Publications

4576

Abstract Objective ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.

Abstract Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.

Abstract Background The appearance of the novel porcine haemotrophic mycoplasma (HM) species ‘Candidatus Mycoplasma haemosuis’ was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of ‘Ca. M. haemosuis’ in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. Results Our novel qPCR assay successfully detected ‘Ca. M. haemosuis’ in all blood samples from the ‘Ca. M. haemosuis’-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. ‘Candidatus M. haemosuis’ was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for ‘Ca. M. haemosuis’. Co-infections with M. suis were evident in all age groups. Conclusion ‘Candidatus M. haemosuis’ infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for ‘Ca. M. haemosuis’ described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of ‘Ca. M. haemosuis’ infections.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati’s blood samples in Midwestern Brazil. Twenty-five samples (25/165—15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with ‘Candidatus Anaplasma brasiliensis’, respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely ‘Candidatus Ehrlichia dumleri’, and Anaplasma sp. closely related to the previously described ‘Candidatus Anaplasma brasiliensis’.

AbstractEleven haplotypes of the bacterium, ‘Candidatus Liberibacter solanacearum’, have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for ‘Ca. L. solanacearum’ by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the ‘Ca. L. solanacearum’ 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of ‘Ca. L. solanacearum’ haplotype F was not detected in this study.

This study presents the molecular detection of Candidatus Rickettsia andeanae and Ehrlichia sp. in Amblyomma pseudoconcolor Aragão, 1908 (Acari: Ixodidae) collected on a large hairy armadillo (Chaetophractus villosus (Desmarest, 1804)). On 12 October 2020, a specimen of C. villosus was found dead on the road in Río Negro province, Argentina. Molecular detection of Rickettsia and Ehrlichia agents was performed amplifying the gltA and 16S rRNA gene, respectively. One tick, determined morphologically and genetically as A. pseudoconcolor, was collected on C. villosus. The rickettsial agent detected in A. pseudoconcolor was identified as Candidatus Rickettsia andeanae. The Ehrlichia sp. strain showed high sequence similarity to different uncultured Ehrlichia sp. detected in horses, capybaras and Ixodes ornithorhynchi from Nicaragua, Brazil and Australia, respectively. The results of this study and previous findings suggest that A. pseudoconcolor may be a potential vector of some Rickettsia and Ehrlichia bacteria of unknown pathogenicity.

Symbiotic associations between bacteria and ciliate protists are rather common. In particular, several cases were reported involving bacteria of the alphaproteobacterial lineage Rickettsiales, but the diversity, features, and interactions in these associations are still poorly understood. In this work, we characterized a novel ciliate protist strain originating from Brazil and its associated Rickettsiales endosymbiont by means of live and ultrastructural observations, as well as molecular phylogeny. Though with few morphological peculiarities, the ciliate was found to be phylogenetically affiliated with Pseudokeronopsis erythrina, a euryhaline species, which is consistent with its origin from a lagoon with significant spatial and seasonal salinity variations. The bacterial symbiont was assigned to “Candidatus Trichorickettsia mobilis subsp. hyperinfectiva”, being the first documented case of a Rickettsiales associated with urostylid ciliates. It resided in the host cytoplasm and bore flagella, similarly to many, but not all, conspecifics in other host species. These findings highlight the ability of “Candidatus Trichorickettsia” to infect multiple distinct host species and underline the importance of further studies on this system, in particular on flagella and their regulation, from a functional and also an evolutionary perspective, considering the phylogenetic proximity with the well-studied and non-flagellated Rickettsia.