Microscopic Analysis of the Chlamydia abortus Inclusion and Its Interaction with Those Formed by Other Chlamydial Species

ABSTRACT

The Chlamydiae are obligate intracellular pathogens that develop and multiply within a poorly characterized parasitophorous vacuole (the inclusion) during growth. Chlamydia abortus is a major pathogen of sheep and other ruminants, and its inclusion development is poorly characterized. We used immunofluorescence microscopy, quantitative culture, and qPCR to examine C. abortus inclusion development and to examine the interaction of C. abortus inclusions with those formed by other species. Antibodies used in these studies include sera from ewes from production facilities that were naturally infected with C. abortus. Multiple inclusions are often found in C. abortus-infected cells, even in populations infected at very low multiplicity of infection. Labeling of fixed cells with sera from infected sheep revealed fibrous structures that extend away from the inclusion into the cytoplasm of the host cell. C. abortus inclusions fused with C. caviae and C. psittaci inclusions in coinfected cells. Inclusions formed by C. abortus and C. caviae did not fuse with inclusions formed by C. trachomatis, C. pneumoniae, or C. pecorum. The ability of inclusions to fuse was correlated with the overall genomic relatedness between species, and with sequence similarity in the inclusion membrane protein IncA. Quantitative PCR data demonstrated that C. abortus grows at a decreased rate during coinfections with C. caviae, while C. caviae growth was unaffected. The collected data add depth to our understanding of inclusion development in this significant zoonotic veterinary pathogen.

INTRODUCTION

Members of the genus Chlamydia are obligate intracellular pathogens of many mammals, birds, and humans. The human pathogen C. trachomatis causes infectious blindness (trachoma) and a variety of sexually transmitted conditions, each of which affects millions of people worldwide (1, 2). Chlamydia pneumoniae, a pathogen of both humans and other animal species, causes respiratory problems in humans and has been associated with atherosclerosis and perhaps other chronic human diseases (3). Chlamydia abortus is a pathogen of sheep and goats, causing serious abortive conditions that can affect large numbers of pregnant sheep. Chlamydia psittaci, C. pecorum, and several other newly identified members of the genus Chlamydia are pathogens of a large number of animal species. Both C. psittaci and C. abortus are zoonotic and can cause life-threatening diseases in human infections (4). More distantly related members of the family Chlamydiaceae are pathogens or commensals in vertebrates and invertebrates.

All examined members of this family develop within a nonacidified intracellular vacuole termed the inclusion. Formation of this unique chlamydial growth niche begins immediately upon chlamydial contact with a host cell (5), and this process continues throughout the infection. One collection of proteins important in vacuole modification are termed inclusion membrane proteins (Incs) (6, 7). Primary sequences of the Inc proteins are highly variable within species and share variable levels of sequence similarity between different species. The function of most Incs remains unclear, but a subset is known to interact with specific host cell proteins, remodeling the host environment to favor the intracellular bacteria (7–10). Some Inc proteins contain domains with features of eukaryotic SNARE proteins, a superfamily that facilitates membrane fusion during cell trafficking (11–13). A specific example of this is IncA, which contains two SNARE-like domains in a carboxy terminal core region that is exposed to the cytoplasm at the inclusion surface (Fig. 1) (14). IncA present on neighboring inclusions forms dimers that facilitate vesicle fusion (13, 15, 16). Clinical C. trachomatis mutants that lack IncA form inclusions that do not fuse with one another (17).

FIG 1.

