Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium ubiquitum mainly differed in copy numbers of secreted MEDLE proteins and insulinase-like proteases and sequences of mucin-type glycoproteins. Recently, Cryptosporidium chipmunk genotype I was identified as a novel zoonotic pathogen in humans. In this study, we sequenced its genome and conducted a comparative genomic analysis.Cryptosporidiosis is a major cause of gastrointestinal diseases in humans and other vertebrates. Previous analyses of invasion-related proteins revealed that Cryptosporidium chipmunk genotype I has gene content and organization similar to C. parvum and other intestinal Cryptosporidium species sequenced to date. A total of 3783 putative protein-encoding genes were identified in the genome, 3525 of which are shared by Cryptosporidium chipmunk genotype I and three major human-pathogenic Cryptosporidium species, C. parvum, C. hominis, and Cryptosporidium meleagridis. The metabolic pathways are almost identical among these four Cryptosporidium species. Compared with C. parvum, a major reduction in gene content in Cryptosporidium chipmunk genotype I is in the number of telomeric genes encoding MEDLE proteins (two instead of six) and insulinase-like proteases (one instead of two). Highly polymorphic genes between the two species are mostly subtelomeric ones encoding secretory proteins, most of which have higher dN/dS ratios and half are members of multiple gene families. In particular, two subtelomeric ABC transporters are under strong positive selection.The genome of Cryptosporidium chipmunk genotype I possesses genome organization, gene content, metabolic pathways and invasion-related proteins similar to the common human-pathogenic Cryptosporidium species, reaffirming its human-pathogenic nature. The loss of some subtelomeric genes encoding insulinase-like proteases and secreted MEDLE proteins and high sequence divergence in secreted pathogenesis determinants could contribute to the biological differences among human-pathogenic Cryptosporidium species.The online version of this article (10.1186/s12864-019-5788-9) contains supplementary material, which is available to authorized users. Cryptosporidium spp. are important apicomplexan parasites, causing moderate to severe diarrhea in humans and various animals. Currently, there are near 40 named Cryptosporidium species and about the same number of genotypes with unknown species status [Cryptosporidium parvum and Cryptosporidium hominis are two major species infecting humans. Other species, including Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium ubiquitum, Cryptosporidium cuniculus, Cryptosporidium viatorum, and Cryptosporidium muris, are less common [s status . Among ts status . Howevers common .Cryptosporidium species differ in host range and public health significance [C. parvum has the broadest host range. In addition to humans, it infects ruminants, equine animals, rodents, and some other animals. In contrast, C. hominis is mostly restricted to humans, nonhuman primates, and equine animals [C. meleagridis has been reported in both mammals and birds [Cryptosporidium species, C. ubiquitum, also has a broad host range, being commonly detected in small ruminants, rodents, in addition to humans [Cryptosporidium chipmunk genotype I, which was initially found in several species of rodents, is a novel zoonotic pathogen, having been reported in humans recently [Cryptosporidium species in humans in rural United States [ificance . Among t animals . As the nd birds , 4, 5. Ao humans , 7. Cryprecently , 9. It id States .Cryptosporidium species [Cryptosporidium species or C. parvum subtype families  indicates that MEDLE proteins could contribute to differences in host specificity [Cryptosporidium spp. [C. parvum, a reduction in the numbers of genes encoding the MEDLE family secreted proteins and insulinase-like proteases was seen in the 3′ subtelomeric regions of chromosomes 5 and 6 of the C. hominis genome [C. ubiquitum and gastric species Cryptosporidium andersoni [Cryptosporidium species differ significantly in sequences of mucin-type glycoproteins [Cryptosporidium species differ significantly in the numbers and sequences of genes encoding mucin-type glycoproteins and insulinase-like proteases, these proteins and other secreted pathogenesis determinants (SPDs) potentially play an important role in tissue tropism also [Results of comparative genomics analysis suggest that members of several secreted protein families, such as MEDLE proteins, insulinase-like proteases, and mucin-type glycoproteins, are potential determinants for differences in host range among  species , 12. Thecificity , 13. Inscificity . Mucin-tium spp. . Compares genome . The ortproteins , 12. As Cryptosporidium species have been sequenced recently, we still have very limited knowledge of genome evolution among Cryptosporidium spp. [Cryptosporidium chipmunk genotype I and conducted a comparative genomic analysis of eight Cryptosporidium species that have been sequenced thus far [Although the genomes of several ium spp. , 17. In thus far , 18–20.Cryptosporidium chipmunk genotype I isolate 37,763 from a naturally infected person in the United States by Illumina sequencing. After filtering out contigs from contaminants among the 298 initial contigs generated using the CLC Genomics Workbench, we assembled a Cryptosporidium genome of 9.05 Mb in 50 contigs (without any scaffolding during the processing), with an estimated 188-fold coverage and an N50 of 320,570 bp. We combined gene prediction results obtained from Augustus, Geneid, and Genemark, leading to the identification of 3783 protein-encoding genes. At the genome level, Cryptosporidium chipmunk genotype I has high nucleotide and amino acid sequence identity to C. parvum , C. hominis , and C. meleagridis  and coding regions (33.6%). The genome of Cryptosporidium chipmunk genotype I has near complete sequence synteny with that of C. parvum and C. ubiquitum  in C. parvum. This rearrangement was observed in both assemblies produced by the CLC Genomics Workbench and the SPAdes assembler. Advanced sequencing using the PacBio technology is needed to confirm the existence of this genome rearrangement. Lower synteny was seen with genomes of C. baileyi and C. andersoni. Cryptosporidium chipmunk genotype I shares almost the same gene density and number of tRNA genes with other Cryptosporidium spp. It, however, has gene content slightly lower than C. parvum and C. hominis, but similar to C. meleagridis, C. ubiquitum, and C. baileyi  and SNF2 (16 genes). ATPases associated with diverse cellular activities (AAA) and ATP-binding cassette (ABC) transporters formed Cluster 4 and 5. We found 21 genes encoding ABC transporters in all three species. Compared with C. parvum and C. meleagridis, one gene encoding AAA proteins was lost in Cryptosporidium chipmunk genotype I (24 AAA proteins). In addition, the Ras proteins, which are involved in- intracellular signaling, formed Cluster 7. Furthermore, the 12 thrombospondin-related adhesive proteins (TRAPs), which are presumably microneme proteins present in all three Cryptosporidium species under analysis [Multiple gene families are present in ers Fig. a. Two ofCryptosporidium spp., Cryptosporidium chipmunk genotype I lacks genes encoding core enzymes of the tricarboxylic acid (TCA) cycle, but possesses enzymes for the synthesis of pyruvate from glucose in glycolysis. Furthermore, a gene for a phosphoenolpyruvate carboxylase (Cch_34.2917) was detected in Cryptosporidium chipmunk genotype I, suggesting that this parasite can convert phosphoenolpyruvate (PEP) to oxaloacetate (OAA).Similar to other intestinal Cryptosporidium spp., Cryptosporidium chipmunk genotype I lacks genes encoding enzymes for de novo isoprenoid biosynthesis. Two genes encoding farnesyl diphosphate (FPP) synthase (Cch_19.1677) and polyprenyl synthase (Cch _17.1265) were detected in Cryptosporidium chipmunk genotype I. These two genes were shown transcribed in C. parvum in vitro [C. ubiquitum [Like other in vitro , but are Cch _17.65 were dCryptosporidium spp. [Cryptosporidium spp. have an alternative oxidase (AOX) and a reduced conventional electron transport system, except for C. ubiquitum, which does not have them and the AOX. Unlike C. ubiquitum, Cryptosporidium chipmunk genotype I and the three major human-pathogenic species possess all enzymes and proteins involved in the ubiquinone biosynthesis  synthase (cgd5_4520 in C. parvum) is lost in Cryptosporidium chipmunk genotype I, indicating that Cryptosporidium chipmunk genotype I cannot convert xanthosine 5′-phosphate (XMP) to GMP. Furthermore, the last gene (cgd1_3860) in chromosome 1 of C. parvum, which encodes a deoxyuridine triphosphate (dUTP) diphosphatase, has an ortholog in C. hominis (Chro.10434), but is absent in Cryptosporidium chipmunk genotype I and C. meleagridis , however, is present in Cryptosporidium chipmunk genotype I (Cch_42.3131).All vo Table . Insteadvo Table . However gene cgd_3860 in Alg genes in asparagine (N)-linked glycosylation was reported in apicomplexans [Cryptosporidium. Similar to C. hominis, C. parvum, C. meleagridis, and C. ubiquitum, Cryptosporidium chipmunk genotype I possesses nine sugars in N-glycan precursors, compared to eight sugars in C. baileyi and five in C. andersoni.A secondary loss of mplexans . The bioCryptosporidium chipmunk genotype I but lost in C. ubiquitum. Similar to other Cryptosporidium spp., genes encoding PIG-W and glycosylphosphatidylinositol deacylase (PGAP1) involved in the acylation and de-acylation of inositol are absent in Cryptosporidium chipmunk genotype I.In glycosylphosphatidylinositol (GPI) anchor biosynthesis, the essential phosphatidylinositol glycan (PIG)-B was detected in Cryptosporidium chipmunk genotype I and other intestinal Cryptosporidium spp. possess similar numbers and components of major protein families, including some of those involved in invasion, such as protein kinases and TRAPs. Cryptosporidium species, however, differ in the number of genes encoding other invasion-related proteins, such as insulinase-like peptidases, MEDLE secretory proteins, and mucin glycoproteins. For example, gastric species C. andersoni and C. muris have fewer genes encoding insulinase-like peptidases  of the insulinase gene family, which has significant sequence similarity to cgd3_4260, was detected at the 5′ end of chromosome 7 (contig_105). Furthermore, all three major human-infecting species, C. parvum, C. hominis, and C. meleagridis, possess MEDLE protein genes, but none of them were observed in C. ubiquitum, C. baileyi, C. andersoni, or C. muris  was seen at the 3′ end of chromosome 4. In the large insertion at the 3′ end of chromosome 5 (contig_35) in Cryptosporidium chipmunk genotype I, Cch_35.2955 is a paralog of Cch_40.3117, Cch_7.3568 and Cch_1.1. Six members  of this gene family were detected in C. hominis but only three  were detected in C. parvum. In contrast, the ortholog of cgd4_3690, which encodes a low complexity protein with a large glycine-rich repeat, was lost in Cryptosporidium chipmunk genotype I. The same is also true for the gene for a cysteine-rich protein with a signal peptide in C. parvum (cgd4_4500), C. hominis (Chro.40511), and C. meleagridis (C_mele_24106.404). Similar to C. hominis and C. meleagridis, Cryptosporidium chipmunk genotype I has only one copy of the paralogous genes cgd8_660_670 and cgd8_680_690. Similarly, orthologs of