Chlamydiales and hemotropic mycoplasma in captive and free-living bats

Background: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasma. This study investigated 475 free-living and captive bats from Switzerland, Germany and Costa Rica for the occurrence of Chlamydiales and hemotropic mycoplasma. Results: Screening for Chlamydiales was performed using a Chlamydiaceae -specific real-time PCR targeting the 23S rRNA gene and a pan- Chlamydiales PCR targeting the 16S rRNA gene resulting in a total prevalence of 31.4%. For sequencing, a PCR with the specifically designed inner primers panFseq and panRseq was performed, and criteria published by Pillonel et al. were used to classify the 19 obtained sequences, resulting in the formation of two groups. Groups one and two shared sequence identities to Chlamydiaceae and to Chlamydia -like organisms, including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively. Analysis for the presence of hemotropic mycoplasma was performed using a universal SYBR Green hemoplasma screening real-time PCR targeting the 16S rRNA gene, real-time PCRs specific for M. haemofelis -like and ' Candidatus M. haemominutum'-like organisms and two conventional PCRs targeting an 871-bp and 1030-bp region of the 16S rRNA gene resulting in a total prevalence of 0.7%. Sequencing and phylogenetic analysis of the 871-bp and 1030-bp region of the 16S rRNA gene were used to classify positive specimens and infer their phylogenetic relationships. Three sequences with identities to other unidentified mycoplasma found in vampire bats and Chilean bats were obtained. Conclusions: Bats can harbor Chlamydiales and hemotropic mycoplasma and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than thought before. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and free-living as well as captive bats can be colonized. In conclusion, bats with a subsequent PCR reaction using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) (41) or b) purified and sequenced by Microsynth using specifically designed inner primers panFseq and panRseq. Sequences were compared to known sequences in the NCBI database by BLAST analysis and phylogenetic analyses were performed using muscle in Seaview (44) with manual correction where necessary to create alignments, and using PhyML in Seaview with default parameters to create phylogenetic trees.

suggest that the different chlamydial species documented in bats might have a pathogenic role for the bats or that these bats act as vectors.
In a study by Hornok et al. (28), 196 individual and 25 pooled fecal samples collected from 19 bat species from Hungary and the Netherlands were investigated for the presence of chlamydial DNA; they all tested negative indicating that the prevalence of Chlamydiae in bats is highly variable in studies from neighboring countries. This observation suggests that, although this new chlamydiae clade may occur in some parts of Europe, colonization or infection of bats is influenced by factors that remain to be defined. Methodological differences may play a part of these factors.
Also a Rhabdochlamydiaceae-like sequence was identified in a sample taken from a free-living bat from Costa Rica, which formed another novel clade with bat samples from Hokynar et al. (9) in the phylogenetic tree. The remaining bat samples, similar to chlamydial sequences from environmental samples retrieved from the NCBI database, originated from free-living and captive bats from Switzerland and Germany. Chlamydia-like organisms are commonly present in water and inside amoebae (29)(30)(31), and have also been observed in ticks (32,33) and fleas (34). Thus, it is difficult to guess, which of these chlamydial putative vectors (amoebae or ticks) is the more likely at play as reservoir and vector.
In previous studies from Chua et al. (7) and Piérle et al. (8), two novel Chlamydia-like organisms belonging to the Waddliaceae family and named Waddlia malaysiensis and Waddlia cocoyoc were isolated from fruit bats from Malaysia and the municipality Cocoyoc in Mexico. These regions have a tropical climate, whereas a temperate climate predominates in Switzerland and Germany. In this study, bats from a tropical climate (Costa Rica) were analysed as well, but compared to the two other studies (n=206 and n=38) our sample size from Costa Rica (n=17) was small. In the bats investigated in this study, no Waddliaceae could be detected. Waddlia malaysiensis was first detected in urine samples from bats (7), which had not been investigated in the current study. Waddlia cocoyoc was detected in DNA from the skin and in infected Vero and BHK 21 cells, but also caused severe lesions in lungs and spleen (8). Therefore, it can be assumed that bacterial DNA would have been detected in these two organs if bacteria were present. The two isolates from Mexico and Malaysia might be bound to the regions and/or the climate. Hence, bacterial DNA was undetectable in the European bat samples in this investigation and are unlikely to get detected in the samples from other European studies.
In this study as well as in the studies from Hokynar et al. and Chua et al., whole genome sequencing was not performed; only short fragments of the 16S rRNA and 23S rRNA genes were amplified, as many of the samples in the present study were only available as FFPE samples because the bats had died in the wild and were subsequently collected and sampled, therefore preventing detailed classification.

