M. hyorhinis. This is the first study using conserved regions of seven members of Vlps and has shown an effective approach toward vaccine development. The proposed vaccine candidate is highly stable and soluble. It elicits an impressive immune response and can be regarded as a hypothetical vaccine candidate in the future. Further laboratory tests and animal inoculation are required to validate vaccine efficacy and safety to combat swine infections.In this study, we designed a multi-epitope vaccine candidate for Mycoplasma hyorhinis (M. hyorhinis) is responsible for infections in the swine population. Such infections are usually cured by using antimicrobials and lead to develop resistance. Until now, there has been no effective vaccine to eradicate the disease. This study used conserved domains found in seven members of the variable lipoprotein (VlpA-G) family in order to design a multi-epitope candidate vaccine (MEV) against M. hyorhinis. The immunoinformatics approach was followed to predict epitopes, and a vaccine construct consisting of an adjuvant, two B cell epitopes, two HTL epitopes, and one CTL epitope was designed. The suitability of the vaccine construct was identified by its non-allergen, non-toxic, and antigenic nature. A molecular dynamic simulation was executed to assess the stability of the TLR2 docked structure. An immune simulation showed a high immune response toward the antigen. The protein sequence was reverse-translated, and codons were optimized to gain a high expression level in E. coli. The proposed vaccine construct may be a candidate for a multi-epitope vaccine. Experimental validation is required in future to test the safety and efficacy of the hypothetical candidate vaccine. M. hyorhinis, M. hyopneumoniae, and M. flocculare [M. hyorhinis resides and colonizes in the respiratory tract of healthy and infected pigs and colonizes most likely due to stress as the mechanisms are not yet well known. The disease is clinically manifested through polyserositis, conjunctivitis, otitis, arthritis, and lameness [M. hyorhinis is also known to be involved in human cancer. The pathogen has been reported to augment the cancer cell migration, invasion, and metastasis significantly [Several species of the genus Mycoplasma infect and produce disease in pigs. Harboring respiratory tracts include occulare . M. hyorlameness . Moreoveficantly , and antMycoplasma hyorhinis belongs to the class mollicutes, lacks cell walls, and has been successfully adopted in host cell environments. Cell membrane surface variations are one of their adaptation strategies to the different host cell environments. These surface variations are due to variable surface lipoproteins (Vlps) possessing a high-frequency phase and size variation resulting in structural repetition at the C-terminal region [M. hyorhinis [l region . This fayorhinis ,6.M. hyorhinis. A remarkable diagnostic and analytical specificity was detected against the pigs inoculated with M. hyorhinis, M. hyosinoviae, M. hyopneumoniae, and M. floccularae [M. hyorhinis. This is not surprising that variable proteins also possess conserved domains, i.e., variant surface glycoprotein (VSG) of Trypnosoma sp. [B. burgdorferi was supposed to act as a protective antigen [Mycoplasma mycoides subsp. caprii lipoproteins stimulate the immune response and may act as immunogenic and protective antigens [Previously, Vlp-ELISA was developed by using unique repeated units at region III of VlpA-G in our laboratory. A total of 247 clinical samples from experimentally infected pigs were tested. The coefficients of variation for the inter-assay and intra-assay were below 10%. The established method did not show any cross-reactivity with other common pathogens infecting pigs and showed up to marked specificity and stability (article published in Chinese). In another study, ELISA was developed based on the chimeric VlpA-G recombinant polypeptide by in vitro synthesizing the Vlp coding genes of soma sp. . Conserv antigen . Another® MycoMAX  [M. hyorhinis and swine circovirus type 2 [M. hyorhinis. Inactivated vaccines are being used to prevent infections. These vaccines include the Boehringer Ingelheim Animal Health’s (BIAH) MHR vaccine and IngelvacON, USA) . Anothers type 2 . Althougs type 2 . The coms type 2 . There iMycobacterium tuberculosis, Mycoplasma pneumonia, Mycobacteroides abscessus, Helicobacter pylori, Campylobacter jejuni, and many others [To meet the limitations of inactivated vaccines, immunoinformatics is an extensively utilized approach to develop peptide vaccines against several bacterial pathogens, including y others ,17,18,19y others . M. hyorhinis infections.The objective of the current study is to use computational biology tools to identify and combine the conserved regions in all seven Vlp members and to test the efficacy of a multi-epitope vaccine construct as a candidate vaccine against M. hyorhinis (ATCC 17981) were obtained from NCBI protein database http://www.ncbi.nlm.nih.gov/protein/ (accessed on 30 July 2022) in FASTA format and multiple sequence alignment was conducted in order to find the conserved regions at N-terminal of each sequence. The IEDB server (http://tools.iedb.org/bcell/ (accessed on 30 July 2022)) was used to identify the B cell epitopes. BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/ (accessed on 30 July 2022)) was used for further confirmation of B cell epitopes using antigenic sequence [Sequences in the representative strain of sequence . Emini Shttp://www.cbs.dtu.dk/services/NetCTL/ (accessed on 30 July 2022)) was used to predict CTL epitopes [http://tools.iedb.org/mhcii/ (accessed on 30 July 2022)) [http://crdd.osdd.net/raghava/ifnepitope/scan.php (accessed on 30 July 2022)) epitope server was used to predict interferon-gamma-inducing epitopes. Positive value meets the criteria for the construction of multi-epitope vaccine.NetCTL 1.2 (y 2022)) . Swine LA candidate vaccine construct consisting of two B cell epitopes, two HTL epitopes, and one CTL epitope was combined using linkers, and an adjuvant β-Defensin was added to enhance the immunity. A vaccine construct with a length of 138 amino acids was designed. http://scratch.proteomics.ics.uci.edu/ (accessed on 30 July 2022)) and VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html (accessed on 30 July 2022)). AllergenFP (https://ddg-pharmfac.net/AllergenFP/) was used to predict allergenicity of vaccine construct. ToxinPred server (https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php (accessed on 30 July 2022)) was used to determine the toxicity of the vaccine construct.Antigenicity was measured using ANTIGENpro (https://web.expasy.org/protparam/ (accessed on 30 July 2022)) was used for the prediction of the entire physicochemical properties of the vaccine construct. Solubility was determined using Protein–Sol server (https://protein-sol.manchester.ac.uk/ (accessed on 30 July 2022)).The Expasy Protparam webserver (http://bioinf.cs.ucl.ac.uk/psipred/ (accessed on 30 July 2022)) while RaptorX Property (http://raptorx.uchicago.edu/StructurePropertyPred/predict/ (accessed on 30 July 2022)) was used for secondary structure prediction using algorithms along with identification of solvent properties [Secondary structure was predicted by using PRISPRED (operties .https://zhanggroup.org/I-TASSER/ (accessed on 30 July 2022)). Structural refinement was conducted using the GalaxyREFINE database ) [https://zlab.umassmed.edu/bu/rama/ (accessed on 30 July 2022)) was used for the verification of 3-D structure. Another method for validating the structure was based on z score using ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php (accessed on 30 July 2022)).A 3-D modeled structure was obtained from I-TASSER online server (y 2022)) . Ramachahttp://tools.iedb.org/ellipro/ (accessed on 30 July 2022)) was applied with all parameters set as default and a 3D model of the vaccine construct was used as input. This tool uses an algorithm for estimating protein shape, protrusion index (PI) of the residues, and clustering. Structural-based computational analysis can determine the linear and discontinuous B cell epitopes. About 90% of the structure consists of discontinuous epitopes synthesized by the protein sequence in the pathogen. Ellipro web tool (https://www.rcsb.org/). Docking was performed by using HADDOCK server (https://wenmr.science.uu.nl/prodigy/ (accessed on 30 July 2022)) [Pdb for TLR2 (id: Q59HI8) was retrieved from the Protein Databank (y 2022)) .http://imods.chaconlab.org/ (accessed on 30 July 2022)) server was used for the molecular simulation of multi-epitope vaccine candidate [iMODs (andidate . https://150.146.2.1/C-IMMSIM/index.php (accessed on 30 July 2022)) was used to obtain the immune profile of the vaccine candidate.C-IMMSIM (C language version of the IMMune system SIMulator) (http://www.jcat.de/ (accessed on 30 July 2022)) web server was used for codon optimization, and SnapGene trail user (https://www.snapgene.com/try-snapgene/) was used to clone the gene sequence into E. coli plasmid pET-30a (+) vectorThe Java Codon Adaption Tool (JCat) , cleavage (0.9251), and TAP (0.5170). Epitopes with good binding affinity can produce a high immune response. The predicted combined score was recorded as 0.5347 .HTL epitopes are presented by MHC class II followed by the recognition of CD4 cells. These cells augment and intensify the immune response. The IEDB MHC II web server with default threshold values was used to predict HTL epitopes. Altogether, two HTL epitopes were predicted and shortlisted based on their antigenicity, non-allergenicity, and non-toxic nature, as shown in The construct of the 138 total amino acids was designed by linking 2 B cell epitopes, 2 HTL, 1 CTL epitope, and an adjuvant . A sequeE. coli, indicates that the protein is highly soluble. All physicochemical properties of the MEV construct meet the criteria of a standard vaccine candidate. Structural residues of the candidate protein are present in coil (51.4%), helix (36.2%), and strand (12.3%) forms. The solubility properties of the polypeptide backbone indicate the nature of the residues as small non-polar (42%), hydrophobic (25.3%), polar (18.1%), and aromatic plus cysteine (13.04%) .By using I-TASSER, a three-dimensional tertiary structure was chosen, having a confidence score (C-score) of −3.62. For the aim of improving the quality of the model and its consistency, we used the GalaxyREFINE webserver. Among the five models generated, model 1 was selected depending on the factors related to the structure, including GDT-HA (0.8098), RMSD (0.734), and MolProbity (3.852). Other factors predicted included Clash score (107.3), rotamers (3.7), and Rama favored (58.8). The tertiary structure was validated by using a Ramachandran plot. It revealed that 74.528% (54.7% before refinement) of amino acids resided in highly preferred regions, while 16.038% (29.245% before refinement) of amino acids resided in preferred regions. We used the ProSA-web tool to validate the 3D model, and a Z-score of −5.12 was calculated .The antibodies produced by B-lymphocytes mediate the humoral immune response. Therefore, B cell epitopes are supposed to reside within the domain of the MEV construct. The vaccine construct harboring B cell epitopes is shown in The affinity of the vaccine construct to the host Toll-like receptors indicates the intensity of immune stimulation. A vaccine construct having good binding with TLR-2 can stimulate a proper host immune response. The designed vaccine construct docked with TLR-2 receptors and showed strong affinity, indicating a robust adoptive and innate immunity response from host. Protein–protein docking was used to predict the interaction of MEV with the TLR2 immune receptor. The 3D structure of TLR2 receptors was retrieved from PDB, and molecular docking was performed. The server generated various docked structures, and the model was picked based on the lowermost intermolecular energy with high binding affinity throughout the docked structures. Moreover, the docked complex structure was analyzed and visualized using PyMOL .