cgd4_10, cgd7_5530, cgd8_4180 and cgd8_5420 were not detected in Cryptosporidium chipmunk genotype I  and 19 are located in the subtelomeric regions .Compared with other related gene Cch_3.573 wasCryptosporidium chipmunk genotype I was compared with the annotated protein-encoding genes of C. parvum and C. ubiquitum. We found 49 highly divergent genes between Cryptosporidium chipmunk genotype I and these two Cryptosporidium species with an amino acid identity below 65%  encode ABC transporters. Among the 20 orthologous genes with the highest dN/dS ratios, 9 (45%) encode proteins with signal peptides, 11 (55%) encode membrane-bound proteins, and 14 (70%) are located in the subtelomeric regions , with the number of predicted protein-encoding genes over a range of ~ 6000–10,000 genes [Plasmodium spp. [Babesia spp. [Cryptosporidium species have shown high genome conservation. The differences in host range among intestinal Cryptosporidium species could be potentially caused by the minor gene gains and losses or sequence polymorphism in SPDs encoded by genes located in subtelomeric regions.The genome organization of 00 genes . Similarium spp.  or Babessia spp. . Thus, cC. parvum, a major reduction in gene content in Cryptosporidium chipmunk genotype I is in the number of subtelomeric genes encoding secreted MEDLE proteins and insulinase-like proteases. Cryptosporidium parvum has two subtelomeric genes for insulinase-like proteases , compared to one in Cryptosporidium chipmunk genotype I , one (cgd5/6_5520–5510 ortholog) in C. meleagridis, and none in C. hominis. The loss of these and some subtelomeric genes encoding secreted MEDLE family proteins in Cryptosporidium chipmunk genotype I  may contribute to its narrow host range. In contrast, the number of genes for mucin-type glycoproteins in Cryptosporidium chipmunk genotype I is similar to that in human-infecting species. Cryptosporidium chipmunk genotype I, C. hominis, C. parvum, and C. meleagridis possess 24 genes encoding mucin-type glycoproteins, whereas gastric species, such as C. andersoni and C. muris, have lost 16 of them, including those encoding gp60, Muc4, and Muc5, which are important in the attachment and invasion of C. parvum [Compared with . parvum .Cryptosporidium chipmunk genotype I is not yet clear. The gene Cch_35.2955, which has three other paralogs in Cryptosporidium chipmunk genotype I, was annotated as a new gene at the 3′ end of chromosome 5. C. parvum has three orthologs  while C. hominis has six . There is also a loss of the cgd8_660_670 ortholog in chromosome 8 of Cryptosporidium chipmunk genotype I. This gene encodes a large low complexity protein in C. parvum and has a paralog (cgd8_680_690) downstream. Likewise, C. hominis has only one member of this multigene family [Cryptosporidium chipmunk genotype I has lost several other genes, such as orthologs of cgd4_3690 (encoding a large glycine-rich repeat low complexity protein), cgd4_4500 (encoding a cysteine-rich protein), cgd5_2960 (encoding a DEAD/DEAH box helicase), cgd5_2980 (encoding another DEAD/DEAH box helicase), and cgd8_4180 (encoding a glycine-rich low complexity protein) in C. parvum. Although the functions of these proteins are mostly unknown, these gene losses could contribute to the narrow host range of Cryptosporidium chipmunk genotype I.The significance of other gene gains and losses in the genome of e family . In addiCryptosporidium chipmunk genotype I and other Cryptosporidium spp. encode secreted proteins and half of the highly divergent genes are located in the subtelomeric regions. These secreted proteins could potentially be SPDs in Cryptosporidium spp., thus play a role in host specificity of Cryptosporidium spp., especially SKSR, FLGN and mucin proteins. Among them, the number of genes encoding SKSR proteins is different between C. parvum IIa and IId subtype families, which have different host preference [C. parvum IId subtype family, 7 paralogous genes encoding SKSR proteins were detected in Cryptosporidium chipmunk genotype I, but the sequence of these genes were divergent from those in C. parvum. The high sequence diversity of mucin-type glycoproteins between human- and animal-infecting species may also contribute to the host specificity and tissue tropism among Cryptosporidium spp. Previously, secretory proteins from dense granules (GRAs), micronemes (MICs), rhoptries (ROPs), and the SRS super-family were identified as potential SPDs in T. gondii, which could be responsible for differences in transmission modes, pathogenicity, and host range among T. gondii strains [Most of the highly divergent genes between eference . As in C strains .C. parvum and C. hominis genomes [Cryptosporidium chipmunk genotype I and C. parvum. ABC transporters are “key components of the cellular machinery for endobiotic and xenobiotic detoxification”, thus may contribute to intrinsic drug resistance in Cryptosporidium spp. [C. parvum IIa (zoonotic) and IIc (anthroponotic) subtype families [cgd2_80 and cgd2_90, are also within the same region (cgd2_70 and cgd2_90) identified as going through positive selection in the present study. These three ABC transporters encoded by genes within the ABC transporter gene cluster (cgd2_60 to cgd2_90) could be potential targets for drug development.The elevated dN/dS ratios for secreted and surface-associated proteins support their function as SPDs. These proteins are apparently under selection, perhaps as a result of high immune pressure due to their importance in invasion and host-parasite interactions. A similar observation was made in comparative analysis of  genomes , 31. Mosium spp. . These gfamilies . InteresCryptosporidium chipmunk genotype I apparently possesses metabolic pathways and invasion-related proteins similar to those in C. parvum, C. hominis, and C. meleagridis. This supports the human-pathogenic nature of Cryptosporidium chipmunk genotype I. The loss of two subtelomeric genes of insulinase-like proteases and four genes of secreted MEDLE family proteins compared with C. parvum are in agreement with the narrowed host range of Cryptosporidium chipmunk genotype I. Sequence differences and selection in genes encoding secreted and surface-associated proteins and ABC transporters could contribute to other biological differences among intestinal Cryptosporidium species. More studies on functional genomics and the basic biology of multiple isolates of Cryptosporidium chipmunk genotype I are needed to confirm some of the conclusions and improve our understanding of the emerging human pathogen.Cryptosporidium chipmunk genotype I isolate 37,763 was collected from one human specimen in Vermont and diagnosed by DNA sequence analysis of the small subunit rRNA gene [http://cab.spbu.ru/software/spades/).RNA gene . OocystsRNA gene . The purCryptosporidium chipmunk genotype I genome and published genomes of C. parvum IOWA isolate [C. hominis, C. ubiquitum [C. baileyi [C. andersoni [Cryptosporidium chipmunk genotype I genome and other four genomes.An alignment of  isolate , C. homibiquitum , C. bail baileyi  and C. andersoni  was consndersoni  with defndersoni  was usedCryptosporidium chipmunk genotype I with the default settings, after training AUGUSTUS and Geneid with the gene model of the C. parvum IOWA genome. Consensus predictor EVidence Modeler [AUGUSTUS 3.2.1 , Geneid  Modeler  was usedCryptosporidium chipmunk genotype I were annotated by using BLASTP [http://www.genome.jp/kegg/), Pfam (http://pfam.xfam.org/) [The predicted genes of g BLASTP  search og BLASTP  and TMHMg BLASTP , respectg BLASTP  was usedg BLASTP  with theam.org/) , and LAMam.org/)  were useCryptosporidium chipmunk genotype I and other Cryptosporidium genomes in CryptoDB (http://cryptodb.org/cryptodb/). Homologous gene families were identified by using OrthoMCL [C. parvum, C. hominis, C. ubiquitum, C. meleagridis, and Cryptosporidium chipmunk genotype I was drawn using VennPainter (https://github.com/linguoliang/VennPainter). The relationship among proteins in Cryptosporidium chipmunk genotype I, C. parvum, and C. meleagridis was visualized with Gephi (https://gephi.org/) with the Fruchterman-Reingold layout based on the result of BLASTP homology analysis, with threshold of protein pairs sharing 30% identity over 100 amino acids. Comparative analyses of metabolism among Cryptosporidium spp. were based on the results of KAAS and data of LAMP. Pfam search results were used in comparisons of transporter proteins and invasion-related proteins among Cryptosporidium species. The nonsynonymous to synonymous substitution (dN/dS) ratios between Cryptosporidium chipmunk genotype I and C. parvum were calculated for orthologous genes using KaKs_Calculator 2.0 [BLASTP was used for sequence similarity searches among OrthoMCL . BLASTP ator 2.0 .Cryptosporidium species and Gregarina niphandrodes were extracted and concatenated to construct a phylogenetic tree. MUSCLE [G. niphandrodes was used as the outgroup.The amino acid sequences of 100 single-copy orthologs shared among . MUSCLE  was used. MUSCLE . Phyloge. MUSCLE  with 100Additional file 1:Table S1. Gene gains and losses in several Cryptosporidium species. Table S2. Major putative invasion- and host specificity-associated genes in Cryptosporidium spp. Table S3. Highly divergent genes among Cryptosporidium chipmunk genotype I, C. parvum and C. ubiquitum. (XLSX 23 kb)


Gymnosporangium spp. are fungal plant pathogens causing rust disease and most of them are known to infect two different host plants (heteroecious) with four spore stages (demicyclic). In the present study, we sequenced the transcriptome of G. japonicum teliospores on its host plant Juniperus chinensis and we performed comparison to the transcriptomes of G. yamadae and G. asiaticum at the same life stage, that happens in the same host but on different organs.Gymnosporangium species showed the expression of a conserved genetic program with the top abundant cellular categories corresponding to energy, translation and signal transduction processes, indicating that this life stage is particularly active. Moreover, the survey of predicted secretomes in the three Gymnosporangium transcriptomes revealed shared and specific genes encoding carbohydrate active enzymes and secreted proteins of unknown function that could represent candidate pathogenesis effectors. A transcript encoding a hemicellulase of the glycoside hydrolase 26 family, previously identified in other rust fungi, was particularly highly expressed suggesting a general role in rust fungi. The comparison between the transcriptomes of the three Gymnosporangium spp. and selected Pucciniales species in different taxonomical families allowed to identify lineage-specific protein families that may relate to the biology of teliospores in rust fungi. Among clustered gene families, 205, 200 and 152 proteins were specifically identified in G. japonicum, G. yamadae and G. asiaticum, respectively, including candidate effectors expressed in teliospores.Functional annotation for the three Gymnosporangium spp. identified gene functions and metabolic pathways particularly expressed in teliospores, a stage of the life cycle that is mostly overlooked in rust fungi. Secreted protein encoding transcripts expressed in teliospores may reveal new candidate effectors related to pathogenesis. Although this spore stage is not involved in host plant infection but in the production of basidiospores infecting plants in the Amygdaloideae, we speculate that candidate