Full genome identity and IncA amino sequence similarity among Chlamydia spp. examined in this work. All alignments are based on the following sequence accession numbers: C. trachomatis D/UW-3/CX (NC_000117); C. psittaci 6BC (NC_017287); C. pneumoniae CWL029 (NC_00092); C. muridarum VR123 (NZ_CP063055.1); C. pecorum E58 (NC_015408.1); C. caviae GPIC (NC_003361.3); C. abortus S26/3 (NC_004552). Panel A: Whole genome alignment for the indicated species. A full alignment was conducted using the Progressive Mauve algorithm and ClustalW (34), with automatic calculations of seed weight and minimum LCB score for colinear genomes. More detailed whole genome trees can be found in references 31 and 32. Panel B: Similarity analysis of the full length IncA polypeptide sequences. Panel C: Examination of sequence similarity within SNARE motifs in each predicted IncA sequence. An N-terminal and C-terminal SNARE motif is present in each IncA sequence (13), downstream of the predicted transmembrane domain of the protein. As is seen in the analysis of the full genomes and the full-length IncA sequence, similarity within the SNARE domains is highest among those species combinations that form cross-species fused inclusions.

Although many aspects of the basic chlamydial developmental cycle are conserved, inclusion structure varies considerably among the different species. An excellent early investigation by Spears and Storz (18) used phase-contrast microscopy to investigate inclusion structure in Chlamydia-infected cells, and all work with immunofluorescence microscopy supports these early investigations (19, 20). On one extreme, wildtype strains of C. trachomatis, C. suis, and C. muridarum form single inclusions within cells and these inclusions fuse with one another, both homotypically (within species) and heterotypically (between species) (21). In contrast, other species, such as C. caviae, form inclusions that predominately divide during the infectious process, leading to inclusions that are multiply lobed (22). Fusion of inclusions across species has not been demonstrated for any Chlamydia spp. outside of the C. trachomatis/C. muridarum/C. suis triad, three species that are closely related in a genus-wide phylogenetic tree (Fig. 1). Attempts to generate fused inclusions between C. trachomatis and species outside of this triad have not been successful (21, 23).

Although C. abortus is an important pathogen of sheep and other ruminants, little is known about its intracellular development. We used both natural antibodies in sera collected from infected sheep, plus monospecific antibodies directed at different chlamydial components, to explore inclusion biology within this species. Analyses included cells infected only with C. abortus, and cells coinfected with C. abortus and other chlamydial pathogens. Microscopic analysis of infections demonstrated limited division of inclusions within cells and, at higher multiplicities of infection (MOI), the fusion of C. abortus inclusions with those formed by C. caviae and C. psittaci. These studies demonstrate that cross-species fusion of inclusions does occur outside of the C. trachomatis/C. suis/C. muridarum group of pathogens, and we propose that such fusion occurs across the genus between pairs of closely related chlamydial species.

RESULTS

Growth of C. abortus in cell culture.

C. abortus growth in vitro was very similar to that of C. caviae (22), with infection forming units (elementary bodies) first appearing at 20–24 hours postinfection (hpi) and increasing in number through 48 hpi (not shown). Monoclonal antibodies to chlamydial developmental form antigens were used to examine C. abortus inclusion structure in vitro. These analyses demonstrated that inclusion shape varied considerably within different infected cells, with data generally consistent with that published by others (18). There was no difference in inclusion structure between a historical isolate (LW203; Fig. 2A and B) or a recent clinical isolate (OP5P5; Fig. 2C and D).

FIG 2.

Chlamydia abortus development within murine McCoy cells. Panels A to D: Cells were fixed with methanol and labeled with anti-LOS antibodies (red) and the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI; blue). Panel A: McCoy cells infected with C. abortus strain LW203 fixed at 17 hpi. Panel B: Similar infections fixed at 51 hpi. Panel C: A recent clinical isolate (OP5P5) infected at a low MOI (0.2 C. abortus EBs per cell), fixed 50 hpi. Panel D: Similar monolayers as those in (C) but infected at an MOI of 2. Panel E: Quantification of C. abortus-infected cells carrying 2 or more distinct inclusions, measured as a function of MOI. The MOI was varied in 10 folds from 0.002 through 2 ifu per cell. Cells were evaluated for the number of individual inclusions using the 40X objective.