−8 is illustrated in Molecular dynamics was performed to determine the complex stability of the vaccine construct during simulation. The motion and stiffness of the predicted residues show that the vaccine construct is stable and suitable. Using the iMOD server, we illustrated the dynamics of atomic movement within the structure of the designed construct. The protein sequence was reverse-translated into a nucleotide sequence using the online web server JCat. The DNA sequence consisting of 5836 bp was improved by using a vector builder to obtain a codon optimization index of 0.93 and 59.9% GC content. Restriction sites for NotI and HindIII were inserted and cloned into PET-30 (a) + using a free trial of SnapGene as illustrated in The immune simulation shows the host’s primary and secondary immune responses after injecting the vaccine. The immune profile confirms the production of antibodies and cytokines. The production of B and T  cells after injecting the antigen shows a highly responsive immune system. Cytokine production shows stimulated T-cell-mediated immunity in response to the vaccine. M. hyorhinis is known to cause several infections in swine and its pathogenesis has been poorly studied to date. Recent research work aided in a better understanding of the pathogenesis of this ubiquitous pathogen [M. hyorhinis have been developed and shown to be helpful in the reduction in lesions, resulting in the decline in infections [® MycoMAX is being commercially used against swine diseases in some countries. Although inactivated vaccines produce a robust immune response, and there is a probability of poor efficacy towards non-vaccine strains, strict storage conditions and exposure to immunocompromised pigs during pregnancy or lactation make the vaccine compromised. A multi-epitope vaccine with animal models has shown advantages over the live or attenuated vaccine because of its efficacy, lower cost, and protection [pathogen ,27,28. Tpathogen . Vaccinefections ,12,30. Aotection ,32. Thesotection . Designiotection . Linear otection ,36,37,38otection ,40,41,42M. hyorhinis has evidence from previous research where unique repeated units in region III of VlpA-G were used to develop ELISA with remarkable specificity and sensitivity [Variable lipoproteins are exposed on the pathogen’s surface, enabling them to directly interact with the host immune response and hence are highly antigenic. Despite the high degree of variability, these Vlps possess conserved domains that can be considered to serve as vaccine candidates. The antigenicity of VlpA-G of sitivity .M. hyorhinis to find CTL, HTL, and B cell epitopes. Epitopes were combined by using EAAK, GPGPGP, and AAY linkers that make the molecule more stable and prevent disruption [We assembled the conserved domains found at the N terminal of variable surface lipoproteins of sruption . Adding sruption . It has sruption ,45. The sruption . Physicosruption ,43. The Mycoplasma sp. Previous studies demonstrated the role of TLR2 in identifying M. hyopneumoniae in porcine alveolar macrophages, hence being the key factor to stimulate an innate immune response [Mycoplasma sp. trigger an immune response via TLR2 receptors [Lipoproteins on the membrane surface are critical antigens of response . It has response . Moreoveeceptors ,50,51. Teceptors . The desM. hyorhinis is a pathogen identified in swine and is known to cause severe illness. Inactivated vaccines currently on the market have not been successful in controlling infections, because of certain limitations. By combining different epitopes, immunoinformatics has been hailed as a potentially fruitful method for developing vaccines. The conserved domains that can be found at the N terminal of VlpA-G in M. hyorhinis were utilized in our research. The MHC-I, MHC-II, and B cell epitopes, in addition to linkers and an adjuvant, were utilized to propose a probable multi-epitope candidate vaccine. The designed proposed vaccine construct was antigenic, did not contain any allergens, and demonstrated excellent stability and binding ability to TLR2 receptors. In addition, the candidate vaccine elicited a robust immune response from both the humoral and cell-mediated types. To validate its safety and efficacy, additional experiments, both in vitro and in vivo, need to be conducted.


Heterorhabditis bacteriophora find and infect their host insects in heterogeneous soil ecosystems by sensing a universal host cue (CO2) or insect/plant-derived odorants, which bind to various sensory receptors, including G protein-coupled receptors (GPCRs). Nematode chemosensory GPCRs (NemChRs) bind to a diverse set of ligands, including odor molecules. However, there is a lack of information on the NemChRs in EPNs. Here we identified 21 GPCRs in the H. bacteriophora genome sequence in a triphasic manner, combining various transmembrane detectors and GPCR predictors based on different algorithms, and considering inherent properties of GPCRs. The pipeline was validated by reciprocal BLAST, InterProscan, GPCR-CA, and NCBI CDD search. Functional classification of predicted GPCRs using Pfam revealed the presence of four NemChRs. Additionally, GPCRs were classified into various families based on the reciprocal BLAST approach into a frizzled type, a secretin type, and 19 rhodopsin types of GPCRs. Gi/o is the most abundant kind of G-protein, having a coupling specificity to all the fetched GPCRs. As the 21 GPCRs identified are expected to play a crucial role in the host-seeking behavior, these might be targeted to develop novel insect-pest management strategies by tweaking EPN IJ behavior, or to design novel anthelminthic drugs. Our new and stringent GPCR detection pipeline may also be used to identify GPCRs from the genome sequence of other organisms.The infective juveniles (IJs) of entomopathogenic nematode (EPN)  Caenorhabditis elegans is a well-known model organism and has tremendously contributed to the advancement in understanding of biology , to orient themselves in response to chemosensory cues such as host exudates, food signals, sex pheromones, and other small molecules . GPCRs, are also known as seven-transmembrane (7-TM) domain receptors, are the largest family and most diverse group of membrane receptors in eukaryotes including EPNs . Most GPC. elegans was the first animal genome to be sequenced, and identification of ca. 1000 GPCRs in the C. elegans genome , a membrane protein topology prediction method based on hidden Markov model (HMM) (https://phobius.sbc.su.se/), an HMM-based signal peptide predictor that detects transmembrane protein topology also (http://www.enzim.hu/hmmtop/), an automatic server for predicting transmembrane helices and topology of proteins based on HMM (https://topcons.cbr.su.se/), a membrane protein topology prediction tool based on a complex algorithm  ; (2) Phoogy also ; (3) HMMd on HMM ; and (4)lgorithm . TMHMM2 https://gpcrhmm.sbc.su.se/index.html), an HMM-based GPCR prediction tool (http://aias.biol.uoa.gr/GPCRpipe/search.php), which detects GPCRs using pHMM library and HMM for GPCRs (https://gpcr.utep.edu/), which determines GPCR based on four different algorithms (https://webs.iiitd.edu.in/raghava/gpcrpred/), a support vector machine (SVM) based approach . Venn diagrams were created to compare and to reflect the number of seven transmembrane sequences and predicted GPCRs made by different combinations of tools.The outcomes of all the bioinformatic tools were then compiled and visualized using the Venny 2.1 software , B (secretin and adhesion), C (metabotropic glutamate), D , E (cAMP receptors), or F (frizzled), as per International Union of Basic and Clinical Pharmacology (IUPHAR) nomenclature (http://218.65.61.89:8080/bioinfo/GPCR-CA), whose algorithm is based on cellular automation (CA)  . GPCR-CAion (CA) .In order to validate the pipeline, the putative GPCRs were then checked for the presence of an extracellular N terminus and an intracellular C terminus using HMMTOP2 and TMHMM2.http://pfam.xfam.org/) (https://www.ebi.ac.uk/interpro/) was also used to analyze GPCR sequences functionally and to classify them into families, and ultimately to predict the presence of signature domains and important sites. We revalidated all these analyses through conserved domain analysis by NCBI CDD search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).We screened all these GPCRs through Pfam (am.org/) , a databam.org/)  protein H. bacteriophora GPCRs were classified into GPCR families according to the families to which their orthologous proteins in C. elegans and Ancylostoma caninum were assigned. For this purpose, orthology was detected between these resultant sequences and other organisms with the widely used reciprocal BLAST approach in WormBase Parasite (https://parasite.wormbase.org/index.html). This is done by using each GPCR sequence detected from H. bacteriophora as a query to the Caenorhabditis elegans proteome and selecting the highest scoring C. elegans protein sequence. This C. elegans sequence was then used as the query in a BLAST search against the proteome of H. bacteriophora. If the C. elegans sequence reciprocally identified the original query sequence as the highest scoring hit with the BLAST, these two sequences were then considered as an orthologous pair, which implies that they may possess similar functions and biological roles and fall into the same GPCR subfamily. H. bacteriophora GPCRs were assigned to families and subfamilies based on their orthologous GPCRs in other model organisms, such as C. elegans and A. caninum. In all BLAST comparisons, we used the BLOSUM62 scoring matrix and a cut-off threshold of 1e-1. Additionally, motif prediction of identified GPCRs was performed in MEME Suite 5.4.1 (https://meme-suite.org/meme/). Motifs were functionally annotated in HHpred (http://toolkit.tuebingen.mpg.de/hhpred) using default parameters. Functional motifs were identified in the GPCR sequences using the MOTIF search tool (https://www.genome.jp/tools/motif/).Putative http://athina.biol.uoa.gr/bioinformatics/PRED-COUPLE2/) (PRED-COUPLE2 (OUPLE2/)  was usedH. bacteriophora, 21 sequences were identified as GPCRs using the pipeline presented in Out of the total 21,699 predicted proteins of The common 69 heptathetical transmembrane sequences were passed through four GPCR prediction tools to identify the GPCRs among these sequences. GPCRHMM, GPCRPipe, GPCR-Pen, and GPCRPred identified 22, 21, 15, and 62 sequences as GPCRs, respectively . InteresH. bacteriophora, published by McLean and coworkers in 2018. Out of these sequences, 13 were found to be similar, with the GPCRs fetched from the earlier version of annotation, published in 2013  images to reveal the features hidden in complex protein sequences. It designated all these GPCRs as Class A Rhodopsin-like GPCR, except Hba_17528, which was identified as Class D Fungal pheromone GPCR . FurtherAdditionally, NCBI conserved domain analysis revealed that four identified proteins were members of the class A seven-transmembrane GPCRs and belonged to FMRFamide (Phe-Met-Arg-Phe)-like receptors and related proteins. Eight of the proteins were rhodopsin receptor-like class A family of the seven-transmembrane GPCR superfamily, which constitutes about 90% of all GPCRs. They include light-sensitive rhodopsin and receptors for biogenic amines, lipids, nucleotides, odorants, peptide hormones, and various other ligands. Six of the proteins were broadly classified under the 7tm_GPCRs superfamily. Among these, Hba_18743 was a serpentine type 7TM GPCR chemoreceptor under the Srsx family, the only family among the various superfamilies of chemoreceptors. Another serpentine type of chemoreceptor GPCR (Hba_17528) was found under the srx family, which is a part of the Srg superfamily of chemoreceptors. Interestingly, Hba_20566 was a pigment-dispersing factor receptor (PDFR), a member of the B1 subfamily of class B seven-transmembrane GPCRs, also referred to as the secretin-like receptor family. Hba_18203 was found to be an FMRFamide receptor and a member of the class A family of seven-transmembrane G protein-coupled receptors. Hba_09978 was a cholecystokinin receptor and came under the class A family of seven-transmembrane GPCRs. This group represents four GPCRs that are members of the RFamide receptor family, including cholecystokinin receptors (CCK-AR and CCK-BR), orexin receptors (OXR), neuropeptide FF receptors (NPFFR), and pyroglutamylated RFamide peptide receptors (QRFPR). Hba_08130 was an amine receptor of the class A family of GPCRs, which include adrenoceptors, 5-HT (serotonin) receptors, muscarinic cholinergic receptors, dopamine receptors, histamine receptors, and trace amine receptors .Lastly, in the 21 GPCR sequences, 18 different motifs were identified, which include 7-transmembrane receptor (rhodopsin family), serpentine type 7TM GPCR chemoreceptor Srw, serpentine type 7TM GPCR chemoreceptor Srsx, frizzled/smoothened family membrane region, 7-transmembrane receptor (secretin family), serpentine type 7TM GPCR chemoreceptor Srx, serpentine type 7TM GPCR