effectors may be expressed as early as the teliospore stage for preparing further infection by basidiospores.This comprehensive comparative transcriptomics study of three  Gymnosporangium japonicum is a major threat to juniper (Juniperus chinensis L.) plantations throughout China as well as in the rest of Asia. J. chinensis is widely planted as a landscaping tree in parks, gardens and Chinese temples, also its wood of high density and decay resistance properties makes it broadly used in construction, furniture, and pulp industries [G. japonicum teliospores causes tree shape malformation and the trunk becomes gradually cracked [Asian juniper trunk rust disease caused by dustries . However cracked , 3.G. japonicum exhibits a typical complex rust life cycle with four spore types and two alternate hosts . Most Gymnosporangium are known to be heteroecious and demicyclic, and all reported telial hosts are gymnosperms, which is different from most rust fungi for which gymnosperms are aecial hosts [Gymnosporangium evidently appears as an undescribed family-level lineage rather than a sub-group of Pucciniaceae [G. japonicum, Gymnosporangium asiaticum and Gymnosporangium yamadae are the three most widespread Gymnosporangium species in Asia and the two later species are the causal agents of Japanese apple rust and Japanese pear rust diseases, respectively [J. chinensis as a telial host, but the parasitic symptoms are formed on different organs of the tree  and we determined the functional annotation of this life cycle stage with an emphasis on the secretome. We also took advantage of previously published transcriptome data in two other Gymnosporangium species at the same life cycle stage to identify lineage-specific gene families compared to other rust fungi, including candidate secreted effector proteins and carbohydrate active enzymes (CAZymes).In this study, we performed an RNA-seq analysis to characterize the transcriptome of G. japonicum teliospores were collected from infected juniper branches in the Jiangsu province (China). Telia were visible in the form of a dense fungal mass of dark orange colour breaking through the bark of the juniper branch  and nucleotide (nt) databases, protein family (Pfam), SwissProt, eukaryotic orthologous groups (KOG), gene ontology (GO), and Kyoto encyclopaedia of genes and genomes (KEGG) databases were selected for similarity searches using Basic Local Alignment Search Tools . A total of 18,872 unigenes showed similarity to at least one database and 2405 unigenes were annotated in all selected databases. The Table G. japonicum unigenes set, which is detailed hereafter. Among these 13,165 unigenes, 41 and 25.1% had a length ranging between 200 and 500 bp, or larger than 2 Kbp, respectively  and searching for transposon-related annotations in the results of the similarity search against nr. Only 75 and 138 unigenes presented similarity to repbase and transposon-related sequences in nr, respectively, indicating that transposons were marginal in the dataset.To obtain a detailed description and a functional annotation of the G. japonicum teliospores , and more than half of these unigenes encode hypothetical proteins with unknown function , two from G. yamadae  and four from G. asiaticum , were retrieved and in particular, one unigene from each species  showed a higher expression level in teliospores. Other GHs from families 16, 17 and 18 showed high expression levels in the three Gymnosporangium spp. , proteases (35 for G. yamadae and 38 for G. asiaticum) and lipases (not found in G. yamadae and 4 for G. asiaticum) using dedicated databases  [Microbotryum lychnidis-dioicae was selected as an outgroup fungal pathogen in the Microbotryomycetes, a sister class of Pucciniomycetes within the Pucciniomycotina [Gymnosporangium spp. transcriptomes and the outgroup genome in Microbotryomycetes were predicted by BUSCO [C. quercuum f. sp. fusiforme clustered with M. larici-populina whereas the Gymnosporangium species were grouped with Puccinia graminis f. sp. tritici; G. japonicum branching more closely to G. yamadae than to G. asiaticum.In order to identify lineage-specific proteins in niaceae) –30. Micrmycotina . A totalby BUSCO  and wereby BUSCO  Fig. 7)GymnosporGymnosporangium, we selected 1905 clusters, representing a total of 6010 proteins, which were only present in the three Gymnosporangium species after the MCL analysis  analysis of predicted proteomes of the seven selected fungal species . Then, tfamilies , 35. To G. japonicum teliospores and take advantage of the previously published transcriptomes of G. yamadae and G. asiaticum teliospores to perform comparison within this genus [G. japonicum, G. yamadae, and G. asiaticum are the three most widespread Gymnosporangium species in Asian areas [J. chinensis as their telial host but they cause rust symptoms on different organs of juniper trees, which indicate major differences in their infection modes [Gymnosporangium transcriptomes with predicted genes in the genomes of C. quercuum f. sp. fusiforme, M. larici-populina and P. graminis f. sp. tritici [Gymnosporangium spp. may exhibit different gene expression patterns and metabolic processes at the teliospore stage on their unique telial host (gymnosperm) than heteroecious rust species [Gymnosporangium species are not overwintering structures, they are produced transiently in spring under favourable conditions and they lead to the production of basidia then basidiospores within a few days [Gymnosporangium have the capacity to infect plants of the subfamily Amygdaloideae, on which important damages can occur. It is crucial to understand the biology of this transient and unique life stage that differ from the corresponding one in macrocyclic rust fungi. So far, our understanding of the molecular processes at play in Gymnosporangium teliospores is still limited.In this study, we report the transcriptome profiling of is genus . G. japoan areas , 36. Theon modes , 6. In o tritici –30. Gymn species , 4. So f species , 21, 22. species . At the few days . BasidioGymnosporangium genes are annotated as proteins of unknown function with no homology in databases, indicating that teliospores undergo largely unknown biological processes. The distribution of unigenes annotated in KOG categories in the three Gymnosporangium teliospores transcriptomes suggests the expression of a conserved genetic program, despite the fact that each Gymnosporangium infect a different organ of J. chinensis. The most abundant cellular categories corresponded to translation, post-translation, signal transduction and energy production which supports that Gymnosporangium teliospores are highly active in spring, likely to prepare for the production of basidiospores and later infection of the aecial host. Plant pathogens like rust fungi secrete effector molecules inside cells of their host plants to manipulate immunity and physiology [Gymnosporangium species, this category is also one of the most represented in expressed unigenes. Teliospores are not infecting living host tissues, thus the presence of a large number of expressed SPs maybe puzzling. However, teliospores lead to the production of basidia and basidiospores within only a few days in favourable conditions [Rust fungi possess large numbers of genes and a majority have no known function, illustrating the need to better describe rust biology through global approaches such as transcriptomics . This isysiology . Secretoysiology –41. Unigysiology . With a nditions , 37 and G. japonicum revealed a hemicellulose cleaving enzyme of the GH26 family and members of the GH16, 17, 18 families. Previous studies showed that the GH26 gene family is expanded in the genomes of M. larici-populina and P. graminis f. sp. tritici [Gymnosporangium species with high expression levels. GH26 genes were previously shown to be highly expressed as well in M. larici-populina telia, urediniospores and during poplar infection [C. ribicola [P. triticina [M. larici-populina and P. triticina teliospores, like multicopper oxidase encoding genes are represented among highly expressed genes of Gymnosporangium teliospores [Gymnosporangium species, indicating that the capacity to infect different organs of J. chinensis may not be reflected in the fungal equipment for plant cell wall decomposition, but rather in the expression or coordination of expression of genes encoding enzymes falling in these categories. Also, it cannot be ruled out that organ specificity may be driven by specific candidate SP effectors, rather than specific enzymatic capacity. Such organ-specific effectors were indeed reported in other fungal plant pathogens [Functional annotation of top highly expressed genes in  tritici . Unigenenfection , 28, 43.ribicola  and highriticina . GH26 isiospores , 21. Altathogens .Gymnosporangium teliospore maturation and basidiospores formation showed that meiosis begins very shortly after karyogamy. Also, meiosis is not interrupted because teliospores of Gymnosporangium germinate without a period of dormancy [M. larici-populina teliospores are not detected in G. japonicum, neither in G. asiaticum or G. yamadae [G. japonicum teliospores. Meiosis anaphase genes are highly expressed in G. japonicum, G. asiaticum and G. yamadae, but not detected in M. larici-populina. For instance, genes encoding the meiotic cell division protein Dom34 and the checkpoint serine/threonine-protein kinase Bub1 are expressed in Gymnosporangium teliospores. Bub1 regulates chromosome segregation [In overwintering macrocyclic rust fungi, ultrastructural microscopy observations showed that meiosis is blocked in prophase I at the diplonema stage and restart after germination in spring, consistent with expression profiling of karyogamy and meiosis-related genes , 12. Ultdormancy . Some ea yamadae  which im yamadae  are founregation  and Dom3regation . These oGymnosporangium group is not confamilial with other members of Pucciniaceae as previous thought and more likely represents a family-level lineage [Gymnosporangium spp. transcriptomes with the genomes of three rust species selected in different taxonomical families of the Pucciniales using predicted proteomes shows that Gymnosporangium stands on an independent branch with more proximity to Pucciniaceae than any other rust families. This comparison also confirms that G. japonicum is closer to G. yamadae than G. asiaticum, consistent with previous phylogenetic findings [C. quercuum f. sp. fusiforme, M. larici-populina, P. graminis f. sp. tritici [Gymnosporangium genus or specific to each Gymnosporangium species. It is tempting to speculate that some of these genes may be related to the telial host that is common to the three species. Future comparison to Gymnosporangium species infecting other gymnosperms will be needed to test this hypothesis and validate host-specific genes. Among highlighted specific genes, SPs represent candidate effectors that may play a role in infection of the aecial host by basidiospores. Identification of effectors is important to support progress in resistance deployment in host plants [G. yamadae causes major damages [Gymnosporangium spp. helps to narrow down the list of specific candidates and will support future effector studies to better understand the biology of infection as demonstrated for other rust species [G. japonicum and its comparison to other Gymnosporangium species bring new valuable information to gain insights into the biology of this genus of