Phenotype of Coxiella burnetii Strains of Different Sources and Genotypes in Bovine Mammary Gland Epithelial Cells

Abstract

Despite the high prevalence of C. burnetii in dairy herds and continuous shedding via milk by chronically infected cows, bovine milk is not recognized as a relevant source of human Q fever. We hypothesized that the bovine mammary gland epithelial cell line PS represents a suitable in vitro model for the identification of C. burnetii-strain-specific virulence properties that may account for this discrepancy. Fifteen C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes (I, II, III and IV). The replication efficiencies of all strains were similar, even though strains of the MLVA-genotype II replicated significantly better than genotype I strains, and bovine and ovine isolates replicated better than caprine ones. Bovine milk isolates replicated with similar efficiencies to isolates from other bovine organs. One sheep isolate (Cb30/14, MLVA type I, isolated from fetal membranes) induced a remarkable up-regulation of IL-1β and TNF-α, whereas prototypic strains and bovine milk isolates tended to suppress pro-inflammatory responses. While infection with strain Nine Mile I rendered the cells partially refractory to re-stimulation with E. coli lipopolysaccharide, Cb30/14 exerted a selective suppressive effect which was restricted to IL-6 and TNF-α and spared IL-1β. PS cells support the replication of different strains of C. burnetii and respond in a strain-specific manner, but isolates from bovine milk did not display a common pattern, which distinguishes them from strains identified as a public health concern.

Keywords: Coxiella burnetii; bovine udder; epithelial cells; genotype; host cell response.

Figure 1

Replication of C. burnetii strains in bovine mammary gland epithelial cells. PS cells were inoculated with C. burnetii strains (strain designations as listed in Table 1 given in bold in all panels; 100 MOI, 24 h), and genome equivalents (GEs) were quantified by icd real-time PCR at 1 d (i.e., 24 h after addition of the C. burnetii suspension) and at 3, 7 and 14 days post-inoculation (d p.i.). GE values determined in technical duplicates in four independent experiments per strain are depicted as box plots (significantly different to 1 d p.i.: a = p ≤ 0.01; b = p ≤ 0.05).

Figure 2

Replication efficiencies of C. burnetii strains in bovine mammary gland epithelial cells grouped by MLVA group (a), by host species (b) and by source sample (c) (only bovine isolates included in the latter panel). Increases in genome equivalents (GEs) between 1 and 14 days post-inoculation are calculated as replication factors and presented as mean values of technical duplicates from four independent experiments per strain. Mean values per group are depicted as black lines (* p ≤ 0.05; ** p ≤ 0.01).

Figure 3

Relative amounts of cytokine-specific mRNA molecules in C. burnetii-infected mammary gland epithelial cells. PS cells were inoculated with C. burnetii strains (strain designations as listed in Table 1 given in bold in all panels; 100 MOI, 24 h). Amounts of mRNA encoding for IL-1β (a), IL-6 (b) and TNF-α (c) were quantified at 7 days post-inoculation, normalized to GAPDH and calculated relative to cell controls (fold increase). The results of four independent experiments per strain, each with technical duplicates, are depicted. The results for different strains are arranged according to MLVA group (open bars indicate bovine milk isolates, hatched bars indicate other bovine isolates; *: p ≤ 0.05; **: p ≤ 0.01).

Figure 4

Host response of C. burnetii-inoculated bovine mammary gland epithelial cells to re-stimulation with E. coli LPS. PS cells were left uninfected, were inoculated with C. burnetii strain Nine Mile I (NMI) or exposed to a heat-inactivated NMI suspension (a) or inoculated with strain Cb30/14 (b). At 7 days post-inoculation, cultures were re-stimulated with E. coli LPS for a further 4 h. Amounts of cytokine-specific mRNA (IL-1, IL-6 and TNF-α) quantified in four independent experiments per strain, each with technical duplicates, are depicted relative to uninfected, E. coli LPS-re-stimulated control cells (values set to 100%; *: p ≤ 0.05; **: p ≤ 0.01).