Prevalence and diversity of Chlamydiales in Swiss ruminant farms

Chlamydia and Chlamydia-related bacteria are known to infect various organisms and may cause a wide range of diseases, especially in ruminants. To gain insight into the prevalence of these bacteria in the ruminant environment, we applied a pan-Chlamydiales PCR followed by sequencing to 72 ruminant environmental samples from water, feed bunks and floors. Chlamydiales from four family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant farms. Parachlamydiaceae were detected in all three types of environmental samples and was the most abundant family-level taxon (60%). In contrast, only one bacterium from each of the following family-level lineages was identified: Chlamydiaceae, Criblamydiaceae and Simkaniaceae. The observed high prevalence of Parachlamydiaceae in water samples may suggest water as the main source of contamination for ruminants as well as their environment due to spoilage. The absence of reported infections in the investigated ruminant farms might indicate that either detected Chlamydiales are of reduced pathogenicity or infective doses have not been reache

A bovine model of a respiratory Parachlamydia acanthamoebae infection

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P.acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 108 and 1010 bacteria per animal. Controls received 108 heat-inactivated bacteria. Challenge with 108 viable Parachlamydia resulted in a mild degree of general indisposition, whereas 1010 bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P.acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interactio

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stag

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020 - the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86-98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred, using a phylodynamic model. We found that transmission slowed 35-63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics

Neglected zoonotic agents in cattle abortion: molecular and serological screening of difficult to grow bacteria

Coxiella burnetii, Chlamydia abortus and Leptospira spp., are three agents that may lead to bovine abortion. The importance of these difficult to grow zoonotic bacterial pathogens lies in significant economic loss in animal production and the public health risk, at least in endemic countries. Routine bacteriological diagnostics of abortion in cattle in Switzerland is regulated by law including screening by serology and staining. However, only few infectious agents are examined using molecular approaches due to the high costs associated with extended analyses. In the present work, we used both serological and molecular methods to assess the possible role of these pathogens in bovine abortion. From 249 studied bovine abortion events, 242 placenta samples, 57 fetal abomasal content and 182 sera were taken from mother cows. The seroprevalence was 15.93%, 38.46% and 21.43% for C. burnetii (ELISA), C. abortus (ELISA) and pathogenic Leptospira spp. (microscopic agglutination test), respectively. Using specific real time PCR, the prevalence of C. burnetii, Chlamydiales and pathogenic Leptospira spp., were of 12.15%, 16.87% and 8.24%, respectively. After direct sequencing of Chlamydiales positive samples, we identified C. abortus in 8.84% of the cases and probable infection with Chlamydia-related bacteria in 5.22% of the cases. Altogether, routine abortion diagnostics did not detect a possible bacterial agent in 96 cases. Extending the spectrum of analysis could assign at least one possible abortive agent in 39 more cases. In conclusion, diagnostic approaches enabling the detection of C. burnetii, C. abortus and Leptospira spp., should be used more commonly due to their zoonotic potential

Neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria

BACKGROUND: Coxiella burnetii, Chlamydia abortus and Leptospira spp. are difficult to grow bacteria that play a role in bovine abortion, but their diagnosis is hampered by their obligate intracellular lifestyle (C. burnetii, C. abortus) or their lability (Leptospira spp.). Their importance is based on the contagious spread in food-producing animals, but also as zoonotic agents. In Switzerland, first-line routine bacteriological diagnostics in cattle abortions is regulated by national law and includes only basic screening by staining for C. burnetii due to the high costs associated with extended spectrum analysis. The aim of this study was to assess the true occurrence of these zoonotic pathogens in 249 cases of bovine abortion in Switzerland by serology (ELISA for anti-C. burnetii and C. abortus antibodies and microscopic agglutination test for anti-Leptospira spp. antibodies), molecular methods (real-time PCR and sequencing of PCR products of Chlamydiales-positive cases), Stamp's modification of the Ziehl-Neelsen (mod-ZN) stain and, upon availability of material, by histology and immunohistochemistry (IHC). RESULTS: After seroanalysis the prevalence was 15.9% for C. burnetii, 38.5% for C. abortus and 21.4% for Leptospira spp. By real-time PCR 12.1% and 16.9% of the cases were positive for C. burnetii and Chlamydiales, respectively, but only 2.4% were positive for C. burnetii or Chlamydiales by mod-ZN stain. Sequencing of PCR products of Chlamydiales-positive cases revealed C. abortus in 10% of cases and the presence of a mix of Chlamydiales-related bacteria in 5.2% of cases. Pathogenic Leptospira spp. were detected in 5.6% of cases. Inflammatory lesions were present histologically in all available samples which were real-time PCR-positive for Chlamydiales and Leptospira spp. One of 12 real-time PCR-positive cases for C. burnetii was devoid of histological lesions. None of the pathogens could be detected by IHC. CONCLUSION: Molecular detection by real-time PCR complemented by histopathological analysis is recommended to improve definitive diagnosis of bovine abortion cases and determine a more accurate prevalence of these zoonotic pathogens