rust fungi poorly explored at the molecular level, as well as for the biology of Pucciniales in general since it focuses on a spore stage that is so far overlooked. Rust genomes are large and highly repetitive, and can be of several hundred megabases in size [Gymnosporangium confusum has been estimated at 893 Mb [Gymnosporangium species is not yet determined but might be substantially large and may require the use of single molecule sequencing technologies, such as PacBio or Oxford Nanopore, to achieve complete sequencing [Gymnosporangium genomics and the next stage will be the sequencing of a representative genome to support progress in understanding pathogenesis of these fungi.Recent phylogenetic analysis revealed that the  lineage . Phylogefindings , 47, 48. tritici –30 revea tritici ; this st tritici , 22, thi damages . Discrim species –52. The t 893 Mb . The genG. japonicum teliospores collected from juniper tree and we conducted a comparative transcriptomic analysis with other Gymnosporangium spp. at the same stage of the life cycle. This stage is mostly overlooked in rust fungi and the complementary information provided here suggests that it is comparable across Gymnosporangium species in term of expressed cellular categories, likely to prepare for production of the next spore stage in the life cycle. Differences in the expression profiles of karyogamy and meiosis related genes in teliospores of Gymnosporangium species and in overwintering macrocyclic rust fungi likely highlight the different timing of related biological processes in these relative species. Comparison of Gymnosporangium transcriptomes to other Pucciniales fungi unravelled lineage-specific protein families, including Gymnosporangium spp. specific candidate secreted effectors expressed in teliospores, that remain to be functionally addressed.We presented the transcriptome analysis of J. chinensis L.) infected by G. japonicum were collected from the Yat-sen Mausoleum, Nanjing, Jiangsu, China, in March 2016. Telia are visible under the form of dark brown teliospore masses under the bark  were constructed with specific 6-bp nucleotide bar-coding tags, using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina  following the manufacturer’s protocols. Tagged cDNA libraries were pooled in equal ratios and used for 100-bp PE sequencing on the Illumina HiSeq2000 platform  at Novogene Bioinformatics Technology Co., Ltd. . Raw reads from fastq files were firstly processed through in-house perl scripts at Novogene Bioinformatics Technology Co. In this step, clean reads were obtained by removing adapters, poly-N, and low-quality reads  and was subjected to a DNase treatment with the DNA-free™ DNA Removal Kit  according to the furnisher’s instructions. The teliospores were ground to powder with sterile mortar and pestle in liquid nitrogen. RNA was stored in DEPC-treated water. RNA quality and quantity were determined with a NanoPhotometer® spectrophotometer  and a Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer . RNA integrity (RIN) was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . Three cDNA libraries of software ;) from tsoftware  with minsoftware  for eachsoftware .In order to determine the functional categories of unigenes, homology searches by BLASTX were performed against public databases , including NCBI nr and nt, KOG, KEGG pathways orthology and, Swissprot and Pfam databases. During assignment to KOG category, we added a KOG category termed “Secreted proteins” that consists of secreted proteins of unknown function as described in .G. japonicum was determined from the proteome using a custom pipeline combining different bioinformatic programs, including SignalP v.4, WolfPSort,Tmhmm, TargetP and PS-Scan algorithms as reported by [G. japonicum (this study) and from G. yamadae and G. asiaticum (previously published by [The secretome of orted by . The preorted by . In ordeished by ), the saished by . For theished by  and the ished by .https://genome.jgi.doe.gov/mycocosm/home; [Cronartium quercuum f. sp. fusiforme G11 version 1.0 [Melampsora larici-populina v2 [Puccinia graminis f. sp. tritici CEL 75–36–700-3 race SCCL v2 [Microbotryum lychnidis-dioicae p1A1 Lamole [Gymnosporangium species : Cronarsion 1.0 , Melampsulina v2 , Puccini SCCL v2 . Microbo1 Lamole . The thrcum from ) were uscum from  using inGymnosporangium transcriptome assemblies and the three selected Pucciniales genomes and in M. lychnidis-dioicae , in order to detect the single copy genes from each assembly. The amino acid sequence of families found in all species were aligned using MAFFT and the alignments was then used to construct a phylogenomic tree using RAxML with 1000 bootstraps and the PROTGAM-MAAUTO substitution model [M. lychnidis-dioicae and visualized in Geneious and labels were manually placed to improve legibility.The program Benchmarking Universal Single-Copy Orthologs  was indon model . The resAdditional file 1: Figure S1. General read sequences and mapping information (top table); transcripts, unigenes and full-length CDS description (bottom table); and unigene length distribution (bottom graph) obtained for the G. japonicum teliospores transcriptome. The same information for the species G. asiaticum and G. yamadae were extracted from [G. japonicum to allow for direct metrics comparison. (PDF 345 kb)ted from  and addeAdditional file 2: Supplementary Data 1. Fasta file containing 40,583 G. japonicum transcript sequences. (FASTA 45632 kb)Additional file 3: Supplementary Data 2. Fasta file containing 30,243 G. japonicum unigene sequences. (FASTA 24636 kb)Additional file 4: Table S1. FPKM values and annotation results for each assembled G. japonicum unigene. (XLSX 7706 kb)Additional file 5: Figure S2. Density distribution profile of FPKM values in the three G. japonicum replicate libraries . (PDF 70 kb)Additional file 6: Table S2. KOG and KEGG annotation and classification of G. japonicum unigenes. (XLSX 93 kb)Additional file 7: Figure S3. Distribution of FPKM values for G. japonicum unigenes. The numbers of unigenes more expressed than each Log10(FPKM) level are detailed. Log10(FPKM) are used as arbitrary separations of highly, moderately and less expressed gene categories. (PDF 5281 kb)Additional file 8: Table S3. CAZymes annotation results for Gymnosporangium unigenes in the dbCAN v2.0. AA, CAZyme with auxiliary activities; CBM, carbohydrate binding module; CE, carbohydrate esterase; GH, glycosyl hydrolase; GT, glycosyl transferase; PL, polysaccharide lyase. FPKM and Log10FPKM values are reported along CAZyme annotation. (XLSX 42 kb)Additional file 9: Figure S4. Distribution of G. japonicum, G. yamadae and G. asiaticum predicted proteins in CAZymes families in dbCAN v2.0. (PDF 27 kb)Additional file 10: Table S4. Reciprocal best Blastp hits results between Gymnosporangium spp. predicted proteomes. GYA, G. asiaticum; GYJ, G. japonicum; GYY, G. yamadae. (XLSX 627 kb)Additional file 11: Table S5. Secreted proteins predicted in G. japonicum, G. yamadae and G. asiaticum. CPL stands for CAZymes, proteases and lipases homology prediction; SP is for swissprot blastp homology search; perc C for the percentage of cysteine residues in the amino acid sequence and NLS stands for Nuclear Localisation Signal prediction. (XLSX 261 kb)Additional file 12: Table S6.Gymnosporangium spp. specific clusters identified by Markov Cluster Algorithm (MCL). Cluster IDs are provided and secretion prediction is indicated. Gymas/GYA, G. asiaticum; Gymja/GYJ, G. japonicum; Gymya/GYY, G. yamadae. (XLSX 276 kb)Additional file 13: Table S7. Clusters of secreted proteins for the three Gymnosporangium species. Cluster ID from the MCL analysis; prot nb, number of proteins in the given cluster; SP nb, number of predicted secreted proteins in the cluster; and detailed protein ID for secreted proteins. Novel secreted proteins with no hit in the NCBI nr database are indicated. (XLSX 13 kb)


Euscaphis konishii. The results show that endocarp reddening is closely related to anthocyanin accumulation, and a total of 86,120 unigenes were assembled, with a mean length of 893 bp (N50 length of 1642 bp). We identified a large number of differentially expressed genes associated with endocarp coloration, including anthocyanin biosynthesis, carotenoid biosynthesis, and chlorophyll breakdown. The genes participating in each step of the anthocyanin biosynthesis were found in the transcriptome dataset, but a few genes were found in the carotenoid biosynthesis and chlorophyll breakdown. In addition, the candidate R2R3-MYB transcription factors and candidate glutathione S-transferase transport genes, which likely regulate the anthocyanin biosynthesis, were identified. This study offers a platform for E. konishii functional genomic research and provides a reference for revealing the regulatory mechanisms of endocarp reddening.Flower and fruit colors are of vital importance to the ecology and economic market value of plants. The mechanisms of flower and fruit coloration have been well studied, especially among ornamental flower plants and cultivated fruits. As people pay more attention to exocarp coloration, the endocarp coloration in some species has often been ignored. Here, we report on the molecular mechanism of endocarp coloration in three development stages of  Ipomoea purpurea), which is the model plant for studying the genetic basis of floricultural traits [Dendranthema morifolium) [Rosa rugosa) [Malus domestica) [Vitis vinifera) [Fragaria × ananassa) [Euscaphis konishii) that belongs to the Staphyleaceae family, which is distributed across Southeast China, Japan, and Korea [E. konishii is closely associated with the capsule craze and seed dispersal. Sequencing the transcriptome of E. konishii pericarp provides a method to reveal the molecular mechanisms of endocarp reddening, and lays the foundation for explaining the capsule craze and seed dispersal mechanisms.In most angiosperms, the flower and fruit colors are not only of vital importance in plant ecology and their ability to attract pollinators, such as insects and birds, and seed-dispersing organisms, but their color is also a crucial trait for both commercial and ornamental value ,2. Flowel traits ,4,5; chrifolium) ,7; and r rugosa) . The frumestica) , grape (inifera) , and strnanassa) , for whinanassa) ,13, so snd Korea , that haE. konishii endocarp pigment content during fruit development and maturation has been studied, and the results show that the endocarp color change from green to red is closely related to the anthocyanin accumulation [E. konishii endocarp is lacking. Anthocyanins, a class of flavonoids, are responsible for red, blue, and black plant pigments. Their biosynthetic pathway is well understood, and most of the genes encoding enzymes in this pathway have been isolated and characterized from many plants [Plant pigments are the key factor affecting fruit coloration, including anthocyanin, carotenoid, and chlorophyll. During fruit development, chlorophyll degradation is accompanied by anthocyanin accumulation or carotenoid retention . Althougmulation ; a compry plants . To datey plants . At leasy plants ,23.E. konishii.In general, chlorophyll degradation is obvious during fruit ripening, and fruit coloration is closely related to the content and proportion of chlorophyll . High coE. konishii pericarp in three development stages, and 86,120 unigenes were assembled for the identification of genes corresponding to the pigment metabolic