Homotypic inclusion fusion or inclusion division varies considerably among different chlamydial species (7, 17, 21). We examined this property in C. abortus-infected cells through the use of immunofluorescence-based evaluation of monolayers infected with different multiplicities of C. abortus. These studies demonstrated that cells with multiple inclusions constituted between 20% and 40% of the examined infected cell population. There was a significant increase in the number of cells with multiple inclusions as a function of MOI (MOI = 0.002 to MOI = 2; one-way ANOVA; P = 0.0004). However, even at MOI greater than one ifu per cell, the percentage of cells with multiple inclusions was always under 0.5 (Fig. 2E). These data indicate that division of inclusions and fusion of inclusions is occurring in C. abortus-infected cells, though neither division nor fusion of inclusions occurs to the extent of that observed with other chlamydiae (Fig. 2) (21, 22).

Sera from Chlamydia-infected animals contain antibodies to antigens not present in the infecting elementary bodies (24, 25). We used sera collected from ewes that were naturally infected with C. abortus to expand the analysis of inclusion structure. These studies demonstrated clearly that C. abortus antigens were recognized in structures that extend away from the inclusions in cells infected in vitro (Fig. 3). In C. trachomatis and C. caviae, these structures are enriched for Inc proteins, have low concentrations of developmental-form-specific antigens, and are more prevalent in cells stressed with beta-lactam antibiotics (26). This was also true with C. abortus (Fig. S1). These labeling patterns suggest that the antisera are recognizing Inc proteins in the C. abortus-infected cells.

FIG 3.

Labeling of C. abortus-infected cells with sera from naturally infected sheep. Immunofluorescence labeling of C. abortus LW203-infected cells fixed with methanol 48 hpi and probed with serum from a naturally infected ewe (panels A, B, D) and anti-MOMP (panel C). Panels A and B represent an identical set of cells examined in different focal planes. Panels C and D show identical cells visualized for the different antigens. The arrows in B and D show Inc-protein-laden fibers that extend away from the inclusion within infected cells. The scale bar in panel C equals 10 microns for all images.

Treatment of Chlamydia-infected cells with beta-lactam antibiotics such as ampicillin leads to the formation of “aberrant forms,” which continue to grow but do not divide. This treatment can also lead to more abundant IncA-laden fibers within cells (23). Immunofluorescence microscopy of ampicillin-treated, coinfected cells revealed typical aberrant morphology for both C. caviae and C. abortus, with enhanced fiber abundance within treated cells.

Fusogenicity of inclusions from different Chlamydia species.

Chlamydia trachomatis inclusions undergo not only homotypic vesicle fusion, but also fusion with inclusions formed by the closely related species C. muridarum and C. suis (23). In contrast, C. trachomatis inclusions do not fuse with those formed by C. caviae or C. pneumoniae (20). We used a coinfection model to determine whether C. abortus inclusions would undergo fusion with those formed by other species. Different combinations of Chlamydia spp. were co-inoculated onto cells, and the resulting inclusion structures were examined via immunofluorescence microscopy with different species-specific antibodies. A majority of the combinations of strains led to inclusions that segregated within cells, with no evidence of fusion of inclusion during infection and, when tested, no apparent detection of an IncA-labeled margin from one species surrounding developmental forms of another. Within these analyses, mixed infections that included any of the species C. pecorum, C. pneumoniae, or C. trachomatis did not demonstrate heterotypic inclusion fusion (Fig. 4A–F). These observations were supported quantitatively by counting fused inclusions in cells coinfected with each of the combinations shown in Fig. 4A–F, which led to no or very few examples of inclusion fusion.

FIG 4.

Examination of cells infected with different combinations of Chlamydia species. (A) C. trachomatis and C. caviae. (B) C. caviae and C. pneumoniae. (C) C. pecorum and C. caviae. (D) C. abortus and C. trachomatis. (E) C. abortus and C. pneumoniae. (F) C. pecorum and C. abortus. The scale bar in D shows indicates 10 μm for panels A-F. Panel G. Microscopic quantification of inclusion fusion in coinfected cells. Infected cells were quantified in three or more experiments, with the exception of combinations between C. abortus and C. pneumoniae, which were analyzed in two experiments. The different species included in the experiments were C caviae (C.c.), C. trachomatis (C.t.), C. abortus (C.a.), and C. pneumoniae (C.p.). Panels H-J: McCoy cells were infected with C. abortus and C. caviae (MOI∼1 for each), followed by incubated for 48 h at 37°C, followed by fixation and examination either with the 40X objective (panel H) or at high magnification (panels I and J). These infected cells were visualized using antibodies to C. caviae IncA (green) and C. abortus MOMP (red), and DAPI was used to label DNA. The scale bar in panel J represents 5 μm for panels I and J.