Presence and diversity of Chlamydiae bacteria in Spinturnix myoti, an ectoparasite of bats

Chlamydia spp. and Chlamydia-like organisms are able to infect vertebrates such as mammals, reptiles and birds, but also arthropods and protozoans. Since they have been detected in bats and bat feces, we expected Chlamydiae bacteria to also be present in the mite Spinturnix myoti, an ectoparasite of mouse-eared bats (Myotis spp.). The prevalence of Chlamydiales in 88 S. myoti was 57.95% and significantly depended on bat host species. In addition, the prevalence was significantly different between bat species living in sympatry or in allopatry. While there was uninterpretable sequencing for 16 samples, eight showed best BLAST hit identities lower than 92.5% and thus corresponded to new family-level lineages according to the established taxonomy cut-off. The four remaining sequences exhibited best BLAST hit identities ranging from 94.2 to 97.4% and were taxonomically assigned to three different family-level lineages, with two of them belonging to the Parachlamydiaceae, one to the Simkaniaceae, and one to the Chlamydiaceae. These results highlighted for the first time the presence of Chlamydia-like organisms and the possible zoonotic origin of Chlamydia sp. in S. myoti ectoparasites of bats, and therefore suggest that these ectoparasites may play a role in maintaining and/or transmitting members of the Chlamydiae phylum within Myotis spp. bat populations. Our results further highlight that the wide diversity of bacteria belonging to the Chlamydiae phylum is largely underestimated

Indication of spatially random infection of chlamydia-like organisms in Bufo bufo tadpoles from ponds located in the Geneva metropolitan area

Occurrence of bacteria belonging to the order Chlamydiales was investigated for the first time in common toad (Bufo bufo) tadpole populations collected from 41 ponds in the Geneva metropolitan area, Switzerland. A Chlamydiales-specific Real-Time PCR was used to detect and amplify the Chlamydiales 16S rRNA-encoding gene from the tails of 375 tadpoles. We found the studied amphibian populations to be infected by "Chlamydia-like organisms" (CLOs) attributable to the genera Similichlamydia, Neochlamydia, Protochlamydia and Parachlamydia (belonging to the family Parachlamydiaceae), Simkania (family Simkaniaceae) and Estrella (family Criblamydiaceae); additionally, DNA from the genus Thermoanaerobacter (family Thermoanaerobacteriaceae) was detected. A global autocorrelation analysis did not reveal a spatial structure in the observed CLOs infection rates, and association tests involving land cover characteristics did not evidence any clear effect on CLOs infection rates in B. bufo. Despite preliminary, these results suggest a random and ubiquitous distribution of CLOs in the environment, which would support the biogeographical expectation "everything is everywhere" for the concerned microorganisms and their amoeba vectors

β-Klotho deficiency shifts the gut-liver bile acid axis and induces hepatic alterations in mice

β-Klotho (encoded by Klb) is an obligate coreceptor, mediating both fibroblast growth factor (FGF)15 and FGF21 signaling. Klb <sup>-/-</sup> mice are refractory to metabolic FGF15 and FGF21 action and exhibit derepressed (increased) bile acid (BA) synthesis. Here, we deeply phenotyped male Klb <sup>-/-</sup> mice on a pure C57BL/6J genetic background, fed a chow diet focusing on metabolic aspects. This aims to better understand the physiological consequences of concomitant FGF15 and FGF21 signaling deficiency, in particular on the gut-liver axis. Klb <sup>-/-</sup> mice present permanent growth restriction independent of adiposity and energy balance. Klb <sup>-/-</sup> mice also exhibit few changes in carbohydrate metabolism, combining normal gluco-tolerance, insulin sensitivity, and fasting response with increased gluconeogenic capacity and decreased glycogen mobilization. Livers of Klb <sup>-/-</sup> mice reveal pathologic features, including a proinflammatory status and initiation of fibrosis. These defects are associated to a massive shift in BA composition in the enterohepatic system and blood circulation featured by a large excess of microbiota-derived deoxycholic acid, classically known for its genotoxicity in the gastrointestinal tract. In conclusion, β-Klotho is a gatekeeper of hepatic integrity through direct action (mediating FGF21 anti-inflammatory signaling) and indirect mechanisms (mediating FGF15 signaling that maintains BA level and composition)