pathways, including chlorophyll degradation, carotenoid biosynthesis, and anthocyanin biosynthesis. This dataset serves as a platform from which to study the regulatory mechanisms of E. konishii endocarp reddening.In the present study, we sequenced the transcriptome of the E. konishii may be closely related to the accumulation of the anthocyanin content. During the pericarp development, the total anthocyanin content increased sharply, but the chlorophyll and carotenoid degradation was accompanied by anthocyanin synthesis  , but considerably longer than the 737 bp for the Litchi chinensis pericarp transcriptome [E. konishii pericarp were usable in this study.The library produced 68.78 G of clean data and the average clean data in the nine samples were 6.78 G. RNA-Seq data were used for the statistical analysis so as to ensure the validity of the transcriptome results. In this study, high-quality libraries with mapping rates higher than 79.74% and Q30 values higher than 92.70% were constructed using the pericarp of loration . A de no1642 bp) , which icriptome  and 508 criptome . These rftp://ftp.ncbi.nih.gov/blast/db/), Pfam (http://pfam.xfam.org/), GO (http://www.geneontology.org/), KEGG (http://www.genome.jp/kegg/), Swiss-Prot (http://www.uniprot.org/), KOG (ftp://ftp.ncbi.nih.gov/pub/KOG/), eggNOG (http://eggnogdb.embl.de/), and COG (ftp://ftp.ncbi.nih.gov/pub/COG/)) using BLAST comparisons  and HMMER comparisons . The results showed that 39,658 unigenes were significantly similar to known proteins in publicly available databases . Among them, 2175 DEGs between green vs. turning, 3935 DEGs between green vs. red, and 936 DEGs between turning vs. red were detected. In order to identify the related genes that are involved in the regulation of endocarp color, the up- and down-regulated genes between the three stages were further analyzed, and the results showed that the down-regulated genes were more abundant than the up-regulated genes in th special DEGs or co-DEGs of the differential stages .Among the 4804 DEGs selected to predict functions by gene ontology (GO) annotation, at least one GO term was assigned to the biological processes, cellular components, and molecular functions categories for the 1129 DEGs between green vs. turning, 2107 DEGs between green vs. red, and 543 DEGs between turning vs. red. The three main categories were further classified into 51 functional groups, including 20 biological processes, 16 cellular components, and 15 molecular functions .E. konishii. Four distinct gene expression patterns were enriched , and selecting the default parameters to predict the transcriptional factors of 4804 DEGs, , two CHI (c69442.graph_c0 and c72737.graph_c0), two F3H (c64532.graph_c1 and c69338.graph_c3), two ANS (c60763.graph_c0 and c73249.graph_c0), and seven UFGT . We also identified one up-regulated flavonol synthase gene , which may be associated with the DFR genes for down-regulation.Twenty- three predicted genes encoding enzymes, including six major enzymes—chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (ANS), and UDP (Uridine diphosphate)-flavonoid glucosyltransferase (UFGT)—involved in the anthocyanin synthesis were identified . With thGST , one MATE (c68306.graph_c2), and two ABC (c64922.graph_c0 and c63386.graph_c0) were up-regulated in the expression during the fruit development stage. Therefore, these up-regulated genes  were candidate key genes for anthocyanin transport.The anthocyanin transport was regulated by a series of genes, and 23 predicted anthocyanin transport genes were identified , includiWe also identified a large number of transcription factors (TF) genes showing a significantly differential expression in the different development stages. A majority of up-regulated or up first and then down TFs, including MYB, hHLH, WD40, WRKY, NAC, ERF, and zinc finger, may regulate anthocyanin biosynthesis . There wArabidopsis thaliana R2R3-MYB TF family members and candidate E. konishii MYB family members , with the function of controlling the flavonol biosynthesis in all tissues [c58440.graph_c0 and c66827.graph_c2 genes. Four homologous A. thaliana genes , which regulate the anthocyanin biosynthesis in vegetative tissues [c72761.graph_c1. AtMYB123, which controls the biosynthesis of proanthocyanidins in the seed coat of Arabidopsis, was clustered together with c61353.graph_c2 [A phylogenetic tree was constructed using 126  members . In this tissues , were cl tissues , were clgraph_c2 .E. konishii, and were used to construct a protein interaction network by using the String Online Tools (https://string-db.org/). Then, we constructed a co-expression network by using Cytoscape. The results show that a total of 204 genes were determined between 235 genes, and the study identified three modules from the network , CHI , F3H (flavanone-3-hydroxylase), FLS (flavonol synthase), ANS (leucoanthocyanidin dioxygenase), and UFGT (UDP (Uridine diphosphate)-flavonoid glucosyltransferase)) involved in the anthocyanin biosynthesis closely interacted with each other. One candidate transport gene  and two transcription factors  strongly interacted with the structure of the genes. The down-regulated DFR (c57877.graph_c0) interacted with two down-regulated MYB genes (c51686.graph_c0 and c63076.graph_c0).A total of 186 transcription factor genes (TFs), 26 synthesis genes, and 23 transport genes may regulate anthocyanin biosynthesis , c64566.graph_c0; one BOH (beta-carotene hydroxylase), c49043.graph_c0; one ZEP (zeaxanthin epoxidase), c68245.graph_c1; and two NCEDs (9-cis-epoxycarotenoid dioxygenase), c64983.graph_c0 and c65617.graph_c0), and one up-regulated gene , related to carotenoid biosynthesis, were identified from the DEG of the E. konishii pericarp transcriptome database, which was far less than the anthocyanin biosynthesis.Carotenoids are essential components of photosystems that confer a yellow to red coloration to flowers and fruits. In this study, the results showed that only six candidate genes , includiE. konishii fruit ripening, which results from rapid chlorophyll degradation. In this study, five candidates that differentially expressed the genes involved in the chlorophyll breakdown were identified from the E. konishii pericarp transcriptome (CLH (c48268.graph_c0) decreased sharply from the green to the red stage, two CLH (c56088.graph_c0 and c69667.graph_c2) were up-regulated genes with low expression in the green stage, but had a high expression in the red stage, and one CLH (c66184.graph_c4) decreased sharply in the turning stage, but was high in the red stage. The expression of unigenes c70181.graph_c0 (MCS) increased in the turning stage, but decreased in the red stage.Degreening is obvious during the criptome , includiIn order to verify the reliability of the RNA-Seq data, nine candidates with differentially expressed transcripts involved in anthocyanin biosynthesis were selected for real-time quantitative polymerase chain reactions (RT-qPCRs) . The RT-Arabidopsis can been divided into “early” genes  and “late” genes  [Arabidopsis are activated by coactivator-independent R2R3-MYB transcription factors and the MBW (MYB-bHLH-WD40) complex, respectively [E. konishii  were strongly related to anthocyanin accumulation  would be regulated by the R2R3-MYB-type gene controlling flavonol biosynthesis (c66827.graph_c2)  were highly homologous with the MYB tanscription factor (c72761.graph_c1) in E. konishii  ,34. The and ANR) ,35. The ectively ,32,34. Tkonishii . The genynthesis . With thmulation , which mraph_c2) , to turnkonishii , and thekonishii . These rGST) strongly interacted with the structure of genes. These results suggest that c48398.graph-c0 (GST) may play a positive role in anthocyanin transport, and may lead to an increase in the vacuolar anthocyanin levels in E. konishii. In addition, two major transporter families (MATE and ABC-C) is closely related to the anthocyanin transport in some plants, such as TT12 from Arabidopsis [Vitis vinifera) [MATE (c68306.graph_c2) and two ABC (c64922.graph_c0 and c63386.graph_c0) were the up-regulated genes for fruit coloration. Thus, the MATE and ABC transport genes may be involved in anthocyanin transport.In cells, the anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum and are then transported and finally accumulated inside the vacuole . In the bidopsis  and grapinifera) . In the E. konishii. We also identified six genes in the carotenoid biosynthesis pathway by using transcriptome data  gene in the E. konishii transcriptome database , with three replicates (The pericarps of plicates . All of v/v mixture of 95% ethanol and 1.5 N HCl) was added, and the sample was extracted for 40 min at 40 °C under a power of 300 W. After filtering, the supernatants were measured for the total anthocyanin content using a pH differential method [The ultrasonic extraction method was used to extract anthocyanin. Approximately 0.5 g of pericarp was collected and quickly ground into powder in liquid nitrogen, before 12 mL of an extract solution  and the RNA concentration was measured using Qubit® RNA Assay Kit in a Qubit® 2.0 fluorometer . The RNA integrity was assessed using the RNA Nano 6000 Assay Kit, part of the Agilent Bioanalyzer 2100 system . Firstly, the strand cDNA synthesis was performed using a Revert Aid First Strand cDNA Synthesis Kit , according to the manufacturer’s instructions, and was stored at –80 °C for RT-qPCR assays.The total RNA was extracted and purified using an RNAprep Pure Plant Kit , according to the manufacturer’s instructions. The RNA quality was checked using a NanoPhotometer® Ultra™ RNA Library Prep Kit for Illumina® , following the manufacturer’s recommendations, and index codes were added to attribute the sequences to each sample. The library generation involved five steps, as follows: firstly, the mRNA was purified and fragmented; secondly, the double-stranded cDNA was synthesized using the fragmented mRNA; thirdly, the sticky ends of the short fragments were repaired with end repair reagents to avoid self-connection; fourthly, the sequencing adaptors were added to the cDNA fragments that were then enriched by PCR amplification; and finally, the quality control of the constructed libraries was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS , according to the manufacturer’s instructions. After the cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000  platform and paired-end reads were generated.The sequencing libraries were generated using NEBNextftp://ftp.ncbi.nih.gov/blast/db/), Pfam , KOG/COG/eggNOG , Swiss-Prot , KEGG , and GO .In order to ensure the accuracy and reliability of the RNA-sequencing data, some of the poor-quality reads were eliminated from the raw reads and only the remaining high-quality reads (clean reads) were used for statistical analysis. The level of gene expression was determined according to the number of fragments per kilobase of exon per million fragments mapped. The genes with a false discovery rate below 0.01 and an absolute value of the log2 (fold change) ≥2 were defined as DEGs. The functional annotation information for these DEGs were obtained using the following databases: NR , according to the manufacturer’s instructions. UBC23  and the neighbor joining method with 1000 bootstrap replicates . https://string-db.org/). Then, we constructed a co-expression network using Cytoscape  [The network was used to construct a protein interaction network by using the String Online Tools  .


