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Chlamydia  ( C. )  pecorum , an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the  ompA ,  incA  and ORF663 genes that potentially differentiate between high-virulence  C. pecorum  isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the  incA  locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence ( P <10 −4 ), as did a high number of amino acids in all  incA  CTRs ( P  = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence ( P  = 0.0001). Correction for  ompA  phylogram position in ORF663 and  incA  abolished the correlation between genetic changes and virulence, demonstrating co-evolution of  ompA ,  incA , and ORF663 towards low virulence. Pairwise divergence of  ompA ,  incA , and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals ( P≤ 10 −5 ), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations.

Host Adaptation of Chlamydia pecorum towards Low Virulence Evident in Co-Evolution of the ompA, incA, and ORF663 Loci