chemoreceptor Srt, frizzled/smoothened family membrane region etc , Fig. 4.12/13) of G-proteins . In this database, 53 proteins from H. bacteriophora were annotated as NemChRs compared to four NemChRs  identified in our study. These four proteins are also listed in NemChR-DB; a total of only seven GPCRs are identified by both our study and Langeland et al., 2021. The reason for the mismatch in numbers of GPCRs identified may be because the present study used highly stringent parameters for identifying a protein as GPCR. Although Langeland et al., 2021 state that 7-TM helices are considered as ideal characteristics for any GPCR , they have also included proteins with 5, 6, 8, and 9 TM domains as GPCR in NemChRs. Their approach is inclusive, whereas we followed highly reductionist approach. In our study, in addition to the 7-TM sequences, four additional, and different, tools were used to search for GPCRs, resulting in a smaller set of sequences being identified as GPCRs as compared to the set identified by Langeland et al. Further, to predict NemChRs, Langeland et al., 2021 and Ancylostoma ceylanicum across various life stages, and GPCRs were identified based on annotations of the sequences resulting from the transcriptomes. It may be suggested that the GPCRs identified using our methodology are accurate and precise due to higher stringency in the filtration criteria as compared to these studies. Our pipeline identified 1,252 C. elegans proteins as GPCRs, a number very close to 1,100 and 1,341 GPCRs reported previously (Langeland et al., 2021 created an integrative database of nematode chemoreceptors called NemChR-DB to facilitate the analysis of NemChRs (eviously , thus vain-silico programs available at public interface to identify transmembrane domains. A comparative study proved that TMHMM2 and HMMTOP2 were more accurate than other programs and Phobius was reported to perform comparably (Further, these filtered sequences were passed through different transmembrane detectors; α-helical transmembrane proteins are the most important class of membrane proteins and constitute almost 20–30% of all the proteins encoded in a genome . From thmparably . TMHMM2 mparably . Along wmparably . Phobiusmparably . Errors Among these 7-TM sequences, 22 sequences were identified as GPCRs by GPCRHMM, a Hidden Markov Model–based GPCR recognition software which identifies TM topology-related features. It captures the variation in amino acid composition and topological segment lengths between GPCR families. It has a bare minimum error rate of identification of GPCR, in comparison to other HMM–based GPCR predictors, including Pfam. It has shown a higher percentage of selectivity and sensitivity over profile HMMs and generic transmembrane detectors on sets of known GPCRs and non-GPCRs . As GPCRC. elegans proteomic dataset. Earlier, C. elegans genome (C. elegans. Based on manual curation and sequence comparisons, C. elegans. The number of GPCRs predicted by all these studies is similar to 1,252 GPCRs identified in our study in the genome of C. elegans, thus confirming the accuracy and preciseness of the pipeline used in the present study.The same methodology of mining GPCRs was applied to the s genome . BargmanInvolvement of GPCRs in a wide array of physiological and pathological processes , and theH. bacteriophora. NemChRs are generally expressed in amphid sensory neurons (M. incognita (The NemChRs identified in this study must be functionally validated for their roles in chemoreception in EPN  neurons  and are  neurons . GPCRs, ncognita . Like Nencognita . Lastly,


Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) repeatedly experience near anoxia and re-oxygenation in their habitats and have adapted to survive AR at tropical temperatures without significant brain injuries. However, these anoxia-tolerant species display contrasting strategies to survive AR, with only H. ocellatum having the capacity to supress metabolism and H. ocellatum mitochondria the capacity to depress succinate oxidation post-AR. We measured oxygen consumption alongside ROS production mediated by elevated succinate in mitochondria of permeabilized cerebellum from both shark species. Although mitochondrial respiration remained similar for both species, the ROS production in H. ocellatum was half that of C. punctatum in phosphorylating and non-phosphorylating mitochondria. Maximum ROS production in H. ocellatum was mediated by succinate loads 10-fold higher than in C. punctatum mitochondria. The contrasting survival strategies of anoxia-tolerant sharks reveal the significance of mitigating ROS production under elevated succinate load during AR, shedding light on potential mechanisms to mitigate brain injury.Anoxia/re-oxygenation (AR) results in elevated unchecked oxidative stress and mediates irreversible damage within the brain for most vertebrates. Succinate accumulation within mitochondria of the ischaemic brain appears to increase the production of reactive oxygen species (ROS) upon re-oxygenation. Two closely related elasmobranchs, the epaulette shark ( WhileHemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are two closely related elasmobranches of the carpet shark family that can tolerate prolonged anoxia at tropical temperatures [C. punctatum rapidly increased its haematocrit, likely representing splenic contraction to supply metabolically active organs with O2 from stored red blood cells [H. ocellatum is capable of metabolic depression and neuronal hypometabolism [H. ocellatum responds to hypoxia pre-conditioning, after which it more readily enters ventilatory and metabolic depression [ex vivo mitochondrial plasticity occurs in the cerebellum from H. ocellatum evidenced by the suppression of succinate dehydrogenase  activity and readjustment of electron transfer pathways in response to exposure to anoxia/re-oxygenation (AR) [C. punctatum.To evade excess oxidative damage, a few hypoxia and anoxia-tolerant species supress ROS production ,14, and eratures . In respod cells . In conttabolism ,19 and dtabolism . Additiopression . Recentlion (AR) . While tH. ocellatum and C. punctatum to AR, we sought to assess the mitochondrial response to elevated levels of succinate and the resultant level of ROS production by cerebella mitochondria of both shark species. Using respirometry coupled with fluorimetry, we tested the hypothesis that mitochondria from H. ocellatum would better manage succinate-derived ROS relative to C. punctatum mitochondria, and that the resulting electron leakage resulting from succinate oxidation would be avoided in H. ocellatum cerebellum. This study aims to address the contrasting strategies displayed by two anoxia-tolerant sharks at the mitochondrial level and is the first to provide insights into mitochondria ROS production in C. punctatum.Based on the contrasting physiological responses of . 2Chiloscyllium punctatum) and 10 adult epaulette sharks (Hemiscyllium ocellatum) were supplied by Sea World  or by Cairns Marine , respectively. Sharks were held under standard condition of temperature, photoperiod and feeding as described for similar individuals in [Ten adult grey carpet sharks . The O2 decline over time due to mitochondria respiration (JO2) was measured in real time and was recorded using DatLab 7 analysis software. The ROS production was measured simultaneously with fluorimeters, as previously described [A substrate-uncoupler-inhibitor titration (SUIT) protocol  was emplescribed , and cora–c). For succinate titration (d–f), fitted curves for respiration and ROS were extracted using the least-squares method based on a dose-stimulation model. Two-way ANOVA with repeated measures tests were then performed to test for main effects by species or succinate concentration. Post-hoc tests with multiple comparison (Tukey correction) were used to assess significant differences chosen as p < 0.05: between species; mitochondria states; and between succinate concentrations. Raw data and statistical analysis are publicly available at the University of Auckland Figshare repository: (doi:10.17608/k6.auckland.22144289) [Two-way ANOVA tests were performed to test for species or substrate/inhibitor effects on respiration and on ROS production rates a–c. For itration d–f, fitt. 32 flux was apparent between C. punctatum and H. ocellatum permeabilized cerebellum . When supported by succinate, OxPhos rates supported around 70% of OxPhos supported by additional NADH-linked substrates in both species (p < 0.0001). Respiratory coupling ratio supported by succinate (RCR) were twice greater than when supported with additional NADH-linked substrates , with no difference between species (p = 0.48). The exogeneous titration of succinate mediated a similar increase in respiration in cerebellar mitochondria of both shark species .No difference in mitochondria OC. punctatum than those from H. ocellatum  with an interaction between species and mitochondria state . In H. ocellatum, ROS production was similar across all mitochondrial states, (p > 0.38), with less ROS production in LeakS relative to LeakPMGS, suggesting that complex I most likely mediated the greatest portion of ROS when mitochondria were not phosphorylating. In contrast, the highest ROS production was observed when C. punctatum mitochondria were in LeakS fuelled by succinate (p < 0.06).ROS production was two- to threefold higher in cerebellar mitochondria from F1,162 = 7.21, p < 0.0001; figure 2e). The ROS production was however threefold higher in C. punctatum mitochondria relative to H. ocellatum mitochondria . In C. punctatum mitochondria, 1 mM succinate was sufficient to mediate the highest electron leakage (p < 0.0001), while in H. ocellatum mitochondria the maximum levels only achieved with a 2.5 higher level of succinate (i.e. 2.5 mM) . While the absolute ROS production may reflect an overall potential for oxidative damage or stress, the normalization of ROS per O2 consumed also represents electron leakage and therefore efficiency of electron transport (figure 2f). Chiloscyllium punctatum mitochondria had a threefold higher electron leakage mediated by succinate oxidation relative to those of H. ocellatum . However, the portion of electron leakage remained constant in both shark species regardless of the succinate concentration .In non-phosphorylating mitochondria, increments of exogenous succinate increased the ROS production from 0.25 mM in both species (main effect . 4 (a)H. ocellatum cerebella, the ETS flux was lower in permeabilized tissue relative to homogenates. Both homogenization and permeabilization preparations facilitate the exchange of soluble molecules between the medium and the cytosolic phase, including succinate to which most plasma membranes are impermeable [Mitochondria from permeabilized cerebella had similar respiration rates in both shark species. OxPhos rates in permabilized cerebella were however three-times greater than those previously measured in homogenized cerebella of these species . In addiermeable . Howeverermeable . Despiteermeable ,25.2JO in the OxPhos state when in the presence of saturating NADH-linked substrates in H. ocellatum and C. punctatum mitochondria, respectively. Here, equally concentrated succinate could sustain 70% of OxPhos when no other additional substrates were present (a). The data also reveal that near-full CII capacity is used when the Q-pool does not compete with NADH-linked inputs, when compared to maximum CII flux [2 utilization when OxPhos is fuelled by succinate, relative to when NADH-linked substrates are also present. Notably, CII mediated respiration transfers of 40% less protons per O2 than NADH-linked-fuelled respiration. Therefore, to achieve similar ATP synthesis rates, more O2 is required when more CII is used to drive OxPhos. In cerebellar mitochondria from C. punctatum, succinate may therefore serve as a fuel reserve that can be used for OxPhos when NADH-linked substrates may become limiting. In contrast cerebellar mitochondria from H. ocellatum turn down OxPhos in the presence of excess succinate.The additive effect of convergent electron transfer from NADH-linked and CII should result in a competition for the Q-pool. In SUIT protocols, the sequential addition of NADH-linked and CII substrates is thought to monopolize a portion of the Q-pool, which then becomes less available to further substrates . In this present a. The daCII flux . In addi (b)C. punctatum mitochondria, the ROS production was the highest in non-phosphorylating mitochondria with succinate (LeakS). In contrast, the ROS production from H. ocellatum mitochondria