Intracranial hemorrhage (ICH) is a catastrophic complication in patients with acute myeloid leukemia (AML). AML cells, especially in the acute promyelocytic leukemia subtype, may release microparticles (MPs), tissue factor (TF), and cancer procoagulant (CP) to promote coagulopathy. Hyperfibrinolysis is also triggered via release of annexin II, t-PA, u-PA, and u-PAR. Various inflammatory cytokines from cancer cells, such as IL-1β and TNF-α, activate endothelial cells and promote leukostasis. This condition may increase the ICH risk and lead to poor clinical outcomes. Here, we present a case under a unique situation with acute ICH detected prior to the diagnosis of AML. The patient initially presented with two episodes of syncope. Rapidly progressive ICH was noted in follow-up computed tomography (CT) scans. Therefore, we highlight that AML should be among the differential diagnoses of the etiologies of ICH. Early diagnosis and timely intervention are very important for AML patients. Intracranial hemorrhage (ICH) is an uncommon complication in patients with acute myeloid leukemia (AML), and coincides with poor clinical outcome. The mortality rate of AML patients developing acute ICH is high and death can occur within days if not diagnosed and treated appropriately. In a study by Owattanapanich et al. , 4.29% .X-ray revealed no significant pulmonary nodules or pneumonia patch. An influenza A + B rapid screening test was performed, which showed negative results. A brain CT revealed a 13 mm lesion with hyperdensity in the left temporal region, with suspected intracerebral hemorrhage  score was E4V5M6 and bilateral pupil size was 2 mm with light reflex. There was no horizontal or vertical nystagmus. The neck was supple with no limited range of motion. The bilateral breath sound was clear without wheezing or crackle, and tachycardia was noted. There was no Babinski sign, decreased muscle power, or unsteady gait. The chest morrhage . Laboratmorrhage .Results from the bone marrow biopsy report showed 90% cellularity. Blasts accounted for more than 90% of all nucleated cells. Hypercellular and monotonous bone marrow was noted with undifferentiated myeloblasts with prominent, convoluted nuclei, and agranular cytoplasm. There were significantly decreased erythroid and megakaryocyte lineages. The immunohistochemical profile was as follows: CD34(+), CD117(+), MPO(+), CD33(+), CD68(–), hemoglobin A(–), Factor VIII(–), CD19(–), CD3(–), TdT(–), and PAX5(–). The peroxidase and alpha naphthyl acetate esterase (ANAE) test was positive and chloroacetate esterase (CAE) test was negative. Acute myeloid leukemia was diagnosed. Broad-spectrum antibiotic and adequate hydration were administered. An antineoplastic agent, hydroxyurea (15 mg/kg/day), and emergency leukocytapheresis were used to control disease progression. Unfortunately, the patient became drowsy with asymmetric pupil size and no light reflex. The follow-up brain CT showed multifocal intracranial hemorrhage in the bilateral cerebral hemispheres with midline shift involving the brain stem . Progres9/L) was reported. In those populations, there was a significantly higher risk of death from rapidly developed fatal ICH. In the study by Chen et al. [In the AML population, intracranial-hemorrhage-induced syncope at initial presentation is an uncommon but fatal condition. The study by Balmages et al.  showed an et al. , a totaln et al. ,6. The hn et al. . Hyperlen et al. . The mecn et al. ,11. The In the study by Dixit et al. , DIC wasp = 0.011), lower hemoglobin (p = 0.004), and blast crisis (p = 0.004) presented with highly probable leukostasis with a high score. A subgroup was given early leukapheresis treatment which showed a benefit (based on a grading score) in preventing leukostasis-related early death. In our case, hyperleukocytosis with thrombocytopenia and blast crisis was noted. Leukapheresis was performed but the treatment was not very effective. Persistent leukostasis and DIC promoted the progression of ICH with multiple focal hemorrhages in day 2 CT scans.Leukapheresis in AML patients with hyperleukocytosis rapidly removes excessive leukocytes by mechanical separation to control complications. One round of leukapheresis could reduce the WBC count by about 10–70% . The effIn this article, we present the rare and unusual case of a patient with AML-induced ICH initially presenting with syncope. In the AML population, DIC and leukostasis play a critical role in ICH. This detailed pathophysiology may improve physicians’ development of therapeutic strategies for AML-induced ICH. Here, we highlight the clinical features and etiologies of AML-induced ICH. Early diagnosis and timely leukapheresis may prevent fatal progressive complications.


Anaplasmataceae in Senegal that infect animals and ticks in three areas: near Keur Momar Sarr (northern region), Dielmo and Diop , and in Casamance .Our study aimed to assess the diversity of the species of 12S rRNA gene. Molecular characterization of species of Anaplasmataceae infecting Senegalese ticks and animals was conducted using the 23S rRNA, 16S rRNA, rpoB and groEL genes.A total of 204 ticks and 433 blood samples were collected from ruminants, horses, donkeys and dogs. Ticks were identified morphologically and by molecular characterization targeting the Rhipicephalus evertsi evertsi (84.3%), Hyalomma rufipes (8.3%), Hyalomma impeltatum (4.9%), R. bursa (1.5%) and R. muhsamae (0.9%). The overall prevalence of Anaplasmataceae infection in ticks was 0.9%, whereas 41.1% of the sampled animals were found infected by one of the species belonging to this family. We identified the pathogen Anaplasma ovis in 55.9% of sheep, A. marginale and A. centrale in 19.4% and 8.1%, respectively, of cattle, as well as a putative new species of Anaplasmataceae. Two Anaplasma species commonly infecting ruminants were identified. Anaplasma cf. platys, closely related to A. platys was identified in 19.8% of sheep, 27.7% of goats and 22.6% of cattle, whereas a putative new species, named here provisionally “Candidatus Anaplasma africae”, was identified in 3.7% of sheep, 10.3% of goats and 8.1% of cattle. Ehrlichia canis and Anaplasma platys were identified only from dogs sampled in the Keur Momar Sarr area. Ehrlichia canis was identified in 18.8% of dogs and two R. e. evertsi ticks removed from the same sheep. Anaplasma platys was identified in 15.6% of dogs. Neither of the dogs sampled from Casamance region nor the horses and donkeys sampled from Keur Momar Sarr area were found infected by an Anaplasmataceae species.Ticks were identified as Anaplasmataceae species that infect animals and ticks in three areas from the northern, central and southern regions of Senegal. To our knowledge, our findings demonstrate for the first time the presence of multiple Anaplasmataceae species that infect ticks and domestic animals in Senegal. We recorded two potentially new species commonly infecting ruminants named here provisionally as Anaplasma cf. platys and “Candidatus Anaplasma africae”. However, E. canis was the only species identified and amplified from ticks. None of the other Anaplasmataceae species identified in animals were identified in the tick species collected from animals.This study presents a summary of  Anaplasmataceae contains the zoonotic intracellular alpha-proteobacteria of the genera Anaplasma, Ehrlichia, Neoehrlichia and Neorickettsia [Anaplasma, Ehrlichia and Neoehrlichia) whereas Neorickettsia are intracellular endosymbionts of a diverse group of the Digenea (Platyhelminthes: Trematoda) [Anaplasma and Ehrlichia species occurs transtadially but not transovarially; therefore, every tick generation must obtain infection by feeding on reservoir hosts [Anaplasma and Ehrlichia are able to cause a persistent infection in the vertebrate hosts, which allows them to be reservoirs of the infection [Anaplasma and Ehrlichia depend on the susceptibility of the infected vertebrate hosts and the availability and abundance of ixodid tick vectors largely interconnected in an epidemiological network [Anaplasma or Ehrlichia can cause death in animals due to co-infection by Staphylococcus aureus or Mannheimia/Bibersteinia, “pasteurellosis” and other opportunistic diseases [Anaplasma and Ehrlichia. For example, the American Anaplasma phagocytophilum strain human-active (Ap-ha) and variant 1 (Ap-v1) [A. phagocytophilum strains are pathogens for cattle, sheep, goats, dogs and cats [A. marginale is a worldwide reported infection. It results in the development of mild to severe anemia [Anaplasma marginale and Babesia spp. together are responsible for economic losses reaching 22 and 57 million USD in Australia and India, respectively [Anaplasma ovis is a neglected agent of sheep and goat anaplasmosis due to the usually subclinical course of the disease [Anaplasma spp. [A member of the order Rickettsiales, the family ckettsia . These vematoda) . In tickir hosts . Anaplasnfection , 5. The  network , 7. The diseases . Animals (Ap-v1)  seem to  (Ap-v1) . Howeverand cats . Bovine e anemia . Anaplasectively , 13. Ana disease . Researcsma spp. , 16.Anaplasmataceae infection and extent to which livestock productivity has been affected remain poorly understood. These bacteria were recorded in many countries in southern Africa but few studies have been conducted in West Africa [Anaplasmataceae infection in animals, it is necessary to include in the analyses multiple samples from different mammalian hosts and vectors.In Africa, the prevalence of t Africa . To bettAnaplasmataceae species infecting and currently circulating in and between cattle, sheep, goats, horses, donkeys, dogs and ticks in this region, to evaluate the genetic diversity of these bacteria and, finally, to carry out their phylogenetic characterization.The current reported statistics about livestock numbers, distribution and economic importance are difficult to evaluate in Senegal. Our main objective was to provide updated information about the local spread and epidemiology of infectious diseases in animals and ticks in three areas near Keur Momar Sarr , Dielmo and Diop  and Casamance . The aim of this study was to provide a detailed overview regarding the presence and the prevalence of All animals and ticks were sampled and collected in June 2013 and June 2014 from three Senegalese areas  using a commercial EZ1 DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA was extracted from 200 µl of blood from all the animal samples. Ticks were recovered from ethanol, rinsed with distilled water and dried on sterile filter paper in a laminar-flow hood. Each tick was cut in half lengthways (the blades were discarded after each tick was cut). DNA was individually extracted from one-half, and the remaining tick halves were frozen at − 80 °C for subsequent studies as previously described .12S rDNA gene. In order to investigate the presence of Anaplasmataceae in Senegalese ticks and in domestic animal blood samples, DNA samples were initially screened by a qPCR targeting the 23S rRNA gene. This qPCR determined that most bacteria belonged to the family Anaplasmataceae [23S rRNA gene, as previously described [Anaplasmataceae species in domestic animals or ticks, one or more DNA samples representative of each Anaplasmataceae species previously identified by the 23S rRNA gene were amplified using primers specifically targeting the 16S rRNA gene for the family