Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci

Abstract

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Figure 1

Chorioallantoic membrane and yolk sac membrane of 10 days embryonated eggs infected with NL_G47. Intracellular inclusions (arrows) in the epithelial cells of the chorioallantoic membrane (A) and yolk sac membrane (B). Inset: higher magnification showing the granular basophilic inclusions in the HE staining. Positive immunolabelling of the intracellular inclusions for chlamydial antigen in the chorioallantoic membrane (C) and yolk sac membrane (D). Sections were photographed with an Olympus BX51 microscope equipped with a high-resolution digital camera and using Olympus’ cellSens software.

Figure 2

Assessment of virulence of C. gallinacea in embryonated eggs. (A) The 50% egg infective dose 50 (EID₅₀) of C. gallinacea NL_G47, NL_F725 and C. psittaci NL_Borg based on IFT of the yolk sac. The difference between EID₅₀ of NL_G47 and NL_Borg was significantly different (*, P < 0.05, Wilcoxon–Mann–Whitney test). For C. psittaci NL_Borg the 50% lethal dose (LD₅₀) was also calculated. The median EID₅₀ or LD₅₀ of the experiments is indicated with a bar. (B–D) Depict the cumulative results of the separate titration experiments per Chlamydia strain. Per dilution, the percentage of eggs that was positive for Chlamydia in the immunofluorescence test (IFT) with mortality, IFT positive without mortality and IFT negative are shown. The total number of eggs per dilution are presented at the top of every bar. These data are also included in Supplementary Table S1. The figure was created in GraphPad Prism 9.0.0.

Figure 3

Phylogenetic analyses of concatenated sequences of Chlamydia. Concatenated sequences were aligned and analysed in MEGA7. Numbers on tree nodes indicate bootstrap values over 75% of the main branches. Horizontal lines are scale for nucleotide substitutions per site. (A) Neighbor-Joining tree of concatenated sequences of 52 ribosomal genes (rMLST) of Chlamydia Type strains as well as three Candidatus species (Ca. C. corallus, Ca. C. ibidis and Ca. C. sanzinia), C. psittaci strain NL_Borg and two additional C. gallinacea strains. All C. gallinacea strains (Dutch strains indicated by an arrow) clustered together in a well-supported and distinct clade with Chlamydia avium as the closest relative. (B) Neighbor-Joining tree of concatenated sequences of 7 housekeeping genes fragments (MLST) of 27 C. gallinacea strains. Shared Sequence types (ST) in clades are indicated by color and STs are denoted by the color key.

Figure 4

Genome comparison of C. gallinacea and C. psittaci. (A) Whole genome BLAST comparison between C. psittaci NJ1 and four C. gallinacea genomes (including Type strain 08-1274/03). The image is created with BLAST Ring Image Generator (BRIG) and the first ring corresponds to the genome that was used for the comparison. (B) CDS for which no homologue (alignment E score higher than 1 × 10⁻³) could be identified in C. gallinacea 08-1274/03 or C. psittaci NJ1. Every colored block in the figure corresponds to a CDS. The different proteins are categorized and colored according to their function and location. The figure was created using the tidyverse package and R v3.6.1^,. (C) Graphical representation of the gene content of the PZs of representative Chlamydia species of avian origin including the Dutch C. gallinacea strains. Arrows represent PZ genes colored according to function (see key). Grey shading scale denotes % nucleotide identity. The image was created with Easyfig.