PCR and sequencing results for Mycoplasmatales
BLAST analysis of the sequences obtained in the current study resulted in the identification of three sequences that were closely related to uncultured Mycoplasma sp. and nine sequences that were closely related to Proteobacteria. The amplification of these Proteobacteria-like sequences is most likely the result of a cross-reaction or unspecific primer binding.
The three sequences related to uncultured Mycoplasma sp. originated from free-living bats from Germany and Costa Rica belonging to the bat species Nyctalus noctula, Vespertilio murinus and Glossophaga commissarisi. Consequently, different species from the bat families Vespertilionidae and Phyllostomidae can harbor hemotropic mycoplasma. The occurrence of mycoplasma could be only confirmed in free-living bats, which is in agreement with the presumed transmission pathways of hemotropic mycoplasma through blood, saliva and arthropods (12-14, 10). The captive bats were isolated from the environment and thus arthropod contact was at least partially excluded or reduced.
Moreover, none of them were sanguivory bats and therefore do not bite other animals living in the enclosure. Consequently, transmission by blood, saliva and arthropods is very unlikely in captive bats.
The phylogenetic tree shows a close relationship between the sequences of the German bats among each other and a sequence from a Chilean bat (25) In all studies, only fragments of the 16S rRNA gene were amplified, sequenced and then combined into contigs where possible to optimize the chances of getting a 16S rRNA gene sequence to analyse.
Whole genome sequencing was not performed. Due to high blood content in the spleen, and samples only available as FFPE samples, methods for DNA analysis in the present study were limited. Therefore, a more detailed description of the analysed sequences and a more precise classification into the mycoplasmal taxonomy were not possible.
According to the classification of mammals, bats belong to the superorder of the Laurasiatheria and therefore are more closely related to carnivores, even-toed and odd-toed ungulates, than to rodents and primates, which belong to the superorder of the Euarchontoglires. While the relationship of other hemotropic mycoplasma represented in the phylogenetic tree ( Figure 1D) roughly corresponds to that of the mammalian classification, Mycoplasma of bats are the only ones to classify quite differently. This is likely due to the high resistance of bats to pathogenic microbes, somehow similar to some rodents (mice, rats). Moreover, this suggests that bats does not get infected by exposure to meat, but rather by exposure to water (and free-living amoebae) as well as to ectoparasites colonizing bats (such as Spinturnix). The latter hypothesis is supported for Chlamydia-like organisms by the recent In conclusion, bats can harbor Chlamydiales and hemotropic mycoplasma and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than thought before. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat the QIAamp DNA FFPE Tissue Kit #56404 (Qiagen) following manufacturer's instructions. DNA quantity and quality of all samples was evaluated with the Nanodrop-1000 (Witec AG, Luzern, Switzerland).

PCR analysis for Chlamydiales DNA
A total of 1021 DNA samples was screened for the presence of Chlamydiales DNA using two different real-time PCRs targeting sequentially the 23S rRNA gene (Chlamydiaceae family-specific) and the 16S rRNA gene (pan-Chlamydiales order-specific).
The Chlamydiaceae-specific real-time PCR targeting the 23S rRNA gene (Chlam23S-qPCR) (36) used primers Ch23S-F, Ch23S-R and probe Ch23S-p (Microsynth, Balgach, Switzerland) described by Ehricht et al. (37). The internal amplification control eGFP amplified with primers eGFP-1-F, eGFP-10-R and probe eGFP-Hex (Microsynth) was added to each reaction (38). The PCR was conducted on a Thermocycler 7500 Fast ABI (Thermo Fisher Scientific). All samples were tested in duplicate and samples with a cycle threshold of <38 in duplicate PCR reactions were considered positive.
Samples positive in the Chlamydiaceae family-specific real-time PCR were then further analysed using three different conventional PCR protocols targeting partial sequences of the 16S or 23S rRNA gene. For the pan-Chlamydiales real-time PCR targeting a partial sequence of the 16S rRNA-encoding gene (16S-pan-qPCR) (41), primers 16S-panCh-F, 16S-panCh-R and probe 16S-panCh (Eurogentec, Seraing, Belgium) were applied in a StepOne Plus real-time PCR system (Thermo Fisher Scientific). All samples were tested in duplicate, and if a single replicate was positive (Ct £ 37), the corresponding sample was considered positive. Quantification was performed using a 10-fold-dilution of a plasmid control tested in duplicate, constructed with the sequence of interest based on the Parachlamydia acanthamoebae 16S rRNA encoding gene, cloned with the TOPO TA Cloning Kit for Subcloning with One Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific). Molecular-biology-grade water was used as a negative control in all PCR reactions.
All PCR primers and probes, the targeted genes and amplicon sizes used in this study are summarized in Table 3. All reaction mix compositions and cycling conditions are shown in Table S1. Instruments, Châtel-Saint-Denis, Switzerland) and PCR products were analysed by gel-electrophoresis on a 1.5% agarose gel.
All PCR primers and probes, the targeted genes and amplicon sizes used in this study are summarized in Table 3. All reaction mix compositions and cycling conditions are shown in Table S1. Sequences were compared to known sequences in the NCBI database by BLAST analysis and phylogenetic analyses were performed using muscle in Seaview (44) with manual correction where necessary to create alignments, and using PhyML in Seaview with default parameters to create phylogenetic trees.

Accession numbers
Many thanks go to the team of the Institute of Animal Pathology, University of Bern, namely Kerstin Hahn, Corinne Gurtner and Marion Schediwy for performing necropsy and organ sampling of the captive bats in Switzerland. We thank Theresa Pesch, Barbara Prähauser, Lea Rohner, Benita Pineroli and Nadine Jahn for their technical assistance with laboratory work, which was partly performed using

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