In contrast, inclusions formed in cells infected by any two of C. abortus, C. caviae, and C. psittaci formed inclusions that interacted more directly. Coinfection of C. caviae and C. abortus at an MOI less than 1 yielded a mixture of singly infected cells and coinfected cells containing both fused and unfused inclusions. In these coinfected cells, most inclusions showed evidence of fusion demonstrated microscopically by C. abortus developmental forms in vacuoles surrounded by a perimeter of the C. caviae IncA-labeled inclusion membrane (Fig. 4H–J). These results were quantified by counting the number of apparently fused inclusions in coinfected cells. In contrast to the other combinations shown in Fig. 4, a high percentage of C. abortus/C. caviae-infected cells demonstrated evidence of inclusion fusion (Fig. 4G).

To confirm the results of our traditional fluorescence imaging and ensure that the C. caviae and C. abortus were colocalizing within the same inclusion, we performed confocal microscopy on coinfected cells. In the confocal images, C. abortus and C. caviae developmental forms colocalize into single vacuoles with the different species often congregating in distinct areas of the inclusion (Fig. 5, Fig. S3).

FIG 5.

Confocal microscopy of methanol-fixed cells coinfected with C. abortus and C. caviae. McCoy cells were infected and incubated for 48 h at 37°C and then fixed with methanol. Cells were triple labeled with antibodies to C. abortus MOMP (panel A), C. caviae MOMP (panel B), and C. caviae IncA (panel C). Panel D is a merged image of the labeling. The arrows indicate two cells that are infected with C. abortus alone and the asterisks are to the right of doubly infected cells. The scale bar in A shows 10 microns for all images.

Chlamydia psittaci is a zoonotic pathogen that is phylogenetically close to C. abortus and C. caviae (Fig. 1). Because of this relatedness, we included this species in qualitative analyses of heterotypic inclusion fusion. We lacked an antibody specific to C. psittaci, and thus coinfections were assessed with antibodies to the genus-common lipooligosaccharide (LOS), plus either anti-C. abortus MOMP (Fig. 6) or anti-C. caviae IncA (Fig. S2). In both coinfection experiments, there was evidence of inclusion fusion with C. psittaci. The images in Fig. 6 show infections with C. abortus LW203 and C. psittaci CP3. Cells infected with each pathogen singly are evident in this figure, as well as two cells carrying both pathogens. All chlamydial developmental forms, regardless of species, are labeled with the anti-LOS, while only the C. abortus is labeled with the species-specific antibody (Fig. 6D; green). The coinfected cells show evidence of inclusion fusion, as the developmental forms from each species are present and colocalize in the septate inclusions.

FIG 6.

Immunofluorescence images of inclusion structure in C. psittaci CP3 and C. abortus LW203-coinfected cells. Cells were infected for 40 h and then fixed with methanol and labeled with (genus-common) anti-LOS and (species-specific) anti-C. abortus MOMP antibodies. Panel A: DAPI labeling. Panel B: anti-LOS. Panel C: anti-C. abortus MOMP. Panel D is a merged image. Cells infected singly with C. abortus (block arrow) or C. psittaci (arrow) are indicated in panel B. The two cells at the top of the images are infected with both pathogens. The scale bar in B represents 10 microns for all images.

Genome sequence and IncA sequence analysis.

