homogenized in broth, made ten fold dilutions and inoculated.
  • Differentiation from bacterial L forms
    • Bacteria that have temporarily failed to form cell walls (L-form) can produce microcolonies similar to those of the mycoplasmas (the failure to form cell wall is often due to the bacteria being exposed to penicillin or other antimicrobial agents that affect cell wall formation).
    • The L-forms may also produce fried-egg colonies like mycoplasmas, but they differ in a number of respects.
    • L-forms resemble the parent bacteria biochemically and antigenically, they are not filterable, the minimum reproductive unit being about 600nm, sterols are not required for their growth, they are non pathogenic and they show nucleic acid homology with parent bacteria.
  • Differentiation between these two can be carried out by
    • Subculturing the suspected bacterial L-form on media without antibacterial substances should cause reversion of the L-forms with the formation of normal sized bacterial colonies.
    • Staining microcolonies with Diene's stain. Mycoplasmal colonies retain the stain indefinitely,whereas bacterial L-form microcolonies tend to decolourise in about 15 mts.
• Growth inhibition test
  • This test is based on the ability of antisera to specifically inhibit the growth of homologus species on solid media.
• Sensitivity to digitonin
  • This test reflects the requirement of cholestrol for growth.
  • Mycoplasma and Ureaplasma species are sensitive to digitonin whereas Acholeplasma are not.
  • Digitonin filter paper discs are commonly used.
• Serological test
  • Rapid plate agglutination test, HI test and AGID: for screening of avian mycoplasmas.

• CFT for CCPP and enzootic pneumonia. FAT, ELISA, LAT, species specific DNA probes are highly useful in the identification of mycoplasmas.