Carassius. This review places emphasis on two Carassius entities with completely different biological characteristics: one globally widespread and invasive Carassius gibelio, and the other C. carassius with a decreasing trend of natural occurrence. Comprehensive biological and cytogenetic knowledge of both entities, including the physical interactions between them, can help to balance the advantages of highly invasive and disadvantages of threatened species. For example, the benefits of a wide-ranged colonization can lead to the extinction of native species or be compensated by parasitic enemies and lead to equilibrium. This review emphasizes the comprehensive biology and cytogenetic knowledge and the importance of the Carassius genus as one of the most useful experimental vertebrate models for evolutionary biology and genetics. Secondly, the review points out that effective molecular cytogenetics should be used for the identification of various species, ploidy levels, and hybrids. The proposed investigation of these hallmark characteristics in Carassius may be applied in conservation efforts to sustain threatened populations in their native ranges. Furthermore, the review focuses on the consequences of the co-occurrence of native and non-native species and outlines future perspectives of Carassius research.Sexual vs. asexual reproduction—unisexual vs. bisexual populations—diploid vs. polyploid biotypes—genetic vs. environmental sex determination: all these natural phenomena are associated with the genus of teleost fish,  Carassius  belongs to a monophyletic group of paleotetraploids from the Cyprinini tribe  and white crucian carp, also known as “gengoro-buna” . However, despite this distinction between several species of Carassius in the literature, taxonomic classification is often inconsistent and often incorrectly referred to by authors  as being native to many European freshwaters, with no records of occurrence in Japan [Carassius cuvieri is phylogenetically distinct from C. carassius and all other Carassius species, representing a basal diverged clade [C. cuvieri include an increased number of gill rakers and endemic to C. cuvieri in Lake Biwa, Japan [Distinct morphological characteristics have emerged in ritoneum ,5. Despi” (sensu ), and an”  and C. auratus auratus , are globally widespread and invasive to parts of mainland Eurasia, where they are highly abundant [C. auratus langsdorfii Temminck and Schlegel, 1846), nigorobuna , okinbuna , nagabuna (C. auratus subsp. 1) and kinbuna (C. auratus subsp. 2) [C. auratus, C. gibelio, and C. langsdorfii as separate independent species [Other se group  with a wse group ,12, diffse group , variousse group , and disse group . Most no species ,5.Carassius, the goldfish (C. auratus), commonly persists in an array of colorful varieties and body morphologies. However, much contention surrounds the taxonomic classification of this species. Identification of C. auratus has been inconsistent for a number of reasons, with some proposing that difficulties in classification are due to: (i) the possibility of multiple independent origins [C. auratus involves either a domesticated form of the goldfish or the entire C. auratus complex including C. (auratus) gibelio, C. (auratus) auratus, C. (auratus) langsdorfii, C. (auratus) grandoculis, C. (auratus) buergeri, C. (auratus) subsp. 1, and C. (auratus) subsp. 2. It is difficult to assign distinct “species” or “subspecies” based on morphological characteristics alone [Carassius auratus complex  [Carassius species has persisted since the first identification of Cyprinus carassius and Cyprinus gibelio by Bloch, 1782. In general, Cyprinus and Carassius are recognized as two separate genera. In part of the natural history Museum für Naturkunde der Humboldt Universität zu , these two specimens were clearly morphologically indistinguishable from C. carassius. In this case, the Cyprinus gibelio type specimen was likely lost and replaced by a specimen of C. carassius [Cyprinus gibelio by Bloch represents a wholly different species or a hybrid between Cyprinus and Carassius. Because a holotype is missing in Bloch’s specimen collection, Kalous et al. [C. gibelio and concluded based on mitochondrial cytochrome b sequences that an additional taxon has likely been grouped under the species C. gibelio. This finding indicates the polyphyletic origin of C. gibelio and sparks doubt if the sister taxon still belongs to C. gibelio. Other alternative classifications have been proposed, such as C. gibelio gibelio or C. carassius gibelio [The most