Previous work in other laboratories has demonstrated that IncA carries SNARE motifs in a core region of the protein that participates in fusion of inclusions (12, 13, 16). We hypothesized that genome sequence similarity, and the similarity within IncA, would correlate with cross-species fusion of inclusions. To explore this, we conducted similarity analyses for both whole genome sequences and for sequences within the IncA polypeptide. Phylogenetic analysis of the whole genome and the full IncA polypeptide mirrored one another, with C. abortus and C. psittaci being most similar, and C. caviae showing linkage to that pair (Fig. 1 A, B). Analysis of amino acid sequences downstream of the IncA transmembrane region and in the SNARE domains within IncA (Fig. 1C) demonstrated a similar phylogenetic linkage among these species. Finally, these analyses demonstrate how C. trachomatis and C. muridarum form linked branches on each tree, which is consistent with the cross-species fusion of inclusions in these species.

C. caviae has a competitive advantage over C. abortus in coinfected cells.

In many inclusions formed in cells infected by either C. abortus and C. caviae or C. abortus and C. psittaci, there were lower numbers of C. abortus developmental forms in the vacuole (Fig. 4J, 5, and 6). These observations led to a hypothesis that C. abortus grew more poorly in coinfected cells than did its partner, either C. caviae or C. psittaci. This hypothesis was tested through the use of a quantitative PCR approach to determine the bacterial load of C. caviae or C. abortus during coinfections in vitro. In these coinfections, the concentration of one species was kept constant, while the titer of the other species was varied. When the MOI of C. caviae was kept constant in the presence of decreasing C. abortus inoculum, there was no observed effect on the bacterial load of C. caviae (Fig. 7A). This indicated that, within our tested range, varying the number of C. abortus developmental forms did not affect the growth of C. caviae. In contrast, decreasing the MOI of C. caviae had a statistically significant effect on the numbers of developing C. abortus in culture (Fig. 7B). In examination of the combined experiments there was no significant difference in effect of C. caviae MOI on C. abortus bacterial load (p > 0.05), however there was a significant difference in resulting Cq values of C. abortus at varying C. caviae MOI (P < 0.05). These data indicate that, while the presence of C. abortus has no effect on the growth of C. caviae during coinfection, high numbers of C. caviae did reduce the productive development of C. abortus.

FIG 7.

Coculture with C. caviae negatively affects the bacterial load of C. abortus within infected cells. qPCR was performed on DNA extracted from coinfections with C. abortus and C. caviae. (A) Bacterial load of C. abortus as effected by changes in starting MOI of C. caviae (B) Bacterial load of C. caviae as effected by changes in starting MOI of C. abortus. The bacterial load was extrapolated from ompA copies per mL for each species. Results are given as mean and standard deviation (± SD) of replicates from a representative experiment.

DISCUSSION

Inclusion biogenesis remains a challenging area of investigation in the chlamydial system. This is especially true with regard to the veterinary chlamydial pathogens. Early work by Spears and Storz demonstrated significant differences in the structure of inclusions in veterinary Chlamydia spp. (18), and work with Inc proteins from veterinary pathogens has been conducted by different laboratories (13, 22, 27, 28). Very little is known about the inclusion development in C. abortus, an important pathogen of small ruminants.

Our primary characterization of inclusion structure is consistent with that described by Spears and Storz, including their demonstration of small secondary inclusions in a subset of cells. We demonstrate that cells with multiple inclusions are common in monolayers, including cells infected at very low MOI. The data support a conclusion that C. abortus inclusions do divide during growth, but not to the extent seen in another veterinary pathogen, C. caviae. As the number of cells with multiple inclusions does not increase linearly with increasing MOI (Fig. 2E), we conclude the inclusions can fuse within these cells as well. Genome sequencing data indicate that all examined chlamydiae, including Protochlamydia spp. (29) encode a number of Inc proteins that localize to the inclusion membrane. This is certainly true for C. abortus (30). It is difficult to demonstrate the localization of proteins to the IM in the absence of specific antisera, but our use of sera from naturally infected sheep shows that non-developmental-form-associated antigens are found in C. abortus-infected cells, consistent with what is known for Inc proteins in other species. In addition, we have previously shown that Inc proteins increase in abundance in cells treated with beta-lactam antibiotics, and this was similarly evident in C. abortus (Fig. S1).

