Diagnostic laboratories routinely receive requests to culture bacterial agents. However, not all types of bacteria are cultivable or grown the same way. Some species are especially difficult to culture under laboratory conditions.
Mycoplasma pneumonia (Mycoplasma hyopneumoniae), a very small bacterium,is one of those fastidious pathogens that challenges and frustrates bacteriologists, mainly due to very slow growth, special media requirements and overgrowth by other bacterial species. Because of these features, and the fact that other diagnostic tools for pathogen detection have been developed (e.g. molecular-based tests like polymerase chain reaction or PCR), bacterial isolation of mycoplasma constitutes a test rarely offered by veterinary laboratories around the world.
Still, mycoplasma is a very important pig pathogen that causes enzootic pneumonia, which is characterized by a chronic dry cough and predisposes animals to respiratory infections caused by secondary bacteria or viruses. Under certain circumstances or in specific clinical cases, obtaining a mycoplasma isolate is desirable – for biological product development, for example – but options to culture this bacterium do not abound.
Committed to Finding Solutions
Recently, at the University of Minnesota Veterinary Diagnostic Laboratory (MVDL), as part of our commitment to offer solutions to the veterinary community, we have dedicated efforts to standardize the bacteriological work with mycoplasmato our laboratory conditions. Basically, we apply proven methodologies for culture and isolation of this swine pathogen and are able to offer bacterial culture and isolation.
Culture consists of growing mycoplasma in broth, using clinical samples as the starting material. The broth is checked to confirm the presence of the bacterium and the absence of other microorganisms (including other swine mycoplasmas) by PCR and by sequencing. Results are reported about four weeks after sample submission.
A second step is isolation, which consists of growing mycoplasma from a single colony. The starting material is a culture of mycoplasma. Then, this culture is plated in solid media and grown back in broth. This process involves several broth passages to adapt the isolate to grow faster under laboratory conditions before plating it to attempt the single colony cloning. Broth purity is reevaluated to confirm the presence of mycoplasma and the absence of other microorganisms. There is a long and variable turnaround time for isolates, depending on the inherent ability of each strain for growth under laboratory conditions but, in general, one isolate can usually be obtained 8-10 weeks after being requested.
The preferred sample to attempt mycoplasma culture is frozen lung tissue. Other samples, such as fresh lung tissue, bronchial swabs or broncho-alveolar lavage fluid can also be used for culture, but the chances of isolating this bacterium are lower.
The ideal sample should include areas of lung consolidation and apparently unaffected areas (Figure 1). Samples should be placed in Whirl-Pack bags for transportation to the diagnostic laboratory, where they will be processed. Figure 2 depicts Mycoplasma pneumonia cultured in broth.
Figure 1. Pictured are lung lesions caused by Mycoplasma pneumonia infection (darker areas of consolidation). Samples for culture and isolation should include affected areas and apparently healthy areas (circled).
Figure 2. Mycoplasma pneumonia is cultured in broth. From left to right, un-inoculated broth (control), and two cultures in different growth stages are shown. Photos by Lucas Santos.
Maria Pieters, DVM, Albert Rovira, DVM, and Tom Molitor.
University of Minnesota Veterinary Diagnostic Laboratory