appeared higher when in LeakS with succinate and NADH-linked inputs (b). Although high leak flux has been shown to decrease the ROS production [H. ocellatum mitochondria. With similar O2 flux, this suggests that electrons appear to be more efficiently channelled in the ETS to O2 in H. ocellatum cerebellar mitochondria. We also note that additional NADH-linked substrate mediate higher proton Leak, as suggested by greater RCR when OxPhos is fuelled by succinate only (c).In mitochondria, ROS production is in part mediated by substrate levels, which alter redox state and mitochondrial phosphorylation state . All of d inputs b. Althouoduction , high ROoduction ,30. Overate only c.C. punctatum mitochondria, the maximal production was reached at 0.1 mM of succinate (d). Succinate concentrations encountered in normoxic brain could be expected to be lower than 0.5 mM [2O2 s−1 mg−1, in phosphorylating mitochondria . In addition, approximately 2.2% of the electrons supplied from succinate oxidation were directed to ROS production, which likely represents a small yet measurable loss of coupling of the energy from electron transfer to proton pumping, previously reported [2JO in C. punctatum mitochondria was altered after AR, as we reported previously [C. punctatum, as observed in [In the brain, succinate has been measured in the range of 0–0.5 mM in normoxia and this increases to several mM during anoxia in vertebrates ,32, incluccinate d. Succinn 0.5 mM , the ROSreported . In addieviously . In accoeviously , a revererved in .H. ocellatum cerebella reached a plateau at much higher (1 mM) succinate concentration, and electron leakage remained constant and low at 0.8% of the total electron flow fuelled by succinate oxidation, thus regardless of succinate concentrations, the mitochondria from H. ocellatum showed less evidence of reverse electron flow or increased ROS production. This indicates that in the eventuality of oxygen-limited succinate accumulation, oxidative damage during re-oxygenation would be averted regardless of succinate levels exceeding 1 mM. This is in accord with the decrease in succinate oxidation rate (CII mediated respiration) observed after AR ex vivo [Conversely, the ROS production from  ex vivo .H. ocellatum benefit from two mechanisms that avert oxidative damage mediated by elevated succinate (i) partial inhibition of CII respiration post AR [Drosophila, which displayed an enhanced Leak, a lower CII activity and a lower mitochondria ROS production [In addition to enhanced active proton Leak rates post AR , mitocho post AR , (ii) looduction ; (ii) choduction ; (iii) soduction ; and (ivoduction  and the oduction .. 5H. ocellatum and C. punctatum during anoxia and re-oxygenation, and how graded excess succinate affects ROS production ex vivo. Mitochondrial responses may not strictly corelate with mitochondrial response in situ, where regulations at higher biological levels could occur. Nevertheless, our findings indicate that C. punctatum had higher levels of ROS production, likely due to higher electron leakage caused by high succinate oxidation rates post AR in vitro. By contrast, H. ocellatum had lower ROS production due to decreased succinate oxidation rates post AR, which enabled conservation of respiration and oxidative phosphorylation capacities. While mitochondrial plasticity in the cerebella of both sharks was assessed in ex vivo settings, the observed mitochondrial response aligns with the contrasting ecophysiological responses of the two species to anoxia and re-oxygenation that are presented in shallow coastal environments. These two distinct strategies used to achieve anoxia-tolerance at the subcellular level among elasmobranchs may assist with the design of potential interventions to mitigate brain damage in other species and even humans via controlling CII activity and unchecked ROS production.This study is the first to investigate the production of ROS in the cerebellar mitochondria of


Depression is the most common comorbidity of rheumatoid arthritis (RA). In particular, major depressive disorder (MDD) and rheumatoid arthritis share highly overlapping mental and physical manifestations, such as depressed mood, sleep disturbance, fatigue, pain, and worthlessness. This overlap and indistinguishability often lead to the misattribution of physical and mental symptoms of RA patients to depression, and even, the depressive symptoms of MDD patients are ignored when receiving RA treatment. This has serious consequences, since the development of objective diagnostic tools to distinguish psychiatric symptoms from similar symptoms caused by physical diseases is urgent.Bioinformatics analysis and machine learning.The common genetic characteristics of rheumatoid arthritis and major depressive disorder are EAF1, SDCBP and RNF19B.We discovered a connection between RA and MDD through immune infiltration studies: monocyte infiltration. Futhermore, we explored the correlation between the expression of the 3 marker genes and immune cell infiltration using the TIMER 2.0 database. This may help to explain the potential molecular mechanism by which RA and MDD increase the morbidity of each other. A meta-A number of evidence shows that a variety of physiological functions of patients with depression are affected, including inflammation , 8, neurIn this study, we found several key cellular signaling pathways and gene networks related to RA and MDD. In addition, we identified three key genes through machine learning, which may can explain the common pathogenesis of RA and MDD. We also studied the immune infiltration of RA and MDD respectively and found that monocyte infiltration may be an important mechanism for connecting RA and MDD. In addition, we used the TIMER 2.0 database and Cibersort algorithm to explore the potential correlation between the expression of this three genes and immune cell infiltration.22.1https://www.ncbi.nlm.nih.gov/geo/), one for MDD and two for RA patients. Details about the dataset are presented in In 2.2To explore the correlation among genes, the WGCNA strategy was adopted . First, 2.3Gene Ontology (GO) provides structured, computable information concerning gene functions . A widel2.4www.string-db.org) was used to establish a network of protein–protein interactions (PPIs) between protein-coding genes  and 95% confidence interval (CI) to quantify its value . The ide2.7https://cistrome.shinyapps.io/timer/) includes 10,897 samples across 32 cancer types from The Cancer Genome Atlas (TCGA) to estimate the abundance of immune infiltrates  calculated. Wilcoxon test was applied to compare proportions of different immune cells between control and patient groups in RA and MDD separately via Base package. Linear fitting and scatterplot was done by ggstatsplot package . It was 33.1Using PCA to analyze the gene expression patterns of different groups of GSE97475 and GSE38206, there are certain differences between the disease group and the control group 3.2A total of 2177 DEGs were identified in GSE169082 using the DESeq2 method, of which 1458 are upregulated and 719 are downregulated. The PCA and volcano plot of RA DEGs are shown in 3.3The intersection of positive module genes and the intersection of negative module genes of GSE97476 and GSE38206 are 179 and 15 genes respectively 5C, D. Thttps://string-db.org/) to find node proteins that can interact with each other. The node proteins are visualized by Cytoscape software , 0.6122(95%CI 0.523-0.702) and 0.942(95%CI 0.909-0.975), respectively. In MDD, the AUC of EAF1, SDCBP, RNF19B is 0.911(95%CI 0.819-1.000), 0.895(95%CI 0.789-1.000) and 0.907(95%CI 0.804-1.000), respectively. The results showed that the higher expression of the three genes, the higher risk of RA or MDD.The nomogram was constructed based on the three candidate hub genes, and a ROC curve was established to assess the diagnostic specificity and sensitivity of the gene set of these three genes and the nomogram  (I to V editions) and International Classification of Diseases (ICD) (from 6th to 11th editions) provide a set of criteria in order to diagnose depression. In spite of this, they are based on the patient’s own description and the clinician’s observation of the patient’s behavior . The DSMThere are many explanations for the high frequency of depression in RA patients, including financial stress caused by long-term treatment of chronic diseases, low self-esteem caused by disability, loss of social role caused by unemployment, persistent pain, fatigue, and low energy can also affect the mood of patients. In addition to these psychological causes, recent studies have proposed an inflammation hypothesis to explain possible biological mechanisms , 30–32. To construct the nomogram and to evaluate the diagnostic value for RA with MDD, we used a bioinformatics analysis and two machine learning methods. The main findings were the identification of three pivotal immune-associated candidate genes  and the development of a nomogram for diagnosing RA with MDD. In the study, peripheral blood samples were used for the MDD and RA datasets; for this reason, we only need to collect peripheral blood from patients with RA and evaluate the expression of the three immune-associated genes to infer the likelihood of MDD in RA patients.ELL-associated Factor 1 (EAF1) was first identified based on its ability to interact with transcriptional elongation factor for RNA polymerase II . In loweMda-9/syntenin, encoded by the SDCBP gene, is involved in a variety of functions, including transmembrane protein transport, neural and immune regulation, exocrine biogenesis and human tumorigenesis , 47. In +T cells and monocytes , is expressed by a variety of hematopoietic cells, including NK cells, CD8onocytes , 54. In ytokines . We specThe inflammation hypothesis of depression is also known as the monocyte/macrophage theory of depression because monocyte/macrophage lineage cells are important producers of inflammatory cytokines . Several++CD16−), intermediate monocyte (CD14++CD16+) and non-classical monocyte (CD14+CD16++)  . The lev+CD16++) –64. Howe+CD16++) , while s+CD16++) . HoweverIn conclusion, we used bioinformatics analysis and machine learning to screen three hub genes shared by RA and MDD, including EAF1, SDCBP and RNF19B, which may help to explain the potential molecular mechanism by which RA and MDD increase the morbidity of each other.https://CRAN.R-project.org/package=tidyverse and https://github.com/slowkow/ggrepel.R scripts for data collation is available for download at https://github.com/YuLab-SMU/clusterProfiler and https://bioconductor.org/packages/org.Hs.eg.db/. R scripts for GEO datasets download is available for download at https://bioconductor.org/packages/GEOquery/.R scripts for enrichment analysis is available for download at https://CRAN.R-project.org/package=factoextra and https://CRAN.R-project.org/package=FactoMineR.R scripts for PCA analysis is available for download at https://github.com/lmcinnes/umap.R scripts for dimension reduction with UMAP is available for download at https://bioconductor.org/packages/limma/ and https://bioconductor.org/packages/DESeq2/.R scripts for Differential gene analysis is available for download at https://bioconductor.org/packages/GWENA/.R scripts for WGCNA is available for download at https://bioconductor.org/packages/ROC/.R scripts for drawing of ROC Curves is available for download at https://bioconductor.org/packages/heatmaps/.R scripts for drawing of heatmaps is available for download at https://CRAN.R-project.org/package=glmnet.R scripts for establishment of LASSO regression model is available for download at https://CRAN.R-project.org/package=randomForestExplainer.R scripts for randomForest analysis is available for download at https://CRAN.R-project.org/package=ggVennDiagram.R scripts for drawing of Venn diagrams is available for download at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97476, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38206, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169082.Publicly available datasets were analyzed in this study. This data can be found here: WJ: Conceptualization, Methodology, Data curation, Software, Formal analysis, Visualization, Writing – original draft. XW: Validation, Investigation, Data curation, Formal analysis, Methodology, Writing – original draft. DT: Project administration, Writing – review and editing. XZ: Project administration, Funding acquisition, Resources, Supervision, Writing – review and editing. All authors contributed to the article and approved the submitted version.