Inclusion fusion is a trait shown directly in C. trachomatis and the closely related C. suis and C. muridarum. There are, however, no reports of heterotypic inclusion fusion outside of those three species. We explored the ability of C. abortus inclusions to fuse with a variety of other chlamydial species. While in most cases there was no evidence of heterotypic inclusion fusion, inclusions formed by C. abortus, C. caviae, and C. psittaci did interact and fuse within coinfected cells. In cells infected with both C. caviae and C. abortus, dense collections of chlamydiae were found within C. caviae-IncA-laden vacuoles, indicating interaction of the two inclusions within infected cells. These results were complemented by confocal microscopy showing both bacteria in the same plane, surrounded by C. caviae IncA. These inclusions most commonly resembled those formed by C. caviae, with multiply lobed, IncA-laden vacuoles present in most cells.

Chlamydia abortus, C. psittaci, and C. caviae are closely related species, on a branch of the tree containing all species except C. trachomatis, C. suis, and C. muridarum. (Fig. 1) (31, 32). Because of the close phylogenetic relationship between these veterinary and zoonotic pathogens, we hypothesized that these species would form inclusions that fused with one another. We conducted limited studies examining the ability of C. psittaci CP3 and VS1 to form inclusions that fused with those formed by either C. caviae or C. abortus. Although our work was challenged by the lack of a specific anti-C. psittaci antibody, inclusion fusion was observed in both C. abortus/C. psittaci and C. caviae/C. psittaci coinfections. These data support a hypothesis that phylogenetically close chlamydial species are more likely to form inclusions that fuse with each other than are distantly related chlamydial species. Chlamydia trachomatis IncA is necessary for fusion of inclusions within the species C. trachomatis and between C. trachomatis and C. muridarum or C. suis (12, 23, 27). Each chlamydial species examined in this work encodes an IncA homolog, and the phylogenetic relationship of the IncA polypeptide, and within SNARE-like domains within IncA (13), mirrors the overall genomic relatedness of these species (Fig. 1). Therefore, cross-species fusion of inclusions is a genus-wide trait within Chlamydia, and the likelihood of fusion of inclusions between two species is related directly to their overall genomic relatedness and the relatedness of IncA.

Our microscopic analysis often qualitatively demonstrated that C. abortus was lower in abundance in cells coinfected with either C. caviae or C. psittaci. This suggested some competitive disadvantage for C. abortus in these coinfected cells. This was examined using a quantitative PCR approach in cells coinfected with C. abortus and C. caviae. We examined this using a coinfection strategy where the relative MOI of each pathogen was altered in cells. The data demonstrated that increasing the amount of C. caviae against a constant number of C. abortus led to decreases in C. abortus genomes within cells, while changing the amount of C. abortus against a constant amount of C. caviae did not affect C. caviae abundance. The processes leading to these differences are unclear but are likely not a function of pathogen growth rate, which was not significantly different between the two species (not shown). Thus, some aspect of intracellular growth in coinfected cells leads to a competitive advantage for C. caviae in these coinfected cells.

Collectively, these experiments add depth to our understanding of inclusion biogenesis in the chlamydiae. Future work will address the possibility of genetic exchange between C. abortus and C. caviae and will work to understand how unique chlamydial antigens recognized in the context of infection might add strength to existing commercial anti-C. abortus vaccines.

MATERIALS AND METHODS

Chlamydia and chlamydial culture conditions.

The list of chlamydial strains used in this work can be found in Table S1 in the supplemental material. All cell culture was performed with McCoy cells incubated in Dulbecco’s Minimum Essential Medium supplemented with 10% fetal calf serum (DMEM10). For propagation, McCoy cells were infected at MOI > 0.5, and incubated 40–72 h, depending on the species. Infected monolayers were frozen at −80°C for 30 min and thawed at 37°C. Cells were harvested using cell scrapers then centrifuged at 225 × g for 5 min. The cell pellet was discarded, and supernatant was centrifuged at 16,000 × g for 15 min to collect the bacteria. Following this centrifugation, the pellet was suspended in 1 mL SPG (0.25M sucrose, 10 mM sodium phosphate, 5 mM -l-glutamic acid, pH 7.2), divided into 100 μl aliquots, and stored at −80°C.