well-known member of  origins , (ii) ge origins , (iii) a origins , or (iv) origins . Typicalcs alone , as manyzations) . Even mozations) ,12. Incoarassius ,22, makis et al.  describe gibelio , but neiC. auratus complex includes all Carassius taxa, except for C. carassius and C. cuvieri, which will be treated as independent species. Other members of the C. auratus complex will be used under the scientific names C. auratus, C. gibelio, and C. langsdorfii. Carassius auratus will be applied only in the context of domesticated/feral goldfish.In this review, we will be keeping with the concept that the Carassius. This work led to the first description of diploidy in goldfish (C. auratus) n” refers to a number of chromosomes in each gamete of extant species, and “x” refers to the number of chromosomes in a gamete of the most recent diploid ancestor of the extant species. Descriptions of the karyotypic diversity in Carassius were further extended by Makino [C. carassius. From this work, it can be concluded that misidentified C. carassius belongs to the taxon C. auratus due to the Japanese origin of the original samples. The karyotype of C. gibelio was first recorded by Cherfas [Carassius is summarised in C. auratus, C. gibelio, C. langsdorfii, C. cuvieri, C. buergeri, and C. grandoculis. Additional information on the numbers of chromosomes and karyotype formulas for diploid C. auratus, C. carassius, and C. gibelio have previously been reviewed in Knytl and Fornaini  male [Carassius tetraploids have not been cytogenetically described. Biologically diploid Carassius biotypes are interestingly considered evolutionary paleo-tetraploids [=4x=100) ,49,50. C=8x≈200) ,45,51,52es) male ,54, or nraploids  because Carassius species to a great extent, especially at the end of the last century [Giemsa staining (G-banding) is the most widely used cytogenetic technique and is commonly used to visualize DNA segments or entire chromosomes by binding to AT-rich regions with high affinity (intense AT bands) and low affinity to GC-rich regions (negative GC bands). Comparatively, C- and CMA century ,35,48,56 century . Convent45S locus composed of 18S, 5.8S, and 28S genes, and minor 5S locus, is formed independently. The 45S locus creates nucleolar secondary constriction, which is called nucleolar organizer region (NOR) present during the M phase of the cell cycle, thus detectable using an rDNA FISH technique  5S and 28S rDNA probes for hybridization with C. carassius chromosomal DNA; Knytl et al. [Esox lucius Linnaeus, 1758) 5S rDNA probes for hybridization on C. carassius chromosomes. The number of positive 5S rDNA loci within the Carassius karyotype is not consistent because of the high number of these loci. Spoz et al. [5S rDNA loci in C. carassius. Knytl and Fornaini [5S rDNA signals in diploid C. auratus and C. gibelio. Zhu et al. [5S rDNA loci in triploid C. gibelio, Knytl et al. [5S rDNA signals in triploid Carassius. The number of intensively stained 5S rDNA loci agreed on the ploidy level in Carassius, i.e., two intensively fluorescent loci in diploids, three in triploids [Carassius biotypes [Carassius biotypes showed different numbers of NORs. Triploid C. gibelio has four [Carassius bears six [Carassius are not consistently specific for each species, but the intensity of these rDNA positive signals could serve as a specific marker for ploidy level determination and for determination of how many times the genome has been duplicated  are widely utilized in many species and may determine a tempo of karyotype evolution focused on repetitive ribosomal DNA (rDNA) loci, microsatellite motifs, ratio of parental genomes within a hybrid genome etc. ,59,60. T species . Therefo species  used thel et al.  used pikz et al. , Knytl ez et al.  and Knytz et al.  found frFornaini  revealedu et al.  revealedl et al.  detectedriploids ,67,68 anriploids . The numbiotypes ,50,67,70has four ,72, tetrears six ,72 or eiplicated .Carassius is the ability to reproduce asexually [Carassius, an embryonic development stimulated by sperm of a heterospecific sexual host gives rise to gynogenetic and clonal progeny consisting of females only; thus, unisexual populations arise [Carassius polyploid males in nature [Carassius males, or an incorporation or expression of paternal genetic material to a polyploid gynogenetic offspring with variable amounts of chromosomes [Carassius males has been up to 23% compared to the incidence of triploid females [Carassius males are common in nature with an occurrence of 1:1 for males and females within the diploid population [C. gibelio