Abstract
BackgroundDuring the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in laboratories that study L. intracellularis, the cultures must be discarded for 4 reasons: 1) Mycoplasma is inevitably concentrated along with L. intracellularis during the passage of L. intracellularis; 2) Mycoplasma inhibits the growth of L. intracellularis; and 3) it is impossible to selectively eliminate Mycoplasma in L. intracellularis cultures. In this study, we observed the contamination of Mycoplasma species during L. intracellularis cultivation among multiple laboratories.ResultsThe presence of a Mycoplasma infection in the L. intracellularis cultures was verified using polymerase chain reaction (PCR), and a sequence analysis of the partial 16S rRNA and 23S rRNA genes was performed. A PCR-based assay using genus-specific universal primers revealed that 29 (85.3%) of the 34 cultures were contaminated with Mycoplasma, including 26 with M. hyorhinis (89.2%), 2 with M. orale (6.9%), and 1 with M. fermentans (3.4%). The Mycoplasma contamination was not the result of infection with material of pig origin. McCoy cells, which are required for the cultivation of L. intracellularis, were also ruled out as the source of the Mycoplasma contamination.ConclusionsIn this study, M. hyorhinis was identified as the most common mollicute that contaminated L. intracellularis cultures. Whether L. intracellularis enhances the biological properties of Mycoplasma to promote infection in McCoy cells is not known. Because the McCoy cell line stocks that were used simultaneously were all negative for Mycoplasma, and the same worker handled both the McCoy cells to maintain the bacteria and the L. intracellularis cultures, it is possible that the L. intracellularis cultures are more vulnerable to Mycoplasma contamination. Taken together, these results suggest that continuous cultures of L. intracellularis must be tested for Mycoplasma contamination at regular intervals.The GenBank accession numbers for the sequences reported in this paper are JN689375 to JN689377.
Highlights
During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem
A total of 26 L. intracellularis cultures contained DNA sequences that matched those of M. hyorhinis (100% sequence identity), whereas 2 cultures and 1 culture contained DNA sequences that matched those of M. orale and M. fermentans, respectively (100% sequence identity)
The McCoy cell line stocks that were used simultaneously were all negative for Mycoplasma. These results suggest that McCoy cells, which are required for the cultivation of L. intracellularis, were not the source of the Mycoplasma contamination
Summary
During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. Mycoplasmas, which are bacteria that lack a cell wall and belong to the class Mollicutes, are found in the physiological flora of both humans and animals, can act as opportunistic pathogens, and are frequent contaminants of cell cultures. Because they frequently contaminate both primary and stable cell lines, species of the genera Mycoplasma and Acholeplasma are a major concern with regard to the regulatory aspects of biosafety [1,2,3,4,5,6]. Most researchers prefer to use McCoy mouse fibroblast cells for the isolation and maintenance of L. intracellularis in vitro [17,18,19,20,21,22,23]
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