Stocks of C. psittaci CP3 and VS1 (32) were prepared in HeLa cells and purified by renografin density gradient centrifugation (33). Because of biosafety concerns, C. psittaci was not expanded or propagated for these studies, and experiments with this species were limited in scope. All coculture work in this study was approved by the Oregon State University Institutional Biosafety Committee.

Nucleotide and protein sequence analysis.

Sequence alignments were performed in Geneious (34) using Geneious global alignment and subsequent phylogeny trees were made with Geneious tree-builder with a Jukes-Cantor genetic distance model and neighbor-joining tree building method. The hydrophobicity plot for IncA was generated in Expasy ProtScale (35) with the Janin hydrophobicity model selected and a window size of 15. Accession numbers for these comparisons are listed in the legend for Fig. 1. Protein statistics were generated in Geneious within the sequence alignments and calculated using the BLOSUM62 scoring matrix for amino acid substitutions.

Collection and testing of ovine anti-chlamydia antisera.

Ewes from two sheep farms in Central and Western OR, USA were examined for serologic evidence of Coxiella burnetii, and remnant sera from these tests were used to examine anti-C. abortus antibody production. These farms have a history of abortion storms due to C. abortus infection that is regularly controlled with aggressive antibiotic therapy and a successful autogenous bacterin-based vaccine. Isolated C. abortus-verified abortions occur routinely each year. Serum from pregnant ewes and ewe lambs (young, not pregnant) were tested in immunofluorescence microscopy and high titer sera were then used in immunofluorescence microscopy. A FITC-conjugated secondary anti-sheep-IgG (Thermo Fisher, Carlsbad, CA) was then incubated on monolayers and inclusions visualized in immunofluorescence microscopy.

Immunofluorescence microscopy of infected cells.

McCoy cells were set onto sterile glass coverslips in 24-well trays. After overnight incubation, these cells were infected with different Chlamydia combinations and incubated at 37°C for between 36 and 60 h. Monolayers were fixed with 100% methanol for 10 min, washed with PBS, and incubated in 2% bovine serum albumin (BSA) as a blocking agent. Fixed, infected cells were then incubated in dilutions of antibodies against different combinations of each of the following antigens: C. caviae MOMP (murine IgG1 monoclonal antibody), C. caviae IncA (murine IgG1) (22), C. pneumoniae IncA (guinea pig antiserum) (20), C. pecorum MOMP (rabbit antiserum: LSBio, Seattle, USA), C. abortus MOMP (murine IgG2b; Santa Cruz Biotechnology; Dallas, USA) and anti-chlamydial lipooligosaccharide (LOS; murine IgG1) (26). Appropriate commercial secondary antibodies were used to differentially label antigens for immunofluorescence. Coverslips were mounted on glass slides using Vectashield Antifade Mounting Media (Vector Laboratories). A Leica LB 100T fluorescence microscope (Leica Microsystems) and Qimaging software (Teledyne; Surrey, Canada) were used to collect and process images.

Confocal images of chlamydial inclusions were captured on a Zeiss LSM780 NLO Confocal Microscope (Zeiss) equipped with a 63X oil immersion lens. Zen 3.0 SR Black software was used to process the images and visually analyze areas of chlamydial co-occurrence. Linear adjustments of brightness and contrast were applied as needed. Confocal microscopy was performed at the Oregon State University Center for Genome Research and Biocomputing.

Bacterial titration.

Ninety-six well plates were seeded with McCoy cells at a concentration of 1 × 10⁴ cells/well and incubated at 37°C overnight. A 50 μL aliquot of the cultured EBs was mixed with 450 μL of SPG and serially diluted 10-fold. The inoculated plate was centrifuged for 1 h at 23°C at 900 × g. Infections were incubated for 48 h, then fixed with methanol for 15 min. Different Chlamydia spp. were labeled with antibodies against LOS and a secondary antibody conjugated with fluorescein isothiocyanate (FITC). Wells containing between 30 and 300 inclusions were counted using fluorescence microscopy and the bacterial concentration was calculated in IFU/mL.