However, the mechanisms and evolutionary implications of this horizontal gene transfer are unclear. Here, we analyzed synteny in phyllogen flanking genomic regions from 17 phytoplasma strains that were related to six ‘Candidatus’ species, including three strains newly sequenced in this study. Many of the phyllogens were flanked by multicopy genes within potential mobile units (PMUs), which are putative transposable elements found in phytoplasmas. The multicopy genes exhibited two distinct patterns of synteny that correlated with the linked phyllogens. The low level of sequence identities and partial truncations found among these phyllogen flanking genes indicate that the PMU sequences are deteriorating, whereas the highly conserved sequences and functions  of the phyllogens suggest that the latter are important for phytoplasma fitness. Furthermore, although their phyllogens were similar, PMUs in strains related to ‘Ca. P. asteris’ were often located in different regions of the genome. These findings strongly indicate that PMUs drive the horizontal transfer of phyllogens among phytoplasma species and strains. These insights improve our understanding of how symptom-determinant genes have been shared among phytoplasmas.Phytoplasmas are obligate intracellular plant pathogenic bacteria that can induce phyllody, which is a type of abnormal floral organ development. Phytoplasmas possess phyllogens, which are effector proteins that cause phyllody in plants. Phylogenetic comparisons of  Rhizobium, there is a plasmid carrying genes that can influence root nodulation transfer among different species and determines the host range for root nodule induction  plays a crucial role in the adaptive evolution of most organisms . In bactnduction . In the ne, cbsA . The modne, cbsA .Candidatus (Ca.) Phytoplasma’ spp.] are obligate intracellular plant pathogenic bacteria in the class Mollicutes. Phytoplasmas are transmitted by phloem-feeding insects of the order Hemiptera and can infect more than 1,000 plant species  can be phylogenetically divided into four groups  and the evolutionary history of these phyllogens differs from that of phytoplasmas, as confirmed by analyses of phytoplasma 16S rRNA gene sequences  of PMUs  and TW strain . Arabidopsis thaliana was maintained as described previously -based gene expression vector system  was usedn in JWB  and PHYL JWB , there were several PMU-associated genes  in their upstream regions. These PMUs could be categorized as type 1 or 2 based on some characteristic genes and synteny , all of which contained phyl-A group phyllogens. These type 1 PMUs also contained hypothetical protein 1 (hp1), hp2, and an hflB-like gene in their upstream regions . Type 2 PMUs were also found in the ‘Ca. P. pruni’ Vc33 strain, which has a phyl-C group phyllogen. The type 2 PMUs contained hp4, hp5, hp6, hp7, smc, and smc-like genes with similarity to smc and hp8 genes in the upstream region and hp9 genes in the downstream region. In the PaWB-Japan, PaWB-China, HP, and RhY strains, the type 2 PMUs exhibited conserved synteny of fliA, himA, hp4, hp5, hp6, hp7, hflB, smc, smc-like genes, hp8, phyllogen, and hp9. The JWB-nky and Hebei-2018 strains had another phyllogen, and its flanking region also contained genes that were characteristic of type 2 PMUs, although synteny was only partly conserved . Additionally, phylogenetic trees based on genes shared by type 1 and 2 PMUs  indicate that the evolutionary history of these genes differs from that of phytoplasmas. In particular, the hflB tree formed two clades, with type 1 and 2 PMU strains  between ‘Ca. P. asteris’ and ‘Ca. P. phoenicium.’ However, genes in the phyllogen flanking region  exhibited low sequence identities (>57%), except in the closely related OY and DY2014 strains  among ‘Ca. P. asteris’, ‘Ca. P. luffae,’ and ‘Ca. P. ziziphi,’ except for the Vc33 strain phyllogen that belongs to a different phyl-C group. However, the genes in the phyllogen flanking regions  exhibited low sequence identities (>32%), except for the closely related HP and RhY strains, and JWB-nky and Hebei-2018 strains . The final subclade comprised strains with complete (De Villa and M3 strains) or draft (LD1 strain) genomes that lacked phyllogens. The TBZ1 strain that contained a phyl-A group phyllogen did not belong to any of these subclades. These results indicate that the strains with phyl-D group phyllogens formed two subclades in ‘Ca. P. asteris’, while the strains with phyl-A group phyllogens formed one clade and one subclade. They also indicate that although strains related to ‘Ca. P. asteris’ retained the same group of phyllogens, at least at the subclade level, the overall relationships were complex.First, intraspecies evolutionary relationships among strains with tritici’ , was also clades . The minCa. P. asteris,’ based on their flanking genomic regions. First, we analyzed the genomic locations of the type 1 PMUs. The PMUs and surrounding genomic regions in the OY, DY 2014, RP166, and AY-WB strains are shown in Ca. P. asteris’  and PaWB-China (67 kbp) strains. Several PMU-associated genes were found in this genomic region in the RP166 and AY-WB strains; however, the syntenies of these genes differed from those of the type 2 PMUs  and the PEY strains (PHYLPEY), as well as the phyl-D group phyllogens of NCHU2019 (PHYLNCHU2019) and the HP strains (PHYLHP). We used the PHYLOY phyllogen of the OY strain (phyl-A group) and the PHYLJWB phyllogen of the JWB strain (phyl-D group) as controls known to induce phyllody  induced phyllody to the same extent as the other homologs  have been linked experimentally with phytoplasma virulence . Among tvia transposable elements (such as transposons) by comparing the genomes of different bacterial species. Thus, this study provides new insight into the mechanisms of HGT of virulence genes.Many studies have reported HGT of bacterial virulence genes based on genomic analyses of individual species , but fewphyllogens lacked tra5 (phyllogens have been acquired. However, the type 2 PMUs of the PaWB-Japan and PaWB-China strains retained their tra5 genes and inverted repeat-like sequences at both ends (Genetic elements may become immobilized due to inactivation or deletion of genes necessary for their transfer . Degenerked tra5 . In addiked tra5 , 2, inclked tra5 . Therefooth ends , 4, suggphyllogens from different species had highly conserved sequences, especially phyllogens from the phyl-A and -D groups (phyllogens were not located within PMUs (phyllogen transfer, PMU sequences may deteriorate completely. Therefore, phyllogens may become fixed in many different phytoplasma genomes after their acquisition, suggesting that they are crucial for their hosts. Previous studies have shown that phyllody symptoms facilitate phytoplasma accumulation/localization within host plants (phyllogens have been strongly conserved throughout evolution suggests that they are critical for phytoplasma survival.Despite the deterioration of PMU-associated genes, D groups . FurtherD groups . Severalhin PMUs  and theit plants , and phyt plants . Althougphyllogens have been identified in strains related to ‘Ca. P. asteris’ (phyl-A, -B, and -D; phyllogens was a complex process, at least for phyl-A and phyl-D. In ‘Ca. P. asteris,’ we identified one subclade comprising strains that retained phyl-A phyllogens and two subclades comprising strains that retained phyl-D phyllogens (phyllogens either moved frequently within the genome or were acquired separately. The PaWB strains had complete PMUs retaining phyl-D, suggesting that these were acquired more recently than the PMUs in the HP and RhY strains. Therefore, the latter hypothesis seems more plausible. We could not determine the acquisition order of the phyl-A and phyl-D group phyllogens in ‘Ca. P. asteris’ due to insufficient genomic data. Therefore, further studies of phyllogen flanking regions and their genomic positions are needed.To date, three different groups of yllogens . In the yllogens , suggestyllogens . Therefoyllogens . PMUs reyllogens . This obphyllogens of ‘Ca. P. asteris’ are most closely related to phyllogens of ‘Ca. P. ziziphi’ (Ca. P. asteris’ and ‘Ca. P. ziziphi’ share common ecological niches. First, the distribution of ‘Ca. P. ziziphi’ is limited to eastern and southern Asia (Ca. P. asteris’ that retain phyl-D (Ca. P. asteris’ and ‘Ca. P. ziziphi’ has been reported in jujube plants (Hishimonus sellatus, a vector of ‘Ca. P. ziziphi,’ can transmit two strains related to ‘Ca. P. asteris’ that retain phyl-D (RhY and MD-China; Ca. P. asteris’ may be acquired from ‘Ca. P. ziziphi’ in the same ecological niche. Further accumulation of information on the synteny of PMUs will reveal how PMUs and symptom-determinant genes located in them are shared among phytoplasmas in the same niche.HGT may occur between organisms that share an ecological niche, irrespective of their phylogenetic relationships . Phylogeziziphi’ . Furtherern Asia , as is tn phyl-D . Second,e plants . Third,