have served as valuable experimental material for artificial crosses with the aim of finding out a mechanism of allogynogenesis/paternal leakage. If conspecific  sperm from triploid C. gibelio inseminates an egg, the responding reproduction mode will be sexual with the generation of bisexual recombinant offspring. If heterospecific sperm from Cyprinus carpio activates C. gibelio egg development, the result is unisexual clonal lineage formed by gynogenesis [C. langsdorfii [C. gibelio [C. langsdorfii [C. langsdorfii retained somatic ploidy by retention of the first polar body [C. gibelio extruded the first polar body, and the somatic ploidy level was restarted using extra DNA endoreplication without cytokinesis during the first mitosis in the stimulated egg [C. auratus complex demonstrates the coexistence of a dual mode of reproduction—sexual (recombinant) and asexual (gynogenetic) with the formation of unisexual  and bisexual (both sexes) offspring [One of the other characteristics of the genus sexually . Gynogensexually . It is gsexually . In Carans arise . An excen nature . The excomosomes . Jiang eomosomes  proved aomosomes . Allogynomosomes . Signifi females , but old females . Diploidpulation . Triploiogenesis ,78. A sigsdorfii . One smagsdorfii  eggs. Anlar body  and an effspring .Carassius females produce eggs with a reduced amount of genetic information in the nucleus. The spawning partners are conspecific males that fertilize eggs (true fertilization), and the resulted offspring is recombinant [Carassius females can produce both gynogenetic and recombinant progenies dependent on heterospecific and conspecific sperm interaction, respectively [Carassius females reproduce gynogenetically [Carassius individual  and subsequent reproductive mode of the examined individual (in what mode its progeny will be produced). Therefore, determination of ploidy level in Carassius can serve as an additional marker helpful for species identification and important marker for determination of reproductive mode.In general, it is possible to conclude that diploid ombinant ,80. Tripectively . Tetrapletically . Exceptietically . TherefoCarassius individuals with different ploidy levels have been performed in order to find out variability or stability in chromosome numbers. Kalous and Knytl [C. gibelio male with 100 chromosomes and triploid female with 159 chromosomes. Resulted F1 offspring possessed 150, 151, 156, 158, and 159 chromosomes. The mode of reproduction was not studied, but there was evident paternal leakage, and European C. gibelio showed high genome plasticity and diversity in chromosome numbers. Variation in the number of chromosomes of Asian Carassius was investigated by Zhou and Gui [C. gibelio and observed the effects in the F1 generation. Progeny contained and intermediate number of chromosomes  [C. gibelio maternal origin, and the GISH technique identified 50 C. carassius chromosomes out of 206 chromosomes of hybrid Carassius female  . Mitochos female .Carassius hybrids were artificially generated and cytogenetically investigated. Carassius Hybrids with 206 and 212 chromosomes contained 156 and 162 C. gibelio chromosomes, respectively [Cyprinus carpio chromosomes  chromosomes. Megalobrama amblycephala was a parental species, and thus, Yi et al. [M. amblycephala genome to the triploid C. gibelio maternal genome. No chromosomal microdissections have been carried out on naturally occurring Carassius, and it would be a prospective challenge for better understanding paternal leakage and determining the parental species of the polyploid Carassius biotypes.Another way to distinguish maternal and paternal genome complements within hybrids is using FISH with whole chromosome painting probes. Individual chromosomes  or morphi et al.  dissectei et al.  providedCarassius biotypes almost all over the world [Carassius expansion entailed negative consequences due to successful competition, interspecies hybridization, and occupation of new areas [Carassius biotypes [Poecilia monacha and P. 2monacha-lucida, respectively. Significantly higher accumulation of parasites was found in prevalent gynogenetic P. 2monacha-lucida [Carassius biotypes. The less common sexual biotypes had higher immune reaction and resistance to parasites [Carassius biotypes might be a trustworthy instance of the Red Queen hypothesis as a driver of Carassius biodiversity, but C. gibelio is still prevalent in natural waters, and thus, the equilibrium is not yet established by the Red Queen hypothesis. We propose the current relationship between co-occurring