Chlamydia coinfections.

McCoy cells were seeded (2 × 10⁵ cells/well) on glass coverslips in a 24-well plate and allowed to attach overnight. These monolayers were inoculated with individual or multiple Chlamydia spp. as indicated (MOI between 0.5 and 1) and centrifuged at 23°C for 1h at 900 × g. Supernatants were removed and fresh media containing 1 μg/mL cycloheximide and/or 100 μg/mL ampicillin was added to the cells. The cells were incubated at 37°C for up to 48 h to allow chlamydial growth. For coculture experiments with C. pneumoniae, monolayers were inoculated singly with C. pneumoniae at T = 0 and the second species after 24 h. Cells were then incubated 48 h prior to fixation.

Fusion of the inclusions were quantitated by identifying differentially stained bacteria within the boundaries of the same infected cell. The number of fused and unfused inclusions were counted at 400X total magnification. Means were calculated for a minimum of three independent experiments for all crosses except those involving C. pneumoniae. Coinfections between C. pneumoniae and C. abortus were quantified in two independent experiments.

For quantitative PCR-based analyses of chlamydial growth in cells coinfected at differing MOI, one species infectivity would be held constant (MOI = 10), while the partner species was infected at MOI varying from 1 through 10. After inoculation, the plate was centrifuged for 1h at 900 × g at room temperature. Supernatants were then removed and replaced with fresh DMEM with 10% FBS, and the plate was incubated at 37°C. Reciprocal experiments were performed with the species having variable and constant MOI switched. After 48 h of culture, the infected cells were lysed by replacing the cell culture medium with 1X PBS and freezing at − 80°C for 30 min. Disrupted cells were then scraped and sonicated to lyse the large cell debris, and cells were then centrifuged at 900 × g for 5 min to pellet the lysed debris. The supernatant containing chlamydial EBs was collected and centrifuged at 16,000 × g for 15 min, and the pellet prepared for genomic DNA extraction. EBs isolated from coinfected monolayers were resuspended in water and treated with RQ1 DNase (Promega, Madison, WI) at 37°C for 30 min. RQ1 stop solution was added at 65°C for 10 min and then supplemented with dithiothreitol (5 mM). Samples were incubated at 56°C for 1 h and total nucleic acid was extracted using the DNeasy blood and tissue kit (Qiagen) per the manufacturer’s instructions. Prepared DNA was stored at −20°C. A set of primers were designed based on unique ompA sequences of C. caviae and C. abortus identified from published reference strains (36); (Table S2 in the supplemental material). The CcOMP1-F primers and CcOMP1-R primers amplify a 107 bp region of C. caviae ompA, while CpaOMP1-F and CpaOMP1-R amplify an 82 bp region of C. abortus ompA. Standard curves were generated by serial dilution of amplified ompA product for each species to contain from 1 × 10¹⁰ to 1 × 10² ompA copies per reaction. Single infections with either C. abortus or C. caviae were conducted in parallel with the competitive infections and qPCR was performed on the prepared genomic DNA to act as positive controls. Each reaction contained template DNA, SYBR GREEN master mix with ROX, and PCR primers. Forty amplification cycles were conducted on an iCycler instrument (CFX Connect Real-Time Systems, Bio-Rad) with an annealing temperature of 57°C and a 1 min extension at 60°C. Melting curves were performed for each experiment. The amplification efficiency was determined for the standard curve and the bacterial load was calculated from genome copies per mL of input bacteria. Graphical representation of the data was produced using GraphPad Prism. Statistical analysis was performed using RStudio (RStudio, Boston, MA). Values were expressed in means as appropriate and ± standard deviation was calculated for experiments. Comparison of means was performed by one-way ANOVA with statistical significance set at a P-value of 0.05.