Burkholderia cepacia complex (BCC) is a Gram-negative bacterial, including Burkholderia contaminans species. Although the plain Burkholderia is pervasive from taxonomic and genetic perspectives, a common characteristic is that they may use the quorum-sensing (QS) system. In our previous study, we generated the complete genome sequence of Burkholderia contaminans SK875 isolated from the respiratory tract. To our knowledge, this is the first study to report functional genomic features of B. contaminans SK875 for understanding the pathogenic characteristics. In addition, comparative genomic analysis for five B. contaminans genomes was performed to provide comprehensive information on the disease potential of B. contaminans species. Analysis of average nucleotide identity (ANI) showed that the genome has high similarity (> 96%) with other B. contaminans strains. Five B. contaminans genomes yielded a pangenome of 8832 coding genes, a core genome of 5452 genes, the accessory genome of 2128 genes, and a unique genome of 1252 genes. The 186 genes were specific to B. contaminans SK875, including toxin higB-2, oxygen-dependent choline dehydrogenase, and hypothetical proteins. Genotypic analysis of the antimicrobial resistance of B. contaminans SK875 verified resistance to tetracycline, fluoroquinolone, and aminoglycoside. Compared with the virulence factor database, we identified 79 promising virulence genes such as adhesion system, invasions, antiphagocytic, and secretion systems. Moreover, 45 genes of 57 QS-related genes that were identified in B. contaminans SK875 indicated high sequence homology with other B. contaminans strains. Our results will help to gain insight into virulence, antibiotic resistance, and quorum sensing for B. contaminans species.The The online version contains supplementary material available at 10.1186/s13568-023-01537-8. We first report the genome characteristics of B. contaminans SK875.The B. contaminans SK875 genome carries virulence and quorum sensing-related genes.The SK875 has 186 unique genes, such as toxin higB, betA, and hypothetical proteinsThe online version contains supplementary material available at 10.1186/s13568-023-01537-8. Burkholderia cepacia complex (BCC) is a group of genetically distinct, phenotypically similar Gram-negative bacteria  centers, while Burkholderia multivorans are the most common species in European CF centers  as a part of their colonization and invasion strategies  . Production is catalyzed by an AHL synthase belonging to the LuxI-family of proteins  and PacBio RS II system . De novo assembly of B. contaminans SK875 was performed using the hierarchical genome assembly process v.2.3. Finally, three chromosomes and one plasmid were produced for the B. contaminans SK875 strain. Rapid Prokaryotic Genome Annotation was used for the genome annotation (Prokka) v.1.10   was conducted using the JSpeciesWS  and Kyoto Encyclopedia of Genes and Genomes (KEGG) categories.Pangenome analysis was conducted to determine genomes’ accessory, core, and unique genes. The BPGA tool was used as the pipeline for pangenome analysis of the five B. contaminans genomes. For this purpose, FASTA assembled files were used as inputs and identified with a comprehensive antibiotic resistance database (CARD) 3.1.1 using DIAMOND. Antimicrobial genes were detected using criteria of perfect and strict hits only, high sequence quality, and coverage, excluding nudging of ≥ 95% identity loose hits to severe. The possible virulence factors in the five B. contaminans genomes were identified by generating orthologous groups with VFanalyzer and then comparing them to reference genomes from the virulence factors database (VFDB)  software v.5.1.1  tool . Genes associated with the CepI/CepR and RpfF/RpfR systems were evaluated by the BlastKOALA search tool v.2.2 in the KEGG database. Sequences were compared using Easyfig version 2.2.5 .The pathway for the QS system in five B. contaminans SK875 strain has published genome sequence in the public database without being deposited in the publicly accessible culture collection. The entire genome of B. contaminans SK875 included three circular chromosomes of 1528467 to 3,618903 bp with GC contents of 65.8 to 66.4% and one plasmid of 200961 bp with a GC content of 61.7%. The entire genome harbored 7625 proteins, 18 rRNAs, and 83 tRNAs. The genomic characteristics of B. contaminans SK875 and the other four B. contaminans strains are presented in Table B. contaminans was 8.59 Mb ranging from 8.17 to 9.00 Mb, and the moderate GC content was 66.31% ranging from 66.06 to 66.53%. Among these strains, B. contaminans FL-1-2-30-S1-D0 strain indicated high GC content (66.53%), and the B. contaminans ZCC strain demonstrated lower GC content (66.06%).B. contaminans SK875 genome, a high proportion of genes in COG functional categories were allocated to the common function prediction only , amino acid transport and metabolism , and transcription  , the biosynthesis of amino acids (ko:01230), carbon metabolism (ko:01200), and two-component system (ko:02020)  and replication (L). In contrast, genes related to transcription (K) were deprived and thus had lower adaptability of them to varying environments  among the B. contaminans genomes. B. contaminans ZCC strain was first isolated from mining soil and is a promising cadmium-resistant strain that enhances the growth of soybeans in the presence of cadmium  , and the accessory genome is composed of 2128 genes (24.09%). The unique genome is composed of 1252 genes (14.18%)  and 186 (2.44%) out of a total of 7625 orthologous genes.Pangenome analysis can be used to investigate the core, auxiliary, and unique genes in pathogenic bacterial genomes  type III secretion system (T3SS) and type VI secretion system (T6SS-I) were commonly found in five B. contaminans genomes. The effector proteins that elude the immune system and cause disease are delivered into host cells with the aid of the T3SS system  was present in the B. contaminans SK875 genome. The effector protein is located on the chromosomal pathogenicity island in chromosome SK875-2, so it can be transmitted to other cells by horizontal gene transfer ), fluoroquinolone/tetracycline (afeF), and aminoglycoside (amrA). Overall, to other Burkholderia species, all strains of B. contaminans possess multiple antibiotic resistance and virulence-related genes . B. contaminans SK875 showed 57 quorum sensing-related genes, and 45 of them were also present in other B. contaminans genomes  and a hypothetical protein (QFR14107.1) were found only in the B. contaminans SK875 strain. In conclusion, five B. contaminans strains indicted a generally high degree of genetic similarity in QS systems and their related genes.Previously, we screened and characterized transposon (Tn5) insertion mutants with longer life spans in mes Fig. . These gBurkholderia species has increasingly been reported as opportunistic pathogens cause respiratory diseases by following ingestion and inhalation (Mahenthiralingam et al. Burkholderia colonizes rather than immediate respiratory tract infections and causes sepsis in patients with respiratory disease (Mahenthiralingam et al. Burkholderia species can produce a wide variety of potential virulence factors, although not all have yet been shown to have a role in the pathogenesis of human disease (Sousa et al. B. contaminans SK875 strain found in the respiratory tract of a pig with a respiratory disease to understand the pathogen characteristics of B. contaminans.B. contaminans SK875 strain and four other strains of B. contaminans worldwide, we found that SK875 strain was most similar to ZCC strain derived from mining soil sample, which was consistent with the results of phylogeny analysis and ANI analysis. This result suggests that B. contaminans SK875 could harbor biological characteristics that are similar to those of ZCC strain derived from Chinese environment.By comparing the B. contaminans genes causes human disease is not well understood, several virulence factors either contributing to growth in adhesion or invasion could be found in the genome of the B. contaminans SK875 strain, such as boaA, boaB, Type IV pili, capsules of synthesis-related and flagella-related genes. Also, B. contaminans SK875 strain had a large number of antibiotic-related genes. Both boaA and boaB genes are related to adherence function through their role as adhesions in vitro, thus conferring the ability to replicate inside macrophage-like cells (Balder et al.  pili genes are important for the virulence of many gram-negative bacteria and also play a role in adherence, a vital virulence mechanism resolved by carbohydrate molecules, pilus, and non-pilus adhesins (Carbonnelle et al. B. contaminans SK875, FL-1-2-30-S1-D0, and XL73 harbor all of these genes, but MS14 and ZCC lacked adherence-related genes, such as boaA or Type IV pili. The lack of these genes in some genomes demonstrates their inability to attach to the host cells to initiate infection (Deng et al. B. contaminans possessed various capsules and flagella genes. This fact indicates that B. contaminans strains have the strategies to infect mammalian hosts.Although the process by which the B. contaminans SK875 has a variety of secretion systems that control the expression of virulence factor genes for bacterial pathogenesis (Rutherford and Bassler cepIR gene of SK875 is able to produce AHL QS molecules, while BsaT3SS system helps to deliver the effector proteins which manipulate host cell functions, thereby evading the immune system and causing diseases into host cells (Kendall  Kendall . Several Kendall . Interes Kendall . From th Kendall .B. contaminans SK875. Comparative genomic analysis revealed that the genomes of B. contaminans were similar regardless of the isolated source, except that B. contaminans SK875 had some QS-related genes different from the other B. contaminans genomes. Therefore, our data will be valuable for understanding genetic characteristics, including the QS system in B. contaminans SK875.In conclusion, genome analysis provided a comprehensive analysis of the disease promising of Additional file 1: Table S1. Genome features of Burkholderia species. Table S2. COG distribution of the genes in B. contaminans SK875. Table S3. KEGG distribution of the genes in B. contaminans SK875. Table S4. List of unique genes of B. contaminans SK875. Table S5. COG distribution of the unique genes in B. contaminans SK875. Table S6. Prophage regions of the B. contaminans genomes. Table S7. The CRISPR loci of the five B. contaminans genomes. Table S8. Putative virulence genes in five B. contaminans genomes predicted by VFDB. Table S9. Antibiotic resistance genes in five B. contaminans genomes.