Anaplasmataceae (828 bp), the RNA polymerase subunit beta (rpoB) gene for the genus Anaplasma (525 bp), and specific primers targeting the heat-shock protein (groEL) gene for the genus Ehrlichia (590 bp)  using the DNA sequencing Big Dye Terminator Kit  as described by the manufacturer. The obtained sequences were assembled using ChromasPro v.1.7 software . The sequences of primers were removed and the newly generated sequences were aligned with other tick or Edit v.3 . The seqin MEGA7 , with thRhipicephalus evertsi evertsi (75.5%), 14 Hyalomma rufipes (6.9%) and 9 Hyalomma impeltatum (4.4%). Twenty-seven ticks were not identified at species level. These ticks included fully-engorged and damaged female ticks (n = 25) and two ticks removed from two dogs identified at the Rhipicephalus sp. level. These ticks were subject to molecular characterization. In addition to ticks not identified morphologically, at least three samples of R. e. evertsi, Hy. impeltatum, and Hy. rufipes identified morphologically were subjected to further molecular identification. Finally, 47 ticks were used for the 12S rRNA gene amplification. Each sequence generated from each amplicon and belonging to the same species were aligned individually using CLUSTAL W; then, gaps and single-nucleotide polymorphisms (SNP) were corrected. The 25 previously unidentified engorged females were identified as follows: 18 Rhipicephalus evertsi evertsi, 1 Hy. impeltatum, 3 Hy. rufipes and 3 R. bursa. The two ticks taken from dogs were identified as R. muhsamae. From the 20 remaining ticks identified morphologically that were subjected to molecular characterization, we did not find any discordance between molecular and morphological identification. The sequences of R. e. evertsi showed 98% identity with R. e. evertsi collected in Zimbabwe (GenBank: AF150052) and Zambia (GenBank: DQ901291–DQ849229). The sequences of Hy. impeltatum showed 99% identity with Hy. impeltatum collected in Niger (GenBank: KX132904). Hyalomma rufipes showed 100% identity with the sequences of Hy. rufipes collected in France (GenBank: KX000618–KX000610) and Italy (GenBank: KC817342). The sequences of R. bursa showed 99% identity with the sequences of R. bursa collected in Italy  and Spain (GenBank: AF150053). Finally, the sequences of R. muhsamae showed 100% identity with R. muhsamae collected in Nigeria (GenBank: KC243829). The phylogenetic tree comparing the sequences of 12S rRNA gene amplified from our ticks to other sequences of the same gene available in GenBank is presented in Fig. All ticks were collected in the Keur Momar Sarr area. A total of 204 ticks were collected; of these, 64.2% 131) were male and 35.8% (73) were female. One hundred thirty-nine ticks were removed from 26 horses, 46 ticks from 15 sheep, 9 ticks from 3 cattle, 6 ticks from 2 donkeys and 2 ticks from 2 dogs  except from dogs. Hyalomma rufipes  were collected from four cattle and 13 horses. Hyalomma impeltatum  and R. bursa  were both collected from three different horses. Finally, R. muhsamae  were removed only from dogs  collected from the same sheep were found positive. In animals, 108/136 sheep (79.4%), 11/29 goats (37.9%), 38/62 cattle (61.3%) and 22/142 dogs (15.5%) were found positive. Sheep, goats and dogs positive for Anaplasmataceae in the qPCR were all from Keur Momar Sarr. For cattle, 29 positives were from the Sine Saloum in the central region of Senegal  and eight were from the Keur Momar Sarr in the northern region of Senegal .All results are summarized in Table 23S rRNA gene, all the Anaplasmataceae-positive samples identified in the qPCR were amplified and sequenced. The sequences generated from each amplicon belonging to the same species were aligned individually using CLUSTAL W; then, gaps and SNP were corrected. Ehrlichia canis was amplified from 2 R. e. evertsi and showed 99% identity with E. canis strain Jack (GenBank: NR076375). Anaplasma ovis was identified exclusively in sheep. The sequences showed 100% with A. ovis genotype KMND Niayes 14 (GenBank: KM021411). Anaplasma marginale was identified from cattle. The sequences showed 100% identity with A. marginale strain Dawn (GenBank: CP006847), Gypsy Plains (GenBank: CP006846), Florida (GenBank: NR076579) and 99% identity with A. centrale strain Israel (GenBank: NR076686). Anaplasma centrale was also identified in cattle blood samples (9.4%) and showed 100% identity with A. centrale strain Israel (GenBank: NR076686) and 99% identity with A. marginale strain Dawn (GenBank: CP006847), Gypsy Plains (GenBank: CP006846) and Florida (GenBank: NR076579). From cattle sampled in the Sine Saloum, we obtained sequences of poor quality in 11/47 cattle. BLAST analysis showed that these sequences had the same homology (98–99%) and belonged either to A. marginale or A. centrale. We removed these poor sequences from further analysis. From sheep, cattle and goats, identical sequences were obtained. They showed 99% identity with A. platys stain ChieGuy88 (GenBank: KM021414) reported from French Guiana, A. platys strain Dog Gard1 (GenBank: KM021412) reported from France and A. platys strain ChieCal05 (GenBank: KM021425) reported from New Caledonia. Finally, from sheep, cattle and goats, another potentially new species was identified. The obtained sequences of this species were identical to each other and showed 93% identity with A. marginale strain Dawn (GenBank: CP006847), Gypsy Plains (GenBank: CP006846) and Florida (GenBank: NR076579), and 92% identity with A. centrale strain Israel (GenBank: NR076686) (Table Using primers that amplify a 485-bp long fragment of the A. platys were found to infect 10/64 (15.6%) of dogs. The sequences were identical to each other and showed 99% homology with A. platys amplified from sheep, cattle and goats in the present study and 99% identity with A. platys stain ChieGuy88 (GenBank: KM021414), A. platys strain Dog Gard1 (GenBank: KM021412) and A. platys strain ChieCal05 (GenBank: KM021425). In addition, 12/64 (18.8%) of dogs were also infected with Ehrlichia canis that showed 100% with the sequences of E. canis amplified from the two R. e. evertsi in the present study and 99% identity with E. canis strain Jack (GenBank: NR076375).From dogs, Anaplasmataceae, the two R. e. evertsi that harbored E. canis and for each different identified Anaplasmataceae species amplified by the primers targeting the 23S rRNA gene, three randomly selected samples were chosen and the DNA was used to amplify a 828-bp long fragment of the Anaplasmataceae family 16S rRNA, an Anaplasma spp. 525-bp long fragment of the RNA polymerase subunit beta (rpoB) gene and an Ehrlichia spp. 590-bp long fragment of the heat-shock protein (groEL) gene. The amplification followed by sequencing result showed that A. ovis 16S rRNA sequences amplified from sheep had 99% identity with A. ovis reported worldwide, whereas the rpoB sequences showed 100% identity with A. ovis strain RhburBas11 (GenBank: KX155495) reported from France and KMND Niayes 14 (GenBank: KX155494) reported from Senegal. Anaplasma marginale 16S rRNA sequences amplified from cattle showed 99% identity with multiple sequences of A. marginale reported from Uganda and with A. marginale strains Dawn (GenBank: CP006847) and Gypsy Plains (GenBank: CP006846). The rpoB sequences of this A. marginale strain showed 99% identity with A. marginale strain Dawn (GenBank: CP006847), Gypsy Plains (GenBank: CP006846) and Florida (GenBank: CP001079). The 16S rRNA sequences of A. centrale showed 99% of identity with A. centrale, A. ovis and A. marginale reported worldwide. The rpoB gene sequence of A. centrale has 99% identity with A. centrale strain Israel (GenBank: CP001759) and 88% identity with A. marginale strain Dawn (GenBank: CP006847). The16S rRNA and rpoB sequences of A. platys amplified from ruminant were amplified from three samples each taken from sheep, goats and cattle. For each gene, the sequences were identical to each other and showed for the 16S rRNA 99% identity with multiple uncultured Anaplasma sp., A. platys (GenBank: KY114935-EF139459), A. platys isolate A.pl. #87 (GenBank: JQ396431), “Candidatus Anaplasma camelii” clone Camel_38 (GenBank: KF843827) and 99% identity with A. phagocytophilum reported worldwide. The rpoB sequences showed 93% identity with A. platys strain Dog Gard1 (GenBank: KX155493) and 89% with A. phagocytophilum strain Norway variant2 (GenBank: CP015376). Sequence analysis of the three genes used in the present study revealed that this species is distinct from A. platys . Due to the absence of additional data on this Anaplasma sp. and the genetic relatedness to A. platys, we refer to this genotype here as Anaplasma cf. platys. A phylogenetic tree based on the concatenated 23S rRNA, 16S rRNA and rpoB genes showed that Anaplasma cf. platys form a separate and well-supported (bootstrap support of 99%) branch on the phylogenetic tree belonging to the cluster of A. platys  were identical to each other and showed for the 16S rRNA 97% identity with multiple A. phagocytophilum reported worldwide, multiple A. bovis sequences reported from China and multiple sequences of uncharacterized Anaplasma spp. reported from China and Malaysia. The rpoB sequence of this species showed 79% with A. centrale strain Israel (CP001759), A. phagocytophilum strain Dog 2 (GenBank: CP006618), JM (GenBank: CP006617) and 78% with A. marginale strain Dawn (GenBank: CP006847) and Gypsy Plains (GenBank: CP006846). Sequence analysis of the concatenated 23S rRNA, 16S rRNA and rpoB genes revealed that this Anaplasma species was distinct from other Anaplasmataceae species considering the lower sequence identity . Because these potentially new species had not previously been reported, we propose the provisional name “Candidatus Anaplasma africae”. The phylogenetic tree showed that “Candidatus Anaplasma africae” form a separate and well-supported (bootstrap support of 100%) branch on the phylogenetic tree situated between Anaplasma spp. and Ehrlichia spp., albeit closer to the Anaplasma spp. clusters . The rpoB sequence showed 99% identity with A. platys strain Gard 1 (GenBank: KX155493) . The groEL sequences of this E. canis strain showed 99% with E. canis isolate D12E (GenBank: JN391407-JN391408) reported from the Philippines and E. canis strain Jake (GenBank: CP000107) reported from the USA . The data collected in the present study are insufficient to conclude or suggest an accidental introduction or for a possible establishment and/or the presence of foci of these ticks in the arid conditions of the northern region of Senegal.The clinical presentation of anaplasmosis depends on multiple factors including the ry tests . Epidemi country . Walker  country  states tortation . This sportation  and Crimortation . In the R. e. evertsi (84.3%), collected from almost all animal species except from dogs. Rhipicephalus e. evertsi is an Afrotropical species. In West Africa, R. e. evertsi is absent on wild ungulates, suggesting that the species was introduced on domestic livestock from East Africa [R. e. evertsi is present in a band extending roughly from 10–16°N and 11°W to 20°E, a region that receives between 400–1000 mm rainfall annually [Rhipicephalus e. evertsi is known in Senegal as a potential vector of Rickettsia africae, R. aeschlimannii, R. conorii and Coxiella burnetii [R. e. evertsi collected from sheep were infected with E. canis. These two ticks