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’.
In Saudi Arabia (SA), the citrus greening disease is caused by ‘Candidatus Liberibacter asiaticus’ (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the ‘Asiatic’ and ‘Floridian’ strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.
The black-eared opossum (Didelphis aurita) is a South American synanthropic marsupial. The presence of opossums in domestic spaces is relevant in the One-Health context since they are hosts of pathogens and ectoparasites that may affect the health of domestic animals and humans. In this study, we aim to determine the occurrence of hemoplasmas and selected tick-borne pathogens in free-ranging black-eared opossums, along with their molecular characterization, hematological and biochemical evaluation and factors associated with infection, in the municipality of Viçosa, State of Minas Gerais, southeastern Brazil. Thirty black-eared opossums were trapped between March 2021 and June 2022. Ectoparasites were collected. Hematological and biochemical analyses were performed. DNA from EDTA-blood samples were analyzed by PCR and qPCR assays. By molecular analyses, ‘Candidatus Mycoplasma haemoalbiventris’ was the most prevalent hemoparasite (73.3%), followed by Hepatozoon sp. (22.2%). Significant differences were observed in the number of platelets, and in the concentration of protein and globulins in the animals infected by ‘Ca. M. haemoalbiventris’ when compared with the negative group. This is the first report of ‘Ca. M. haemoalbiventris’ infection in D. aurita.
It was shown that the SAP11 effector of different Candidatus Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches’ broom and crinkled leaves. Some SAP11 exclusively localize in the nucleus, while the others localize in the cytoplasm and the nucleus. The SAP11-like effector of Candidatus Phytoplasma mali strain PM19 (SAP11PM19) localizes in both compartments of plant cells. We show here that SAP11PM19 can destabilize TCPs in both the nucleus and the cytoplasm. However, expression of SAP11PM19 exclusively in the nucleus resulted in the disappearance of leaf phenotypes while still showing the witches’ broom phenotype. Moreover, we show that SAP11PM19 can not only destabilize TCPs but also relocalizes these proteins in the nucleus. Interestingly, three different transgenic Nicotiana species expressing SAP11PM19 show all the same witches’ broom phenotype but different leaf phenotypes. A possible mechanism of SAP11-TCP interaction is discussed.
The metagenome of foulings from sulfidic spring “Serovodorodny” (Tatarstan, Russia), where members of the genus Thiothrix was observed, was sequenced. Representatives of the phyla Gammaproteobacteria, Cyanobacteria and Campilobacteriota dominated in the microbial community. The complete genome of Thiothrix sp. KT was assembled from the metagenome. It displayed 93.93–99.72% 16S rRNA gene sequence identity to other Thiothrix species. The average nucleotide identity (ANI) и digital DNA-DNA hybridization (dDDH) showed that the genome designated KT represents a new species within the genus Thiothrix, ‘Candidatus Thiothrix sulfatifontis’ sp. nov. KT. The taxonomic status has been determined of the strain Thiothrix sp. CT3, isolated about 30 years ago and not assigned to any of Thiothrix species due to high 16S rRNA gene sequence identity with related species (i.e., 98.8–99.4%). The complete genome sequence of strain CT3 was determined. The ANI between CT3 and other Thiothrix species was below 82%, and the dDDH values were less than 40%, indicating that strain CT3 belongs to a novel species, Thiothrix winogradskyi sp. nov. A genome analysis showed that both strains are chemo-organoheterotrophs, chemolithotrophs (in the presence of hydrogen sulfide and thiosulfate) and chemoautotrophs. For the first time, representatives of Thiothrix showed anaerobic growth in the presence of thiosulfate.
‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.
The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods.
Human ehrlichiosis are scantily documented in Uruguay. The aim of this study was to investigate the presence of Ehrlichia spp. in Haemaphysalis juxtakochi and in a gray brocket deer (Mazama gouazoubira) from Uruguay. The presence of Ehrlichia DNA was investigated in free-living H. juxtakochi in five localities of southeast and northeast Uruguay, as well as blood, spleen, and ticks retrieved from a M. gouazoubira. Ehrlichia spp. DNA was detected in six out of 99 tick pools from vegetation, in the spleen of M. gouazoubira, and in one out of five pools of ticks feeding on this cervid. Bayesian inference analyses for three loci (16S rRNA, dsb, and groEL) revealed the presence of a new rickettsial organism, named herein as “Candidatus Ehrlichia pampeana”. This new detected Ehrlichia is phylogenetically related to those found in ticks from Asia, as well as Ehrlichia ewingii from USA and Cameroon. Although the potential pathogenicity of “Ca. E. pampeana” for humans is currently unknown, some eco-epidemiological factors may be relevant to its possible pathogenic role, namely: (i) the phylogenetic closeness with the zoonotic agent E. ewingii, (ii) the evidence of H. juxtakochi parasitizing humans, and (iii) the importance of cervids as reservoirs for zoonotic Ehrlichia spp. The molecular detection of “Ca. E. pampeana” represents the third Ehrlichia genotype described in Uruguay.
Rubbery taproot disease of sugar beet (RTD), associated with ‘Candidatus Phytoplasma solani’, appeared in 2020 on an epidemic scale in northern Serbia and southern Slovakia, situated at opposite edges of the Pannonian Plain. In the affected locations where the disease was assessed, symptomatic sugar beets were analysed for phytoplasma infection. Additionally, multilocus sequence analyses of ‘Ca. P. solani’ strains on epidemiologically informative marker genes (tuf, stamp and vmp1) were performed. Symptomatic sugar beets from other countries of the Pannonian Plain (Croatia, Hungary and Austria), one sample from Germany, and red beets from Serbia were included in the analyses. ‘Ca. P. solani’ was detected in sugar beet in all assessed countries, as well as in red beet. Molecular analyses revealed the high genetic variability of ‘Ca. P. solani’ with the presence of all four tuf-types (a, b1, b2 and d), 14 stamp genotypes (seven new) and five vmp1 profiles (one new). The most common multilocus genotype in Serbia, Slovakia, Croatia, and Hungary was dSTOLg (tuf-d/STOL/V2-TA). It was dominant on sites with epidemic RTD outbreaks in the Pannonian Plain and in several sugar beet fields with non-epidemic RTD occurrence suggesting the prevalence of a particular epidemiological pathway during the epidemic’s phases.