Plasmodium vivax, one of the major species associated with human malaria, continues to be a major public health problem in many parts of the world. Numerous studies related to vivax malaria have described quantitative haematological findings , but diverse morphological changes of parasite forms within infected red blood cells (iRBCs) have been mentioned only in few studies. Here we report a case of a 13-year-old boy who presented with fever, significant low platelet counts and hypovolaemia that created a diagnostic dilemma. Detection of microgametocytes by microscopic examinations, further confirmed by multiplex nested PCR assays and response to anti-malarials, helped to make the diagnosis. We present an atypical case of vivax malaria with a review of morpho-variations of iRBCs and have summarized the characteristics that aid in creating increased awareness among laboratory health professionals and public health workers. No supporting data, software or code was generated or re-used.Plasmodium falciparum and Plasmodium vivax co-endemicity, vivax malaria is relatively more widely distributed, causing singnificant morbidity and mortality [P. vivax as intrinsically benign, but such a description may no longer be viewed as a paradigm of vivax malaria [P. vivax infects reticulocytes and young red blood cell (RBCs) [P. vivax malaria from eight different countries revealed an association of severe malaria, severe anaemia, acute respiratory distress syndrome (ARDS) and cerebral malaria [P. vivax malaria can be attributed to long duration of the hepatic stage that allows the parasite to remain in the host even after interruption of transmission and successful treatment of the primary infection [In geographical areas with ortality . In the  malaria . P. vival (RBCs)  and rarel (RBCs) . Classic malaria . Severe nfection . Furthernfection .P. vivax observed during microscopic examination of stained peripheral blood smears include (i) crisis forms, (ii) pseudo-parthenogenesis forms, (iii) equatorial trophozoites, (iv) phagocytosed parasites, (v) malaria pigment-containing phagocytes, (vi) microgametocytes with or without ex-flagellation, (vii) multiple invasions of iRBCs and (viii) macrogametocytes. Crisis forms appear as blackish-brown pigmented and are characterized by an extra-erythrocytic bluish amoeboid structure of the parasites. These are disintegrating forms of parasites and have been reported from India, often in co-infections with lymphatic filariasis [Various morphological changes in infected RBCs (iRBCs) due to lariasis . The undlariasis .P. vivax malaria were first described by Garnham et al. in 1966 where the simultaneous presence of both asexual and sexual forms were detected in the same iRBC [et al. in 1997 [‘Pseudo-parthenogenesis forms’ in ame iRBC . These fame iRBC . In equa in 1997 .et al. in 1986 [et al. in 1983 using dark-field microscopy [Intra-leucocytic malaria pigment was first observed by Meckel in viscera and blood of patients who died of pernicious fever . Such fi in 1986  using pocroscopy . MalariaPhagocytosed malaria parasites have been reported in patients with extremely acute pernicious malaria. Almost a century ago, Golgi depicted circulating leucocytes containing malarial parasites either well preserved or in various stages of disintegration .in vitro ex-flagellation [P. falciparum malaria [Plasmodia species have been reported [Plasmodia occur as thin filamentous structures with an oval-shaped dark blue nucleus [et al. [P. falciparum malaria, have also been reported in P. vivax. As many as five rings in a single iRBC in a neonate just 15 h after birth [More commonly reported variations are microgametocytes with or without ex-flagellation. Such events occur often in definitive hosts but are rarely seen in peripheral blood smears of intermediate hosts. A heat-stable molecule derived from mosquito head and gut, namely mosquito ex-flagellation factor (MEF), is believed to be vital in inducing ellation . During ellation . This fo malaria . At the reported . The occreported . With de nucleus . In cont [et al. . Multipler birth  and singer birth .–3 blood; normal: 1.5–4.5 mm–3 blood), low haematological parameters of haemoglobin , haematocrit , mean corpuscular volume  and mean corpuscular haemoglobin . Blood urea  and random blood sugar  levels were significantly raised. Peripheral blood smear examination was positive for P. vivax. Other tests such as Dengue  NS1, scrub typhus card tests, Widal test and COVID 19 reverse transcriptase (RT)-PCR were negative. Despite thrombocytopaenia, the child had no bleeding manifestations and was administered a first dose of intravenous artesunate and intravenous fluids.A 13-year-old boy was referred to our hospital with complaints of high-grade fever, non-bilious, non-bloody projectile vomiting, light headedness, generalized weakness, reduced appetite and passing black-coloured urine. Prior to his admission, the child was febrile (40 °C) with blood pressure of 110/40 mmHg , along pO2) was well maintained. Liver was enlarged up to 2 cm below the right costal margin. Other systemic examinations were within normal limits. Upon laboratory investigations, his haemoglobin level was 11 g dl−1, with a low haematocrit (34.2 %), mean corpuscular volume (71 fl) and mean corpuscular haemoglobin (22.9 pg) values with platelet count of 36 000 mm–3 blood. Serum direct bilirubin and liver enzymes were elevated. Giemsa-stained peripheral blood smears revealed normal sized erythrocytes with microgametocytes containing malarial pigments  , with high pulse rate (110 bpm) and facial swelling. There was reduction in urine output of around 150 ml per day, but the oxygen saturation  . Buffer P. vivax infection can manifest clinically as that of P. falcipraum infection and has been previously reported in the literature. A case series [P. vivax malaria simulating that of P. falciparum infection. In each case, blood pressure was 80/50 mmHg, haemoglobin 9 g dl−1, platelets 70 000 mm–3 blood, and both chloroquine and artesunate was continued along with other supportive medications, which resulted in improvement of vital signs over the next 48 h in these patients. In another case, severe P. vivax malaria mimicked sepsis in a neonate, with low hemoglobion (4.4 g dl−1), hematocrit (13.1 %) and platelet count (95 000 mm–3 blood). The child improved with administration of injectable artesunate [P. vivax proteins such as Pv RBP1 and Rp RBP2 that are thought to restrict P. vivax to reticulocytes [Severe and complicated e series  describetesunate . Two othtesunate . Such evulocytes .P. vivax have been increasingly reported, suggesting a diversity of P. vivax isolates in different parts of the world [P. vivax cases in duffy antigen receptor for chemokines (DARC)-negative individulas further adds to the changing paradigm [P. vivax isolates supported the distinctiveness of transmissibility and spectre of P. vivax related manifestations [P. vivax infection can be identified with the help of latency, patency and relapse. The periodicity of P. vivax varies systematically in different regions, indicating adaptations in the variability of iRBCs. Presentations mimicking dengue in the present case with significant thrombocytopaenia and without haemorrhagic manifestations is another facet, although is not unusual. Malaria and dengue co-infections have been reported most frequently from the Indian subcontinent and often cause misdiagnosis and/or misinterpretation of mono-infections [P. vivax malaria indicate interactions among host, parasite and external factors, of which parasite virulence seems to play a major role [P. vivax polymorphisms altering cytokine production are other important factors [P. vivax shows its distinctiveness with underlying pathogenetic mechanisms. The recognition of such morphological changes of iRBCs is important to avoid diagnostic dilemmas.In recent years, severe forms of malaria due to he world . The groparadigm . In thisstations . P. vivafections . Variatijor role . Anti-ma factors . PeripheWhat makes our case interesting is that our patient had a very low thrombocytopaenia with no bleeding diathesis but with severe hypovolaemia without manifesting with any features of shock or sepsis. In addition, a collective investigative algorithm was able to conclusively achieve a diagnosis.


Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens . We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored ( Sorex) and three species of white-toothed shrews (genus Crocidura) are described [Sorex shrews prefer cool and moist, forest-covered habitats, while Crocidura shrews are found in dry and arid, more open spaces and can be commensal [Sorex araneus).Shrews are small insectivorous mammals belonging to one of the largest mammalian families, the Soricidae . Currentescribed . They diommensal ,9. The mCrocidura suaveolens ) and the bicolored white-toothed shrew ) are sympatrically found mainly in southern and eastern Europe [Crocidura russula ) expands from northern Africa through the Iberian Peninsula and France into Germany [C. russula within Germany, have been described. In areas newly colonised by C. russula, competition with the smaller C. leucodon and C. suaveolens has led to their local extinction [The exact distribution ranges of these shrews are scarcely described, especially for white-toothed shrews. The lesser white-toothed shrew  [Erinaceus europaeus, Linnaeus, 1758), are well known major carriers of Leptospira spp. [The role of shrews as carriers for zoonotic pathogens is still understudied ,20, and la virus ,22. Shreviridae) ,24. Otheira spp.  and arthira spp. ,27,28. ILeptospira spp. are obligate extracellular bacteria belonging to the phylum Spirochaetes. They are distributed worldwide and are associated with different reservoir host species, of which small mammals are the most important [Leptospira spp. varies from mild flu-like symptoms to severe forms such as kidney organ failure (Morbus Weil) or encephalitis [Leptospira spp. prevalence in small mammals in Germany have mainly focused on rodents and Sorex shrews [Leptospira kirschneri in C. russula and C. leucodon [Leptospira alstonii was isolated from invasive C. russula in Ireland, with previous isolates only originating from non-mammal hosts from China, Japan and Malaysia [Leptospira spp. in lesser and bicolored white-toothed shrews.mportant . The bacphalitis . Studiesx shrews , with thleucodon ,32. InteMalaysia . Little Anaplasma phagocytophilum, Babesia spp. and Neoehrlichia mikurensis are tick-borne pathogens transmitted by hard ticks, mostly of the genus Ixodes [Bartonella spp., most of which are considered zoonotic [Felis catus) and game). The detection of Bartonella spp. in shrews was described for Sorex spp. in Germany [Bartonella strain, named Bartonella florenciae, was previously isolated from the spleen tissue of a C. russula from France [s Ixodes , causings Ixodes . High prs Ixodes ,37, but zoonotic , are Gram France ,39.Coxiella burnetii, is a globally distributed Gram-negative bacterium that causes infertility and abortions, mainly in ruminants , and is excreted in great numbers with birth materials and, to a lesser extent in milk, faeces and urine. Farmers, veterinarians and abattoir employees are high-risk groups for infection. Numbers on reported human infections have fluctuated between 55 and 416 cases per year in Germany since 2001 [Dermacentor marginatus) can shed C. burnetii in their faeces and transmission could potentially occur through inhalation of faecal dust rather than by the tick bite [C. burnetii. A seroprevalence of 19% was previously reported for rodents in the UK [Sorex spp. had no antibodies against C. burnetii [The causative agent of “Q-fever”, nce 2001 . Ticks  and humans (feverish multi-organ failure). Germany is considered to be free of bovine, ovine and caprine brucellosis. To maintain this status, its potential reintroduction by wildlife should be closely monitored. However, reported human cases are increasing [Brucella species, Brucella microti, was isolated from common voles in central Europe [Brucella spp.-positive, but so far no data are available on the presence of this pathogen in Crocidura spp. from Germany.creasing . Severall Europe  and has l Europe ,49. Prevl Europe  were BruLeptospira spp. in white-toothed shrews and (iii) evaluate white-toothed shrews as reservoirs for arthropod-borne pathogens and compare the findings to European hedgehogs.As data on the current distribution of greater, lesser and bicolored white-toothed shrews in Germany are incomprehensive and knowledge on their role as carriers for pathogens with zoonotic potential is limited, the objectives for this study were to (i) contribute to the current knowledge on the distribution of white-toothed shrews in Germany, (ii) detect and characterise Shrews from Germany, Luxembourg, Austria and Slovakia were collected between 1999 and 2021 . The majNucleic acids were extracted from kidney and spleen tissue using a Nucleo Mag Vet Kit  and a KingFisher™ Flex Purification System  according to the manufacturer’s instructions.cytochrome b gene and sequence comparison to GenBank entries as previously described [Species identification for each shrew was performed based on the molecular analysis of the almost-complete escribed .Leptospira spp. DNA with a real-time PCR (qPCR) targeting the lipl32 gene , encoding for an outer membrane lipoprotein [Leptospira-positive. As positive control, DNA of a laboratory strain of L. kirschneri serovar Grippotyphosa was used [C. leucodon samples were investigated previously by conventional lipl32 gene PCR [Kidney-derived DNA was screened in pools of two for the presence of oprotein . Positivwas used . Three CglmU (amplicon size: 650 bp), pntA (621 bp), sucA (640 bp), tpiA (639 bp), pfkB (588bp), mreA (791 bp) and caiB (650 bp), was performed for samples with a Ct value < 36 following the scheme from Boonsilp et al. [Multilocus sequence typing (MLST) of seven target genes, p et al.  considerp et al. .secY gene (657 bp) was performed to determine Leptospira species as previously described [L. interrogans serovar Icterohaemorrhagiae was used [For samples with a Ct value > 36 or with incomplete MLST results, a conventional PCR targeting the escribed . As posiwas used .https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 7 August 2022). The obtained sequences were uploaded to GenBank (accession numbers: OQ865429–OQ865435).PCR products were prepared with DNA Gel Loading Dye (6x)  for gel electrophoresis in 2% agarose, and gels were stained with HDGreen Plus DNA Stain . Amplification products were visualised by UV light using the UVP GelSolo streamlined gel documentation . The samples were purified for sequencing using a NucleoSpin Gel and PCR clean-up kit  as recommended by the manufacturer. The sequences were trimmed using Bionumerics v.7.6.1.  