were removed from the same sheep. To the best of our knowledge, this is the first identification of E. canis in R. e. evertsi. In previous studies, A. platys, E. ruminantium and A. ovis were also reported from R. e. evertsi in South Africa and Ethiopia [E. canis has never been reported from sheep. Rhipicephalus e. evertsi is a two-host tick species with larvae and nymph infesting the first host and adults infesting the second host [R. e. evertsi was shown experimentally to transmit “E. ovina” to sheep by adult ticks. “Ehrlichia ovina” is a not a completely described Ehrlichia species but it is reported to infect domestic ruminants from Turkey and the Caribbean islands [16S rRNA sequences of “E. ovina” (GenBank: AF318946) showed 100% identity with multiple E. canis sequences reported worldwide [gltA sequence (GenBank: KP719095) showed 99.9 % (two mismatches) identical to that of E. canis from Italy [E. ovina” are not readily available and their taxonomic position needs to be analyzed for other genes including groEL [rpoB [E. ovina” as a species or to combine it with E. canis, as well as to confirm and consider R. e. evertsi as a competent vector for E. canis.The most abundant tick species found was t Africa . This spt Africa . In Afriburnetii , 29, 30.Ethiopia –33. Howeond host . Interes islands , 35. Theorldwide , whereasom Italy . NonetheEL [rpoB . These pHyalomma rufipes ticks were removed only from cattle and horses. This species represents 8.3% of the overall tick species collected. Hyalomma rufipes is most common in dry areas and distributed in almost all African countries [R. aeschlimannii and C. burnetii [A. marginale was found in these ticks [Hy. rufipes tested here were infected by Anaplasmataceae species.ountries . This tiountries . In Seneburnetii . In Soutse ticks , 38. NonHyalomma impeltatum was collected exclusively from horses at low occurrence (4.9%). In Senegal, Hy. impeltatum is more frequently encountered in cows than in sheep [R. aeschlimannii [Ehrlichia chaffensis in Nigeria [Ehrlichia chaffensis is an emerging bacteria in Africa, and until now, this species has been reported in three countries in Africa including Nigeria, Uganda and Cameroon [Rhipicephalus sanguineus and Hy. impeltatum seem to be potential vectors of E. chaffensis in Africa [Hy. impeltatum collected were infected by an Anaplasmataceae species. However, E. chaffensis has never been reported from humans or other animals in Senegal. Finally, only two R. muhsamae (0.9%) were collected exclusively from dogs. Rhipicephalus muhsamae is an Afrotropical species found mainly in forest savanna mosaic and less abundant in tropical and subtropical moist broadleaf forest [Rickettsia conorii has been detected in R. mushamae ticks removed from cattle in the Central African Republic [R. mushamae collected were infected by Anaplasmataceae species.in sheep . This Aflimannii  and inte Nigeria . EhrlichCameroon , 40. Rhin Africa , 41. In f forest . RickettRepublic . None ofAnaplasmataceae infection in the sampled animals was 41.1%. Anaplasma ovis was the common species found in sheep (55.9%). The infection prevalence found in the present study was higher compared to that previously reported in Senegal in 2013 (31.6%) [A. ovis in sheep [A. ovis lead to the development of an acute disease phase characterized by fever, progressive anemia, icterus, weight loss, milk yield decrease and sometimes death [A. ovis has been reported from sheep in Ethiopia, South Africa, Tunisia [Rhipicephalus e. evertsi was previously reported as an important vector of A. ovis in Africa [R. e. evertsi collected here was infected by A. ovis. Anaplasma marginale and A. centrale were identified in 19.4 and 8.1% of cattle, respectively. These two pathogens have never been reported in Senegal but have been reported from many other countries in Africa including Algeria, Angola, Botswana, Egypt, Ivory Coast, Kenya, Morocco, Mozambique, Nigeria, South Africa, Sudan, Tanzania, Tunisia, Uganda and Zambia [Rhipicephalus and Amblyomma [Anaplasma marginale is associated with mild to severe anemia. Cattle infected with this bacterium developed various clinical signs including fever, decline in milk production, temporary infertility, and some animals developed an acute disease that manifested with gastrointestinal and neurological signs associated with the development of icterus seen during early convalescence. Mortality rates can reach 50–60% in adult cattle [Anaplasma centrale is less pathogenic and has been used as a live blood vaccine to protect against bovine anaplasmosis caused by A. marginale [Four hundred thirty-three blood samples were collected from different animals including sheep, goats, cattle, horses and dogs. The overall prevalence of  (31.6%) . Sheep din sheep 5.9%. Thein sheep . Complices death . In Afri Tunisia , 44, 45  Tunisia . Rhipicen Africa , 38. Howd Zambia –54. The mblyomma . The premblyomma . Infectit cattle . Anaplasin sheep .9%. The Anaplasma cf. platys found in this study was gentically close to the dog pathogen A. platys. Many genotypes close to A. platys, commonly named Anaplasma platys-like bacteria, were reported from animal families other than dogs [Anaplasma platys-like is considered an emerging bacterium in many countries of the world. The same bacterium was reported previously from two sheep in Senegal [Anaplasma cf. platys  was reported from cattle in Algeria and Tunisia, from sheep in South Africa and goats in China [Anaplasma sp. in ruminants and platelet-tropic in cats [A. platys (nucleotide homology was 99% for rrs and rrl); however, the rpoB gene showed 93% identity with A. platys. Anaplasma cf. platys is located independently in a separate group in the phylogenetic tree based on the concatenated genes rrl, rrs and rpoB . However, the prevalence of A. platys was higher than what was reported in the same study (5.9%) [Ehrlichia canis infection in dogs induces monocytotropic ehrlichiosis and results in various signs depending on the disease stages [A. platys induces a recurrent thrombocytopenia that can be resolved in the absence of other infecting agents or complicating factors [E. canis and A. platys in Africa is poorly known; however, these two species are apparently ubiquitous throughout the African area where the R. sanguineus tick group is spread [y (5.9%) . Ehrliche stages , whereas factors , 63. Thes spread , 64.E. canis [Rhipicephalus sanguineus (s.l.) in the tropical and subtropical region are active throughout the year and apparently have no seasonality [R. mushamae ticks were collected from two dogs. To the best of our knowledge, A. platys has never been associated with R. mushamae. Ehrlichia canis has been reported once from R. mushamae in Mali [R. sanguineus (s.l.), A. platys was amplified from R. e. evertsi salivary glands in South Africa [R. camicasi in Kenya and Ivory Coast [Nonetheless, dogs from the Central African Republic and Reunion Island were all negative for E. canis . Rhipicesonality . In our  in Mali . In Afrih Africa  and R. cry Coast .Anaplasmataceae infection. In addition, none of the Anaplasmataceae species identified from ruminants and dogs were identified in ticks, except for E. canis identified in dogs and R. e. evertsi collected from sheep. Anaplasma cf. platys and “Ca. Anaplasma africae” were found in sheep, goats and cattle. Despite the fact that R. e. evertsi were collected from different ruminant animals, none of these ticks were found positive for any Anaplasmataceae species. Anaplasma phagocytophilum was also not found in the present study. Anaplasma phagocytophilum has already been identified in sheep in Senegal, although all of its known vectors are absent in sub-Saharan Africa [A. phagocytophilum in Africa has been reported from Morocco, Algeria and Tunisia [Ixodes ricinus has been identified in some foci in the northern regions of these countries [Ambyomma flavomaculatum collected from two different specimens of lizard (Varanus exanthematicus) imported to Poland from Ghana [A. cohaerens, A. lepidum and A. variegatum in Ethiopia [Hy. marginatum in Tunisia [A. phagocytophilum, suggesting the implication of other tick species in the epidemiology of A. phagocytophilum in sub-Saharan Africa.None of the 64 horses tested were found positive for an n Africa . Evidenc Tunisia , 54, 67,ountries . This inountries , 69, andom Ghana , from A.Ethiopia , 72 and  Tunisia . In our Anaplasmataceae species infecting animals and ticks and determine a possible epidemiological Anaplasmataceae-host-tick connection. The present work indicates that ruminants and dogs in the northern and central areas of Senegal are a reservoir for multiple Anaplasmataceae species. The prevalence of A. ovis and A. marginale was high in sheep and cattle, respectively. Molecular analysis revealed an interesting diversity of Anaplasmataceae infections in ruminants and dogs including a potentially new species infecting ruminants. However, except for E. canis, none of the other Anaplasma spp. found in the present study was amplified from ticks. Nevertheless, further studies are needed to ascertain the Anaplasmataceae-host-vector connections in sub-Saharan Africa as well as to decipher the zoonotic potential of newly identified genotypes and their significance for animals and public health.The present study aimed to identify


Corynebacterium ulcerans infection is emerging in humans. We conducted phylogenetic analyses of C. ulcerans and C. diptheriae, which revealed diverse diphtheria toxin in C. ulcerans. Diphtheria toxin diversification could decrease effectiveness of diphtheria toxoid vaccine and diphtheria antitoxin for preventing and treating illnesses caused by this bacterium. Corynebacterium ulcerans is a rod-shaped, aerobic, gram-positive bacterium closely related to C. diphtheria. Some strains of C. ulcerans can produce diphtheria toxin, which causes respiratory diphtheria in humans and animals. Reports of human infections with C. ulcerans have increased during the past 20 years, and C. ulcerans is a recognized emerging human pathogen . Then, we performed phylogenetic analyses by using MEGA 7.0 (https://www.megasoftware.net). A report evaluating the differences in the amino acid sequences of the diphtheria toxins in C. diphtheriae and C. ulcerans strains with some sequence variability among the strains in each species (C. diphtheriae strains were identical, but C. ulcerans strains were diverse (C. ulcerans tends to acquire mutations more frequently than C. diphtheriae. Two possible explanations for this phenomenon are that C. ulcerans is maintained by various animals, increasing its diversity compared with C. diphtheria, which is believed to infect only humans; or that C. ulcerans has a phage-independent pathway to acquire the diphtheria toxin–encoding gene, as reported (We found that the 16S rRNA gene sequences divided into separate  species , panel A diverse , panel BC. ulcerans have occurred in unvaccinated or inadequately vaccinated persons. However, a fatal case was reported in a person who received a diphtheria vaccination booster ≈10 years before disease onset (C. ulcerans diphtheria toxin gene is of note because accumulation of these gene mutations potentially could lead to decreased effectiveness of the diphtheria toxoid vaccine for prevention and diphtheria antitoxin for treatment of toxigenic C. ulcerans disease.Most severe human cases of disease caused by toxigenic