and compared to available data in GenBank with the Basic Local Alignment Search Tool (BLAST)  subunit (nuoG) with an amplicon size of 346 bp [B. henselae Marseille strain was used as positive control. Positive samples were further analysed by PCR targeting the gltA gene (amplicon size: 378 bp) [Anaplasma phagocytophilum DNA targeting the msp2 (major surface protein 2) gene (amplicon size: 77 bp) [Neoehrlichia mikurensis DNA targeting the groEL gene (amplicon size: 99 bp) as previously described [A. phagocytophilum culture and DNA from a N. mikurensis positive yellow-necked field mouse (Apodemus flavicollis) from Leipzig, Germany, that was trapped in 2016 [Babesia spp. DNA by conventional PCR targeting a fragment (411–452 bp) of the 18S rRNA gene [Coxiella burnetii DNA and Brucella spp. DNA, all individual spleen-derived DNA samples were screened using a qPCR targeting the multicopy insertion element IS1111 [bcsp31 gene [The presence of f 346 bp . DNA fro 378 bp) ,58. Posi: 77 bp)  and Neoeescribed . As posi in 2016 , respectRNA gene . For thet IS1111  or the bp31 gene , respectLeptospira spp. were determined using the Clopper and Pearson method with an alpha value of 0.05. For the prevalence of Leptospira and the sex of different Crocidura species, Fisher’s exact test was used to test independence. Tests were considered to be significant if p (probability) < 0.05.All statistics were performed in the GraphPad Prism Software v. 4.0 . Mean prevalence and confidence intervals (95% CI) for Maps were generated using Karten-Explorer v. 2.21 (Friedrich-Loeffler-Institut (FLI), Bundesforschungsinstitut für Tiergesundheit Copyright © 2022, Greifswald, Insel Riems, Germany). The German federal states were grouped into four regions: southwest, northwest, northeast and southeast, for the evaluation of the geographical distribution of white-toothed shrews .In total, 341 shrews were collected between 2002 and 2021 in Germany: 235 greater white-toothed shrews , 83 bicolored white-toothed shrews  and 23 lesser white-toothed shrews  .Crocidura russula was the most abundant species, especially in the western parts of Germany—northwest: 99% (n = 103) and southwest: 100% (n = 9). Only one C. leucodon (1%) was collected in the southeast of Lower Saxony close to the Harz mountain range (C. russula (n = 77), 13.6% C. leucodon (n = 15) and 16.4% C. suaveolens (n = 18). Crocidura russula was still the predominant species in northeast Germany, but it was not collected in the state of Brandenburg (BB), which is far northeast, where mainly C. suaveolens was found . In the southeast, especially in the south of Bavaria, C. leucodon was the most prominent , and C. russula  was mainly found in Franconia and further north. Of all the collected white-toothed shrews from the southeast 4.2% were C. suaveolens (n = 5) , northwest (n = 103), northeast (n = 110) and southeast (n = 118) of Germany . Crociduin range . In the  (n = 5) . The speve sites , and C. Slovakia .Leptospira spp. DNA was detected in kidney samples from 28 out of 227 C. russula  and three out of 81 C. leucodon  samples from Germany . Thus, the prevalence was significantly lower in C. leucodon and C. suaveolens compared to C. russula (p = 0.003). Out of the 28 lipl32 qPCR-positive C. russula, six were identified as Leptospira kirschneri by sequencing the secY PCR product. MLST was successful for an additional six individuals (C. russula) and were determined to be the same sequence type: Leptospira kirschneri ST 100. The sequencing of the secY PCR product of the lipl32 qPCR-positive C. leucodon was not possible, which was most likely due to the poor sample DNA quality. There was no significant difference in the prevalence between female  and male C. russula  (p = 0.337).All of the Leptospira spp. DNA-positive individuals originated from 15 trapping sites from across Germany (Leptospira kirschneri at the different sites varied between 5.6% and 40% (mean x = 25%); sites with less than four individuals were excluded . The hedgehog investigation revealed that four of the 42  animals were lipl32 qPCR-positive, which were further characterised as L. kirschneri (ST 100) and L. interrogans (ST 24). Germany . The preC. russula, 80 C. leucodon and 21 C. suaveolens from Germany detected Neoehrlichia mikurensis DNA in two female C. russula  samples, one from southeast Germany and the other one from northwest Germany  and 21 C. suaveolens  tested positive for N. mikurensis DNA. None of the investigated shrews were positive for Babesia spp., A. phagocytophilum, Bartonella spp., Brucella spp. or C. burnetii DNA  animals. Three of the 42  hedgehogs tested positive for Bartonella spp., two being typed as B. clarridgeiae strain 73 and one as uncultured Bartonella spp. None of the 42 hedgehogs tested positive for N. mikurensis, Babesia spp., Brucella spp. and C. burnetii DNA.The PCR screening of spleen samples of 213  Germany . None ofetii DNA . The shrCrocidura spp. in Germany. The latest comprehensive survey on the distribution of white-toothed shrews in Germany covered only southeast Germany (Bavaria) [The collection of 341 white-toothed shrews allowed, albeit with limitations due to the heterogenous sampling, an update on the current distribution of Bavaria)  and was C. russula expands from the western European countries into central Germany and is slowly expanding further east [C. russula in our study in western and southeastern Germany coincided with the easternmost expansion into Franconia, Bavaria [C. russula occurred, C. russula predominated over the other two species, which may have led to the local extinction of C. suaveolens as they are considered parapatric species [C. russula and the smaller C. suaveolens or due to differences in adaptations to synanthropic habitats and climate conditions, as C. russula copes better with drier, hotter summers, and therefore, out-competition is still under debate [C. leucodon, as C. russula was primarily found in former typical C. leucodon habitats [C. russula and the replacement of C. leucodon has also been observed in Switzerland [C. russula, it being found in the northwest of Austria, while in the east of Austria so far only C. leucodon and C. suaveolens were collected. We primarily detected C. suaveolens in the northeastern part of Germany, supporting the westwards expansion trend described by Jentzsch and Trost [Crocidura suaveolens were sporadically found in the southeast, but not at all in the western parts of Germany. Similarly, the absence of C. leucodon from the southwest was consistent with previous reports describing a decline in C. leucodon occurrence in the western half of Germany [C. leucodon and C. russula versus C. leucodon and C. suaveolens was almost equally frequent (n = 5 vs. n = 4); however, C. suaveolens and C. russula were only collected together at one site in northeastern Germany. Between 1995 and 2010, the co-occurrence of all three species was described for east Thuringia [C. russula has expanded at a pace of 15 km/year, which is much faster than described for continental Europe.Over the past decades, multiple studies ,67,68,69her east ,73,74. T Bavaria . In regi species ,18,74. Wr debate ,18. The habitats ,76. The tzerland  and Austtzerland . Althougnd Trost . Crocidu Germany ,76. Infohuringia  or simplhuringia . Anthrophuringia ,81,82, ahuringia . Since tLeptospira prevalence were mainly focused on rodents and soricine shrews. Depending on the shrew species and geographic region, previous studies describe a mean Leptospira prevalence of 3.0% , 6.8%  and 15.5%  [In regard to small mammals, previous studies of araneus) .Leptospira in crocidurine shrews in central Europe is scarce. Leptospira spp. was detected in C. russula already in the 1970s [Leptospira kirschneri was found in Crocidura russula [Crocidura leucodon [Leptospira kirschneri in 28 C. russula and two C. leucodon with a mean prevalence of 25% (5.6–40%) at 15 trapping sites. Leptospira spp. was irregularly distributed in Germany, as demonstrated by its absence in white-toothed shrews from Saxony  [The current knowledge on he 1970s . In Germ russula  and Crocleucodon , but no s study, ). The iral hosts ; crocidu invader  and may e et al.  detectedvaticus) .Leptospira spp., and it is widely used to evaluate the spread of a specific pathogen within a population to distinguish detection in maintenance hosts from spill-over and host-switch events. In small mammal populations, different sequence types are seen within the same species and the same ST in different animal species. Common shrews from various locations in Germany have been shown to carry Leptospira kirschneri of two different sequence types  as well as Leptospira borgpetersenii of ST 146 [Leptospira kirschneri ST 110 is strongly associated with voles of the genus Microtus and is the most common source of leptospirosis outbreaks in strawberry pickers in Germany [Leptospira kirschneri ST (ST 100) in all the C. russula samples from the different trapping sites across Germany, suggesting a possible host species specificity and may identify C. russula as maintenance host rather than spill-over host. However, this ST was also found in a European hedgehog (this study) and was previously isolated from a Portuguese house mouse (Mus musculus) [Apodemus agrarius) in central Europe [Leptospira isolate 15-LE00367-0 [MLST allowed us to determine the ST of f ST 146 . Leptosp Germany . Interesusculus) . This STl Europe , which cl Europe  and is al Europe . FurtherE00367-0 ) from GeA. phagocytophilum in one of six C. russula samples [A. phagocytophilum, Babesia spp. and N. mikurensis in any C. russula sample [A. phagocytophilum in any white-toothed shrew. Anaplasma phagocytophilum is present in the small mammal community in Germany, as confirmed here by the prevalence of about 10% in European hedgehogs  [N. mikurensis DNA in two C. russula samples at different urban sites in northwestern and southeastern Germany, a finding that seems to be in contradiction to the assumption of previous studies that insectivores do not play a role in the transmission and maintenance of N. mikurensis [Bartonella spp. from soricine shrews in Germany revealed host-specific Bartonella taylorii-associated strains [Bartonella spp. have only been detected in Crocidura spp. outside of Germany [Bartonella refiksaydamii in the blood of a lesser white-toothed shrew from northwestern Turkey by Celebi et al. [Bartonella spp. DNA in any of the white-toothed shrews, but we identified the Bartonella clarridgeiae strain 73 and an “uncultured Bartonella spp.” in the hedgehogs. Bartonella clarridgeiae is commonly present in cats [Ctenocephalides felis) and was once found in an asymptomatic blood donor in Brazil [Babesia spp. and Coxiella burnetii is ill-defined. In our study, we did not detect Babesia spp. DNA in any of the crocidurine shrews or hedgehogs, even though Bown et al. [Babesia microti prevalence of 30.3% in common shrews occupying the same habitat as field voles . Despite reports of a high seroprevalence for C. burnetii in rodents [C. burnetii, shrews and hedgehogs do not seem to play a role as reservoirs. Fleas collected from C. suaveolens were tested for the presence of C. burnetii and rickettsiae, but they did not contain any respective DNA [Brucella spp. in soricine shrews [A high prevalence of tick-borne pathogens has been described for common shrews ,37, but  samples , whereasa sample . Even ths study, ) and in kurensis . The det strains . So far, Germany ,96, e.g.i et al. . In this in cats , is trann Brazil . The roln et al.  reported rodents , all of tive DNA . Previoue shrews  could noIxodes ricinus and Dermacentor reticulatus could be collected from C. leucodon and C. suaveolens trapped in Slovakia, the numbers were much lower than those from sympatric rodent species [Little is known about ectoparasites on shrews, but a white-toothed-shrew specific “ectoparasite milieu” ,100, red species .Leptospira spp. and arthropod-borne pathogens. However, our study was limited by its sample size and sampling approach, heavily relying on the cooperation of the public. In the future, a more systematic and longitudinal study, ideally in a One Health setting, is needed to evaluate the potential infection risks of shrews and hedgehogs. The short life expectancy and high turnover rate of local shrew populations, including frequent extinction and fast recolonization events as described for C. russula [This study provides an update on the current distribution of white-toothed shrews in Germany. Altogether, white-toothed shrews seem to play a minor role in the transmission of  russula , potenti