invasive and native Carassius as the birth of the existence of the Red Queen hypothesis.The gynogenetic reproduction mode and production of all female populations without the need for coupling with conspecific males caused rapid invasivity and expansion of polyploid he world ,102,103.ew areas ,105. Theew areas . Herpesvbiotypes ,110,111.a-lucida . Signifiarasites . The coeCarassius is a typical example of both possible ways of sex determination. The genetic sex determination system is heterogametic for males (XY) and homogametic for females (XX). The XX/XY was evidenced in C. auratus using a crossbreeding experiment of hormone-treated (sex-reversed) with normal individuals followed by analysis of the progeny sex ratio [C. auratus was identified cytogenetically using the C-banding method. Intensive heterochromatic blocks were found on the short arms of the second largest submetacentric chromosome pair in a diploid female and only one of these two heterochromatic blocks has been found in a diploid male. These cytogenetic findings confirmed the XX/XY sex determination system [C. auratus male, indicating male heterogamety and a far smaller Y chromosome than X. Additionally, Zan [Carassius female. However, C-banding revealed ten additional chromosomes, and there is no justified conclusion why three C-banded chromosomes were determined as three X chromosomes. Neither C-banding differences in number and position between sexes have been found in European C. carassius [Carassius auratus complex [C. auratus Y chromosome. This sex region of the length of ∼11.7 Mb mapped on linkage group 22 involves male-specific genetic markers. Some of these markers were found in all XY males and were absent in all but one XX females/sex-reversed XX males. Even though sex-linked markers were tested on a less diverse C. auratus artificial population [C. auratus complex.Sex determination, i.e., whether an embryo develops into a male or female individual, can be governed environmentally by temperature  or genetically by a cascade of influential sex-determining genes located on sex chromosomes (genetic sex determination). Sex determination in fish is a diverse process with various mechanisms of both genetic and environmental sex determination (reviewed in ). Carassex ratio . Male hen system . Zan [28arassius  nor in J complex  revealedpulation , the resCarassius is given by the temperature of the environment in which the embryo develops [hypoxia-inducible transcription factor-1 gene, and this gene may be involved in sexual differentiation and an ability to display an extreme anoxia tolerance of C. carassius and C. auratus [C. auratus with both testes and ovaries were found in Western Siberia [Carassius hermaphrodites has probably been caused by wide-ranged oscillation of water level, salinity and temperature. The Siberian area, where C. auratus hermaphrodites have been caught, is characterized by (1) long-lasting regression of water level, (2) high variability of water salinity and (3) fluctuation of water temperature in spring (spawning time of Carassius) from 10 °C at night to 30 °C in the day time [Carassius. Temperature can likely influence hermaphroditism, and thus, the sex of Carassius can be skewed towards one favorable sex.The other mechanism of sex determination in develops ,115,116.develops . Moreove auratus . Apart f Siberia . A naturday time . No moleCarassius benefit from the establishment of new genome combinations with the potential for clonal reproduction [Carassius ancestors.Hybridization has often been viewed as a destructive force leading to hybrid sterility or non-viability, but it is increasingly being recognized as a potential creative force in evolution. Novel hybrids can benefit from rapid adaptation to new environmental conditions and the potential to exploit ecological niches where a primary reproductive barrier would have existed between hybrids and parental species ,120. Foroduction . In accooduction , novel hoduction . CytogenDiffering modes of reproduction are natural phenomena, which are hypothesized to be associated with the transition between genetic and environmental sex determination . The useC. cuvieri and C. cuvieri x C. auratus hybrids was where melanin synthesis was suppressed via the disruption of a tyrosinase gene [Carassius genes. Thus far, a single candidate master sex-determining gene has been proposed, the anti-müllerian hormone gene (amh) [Once identified, FISH can be used to map the physical location of sex-determining genes on chromosomes ,128. Furase gene . CRISPR/ase gene , and sucne (amh) . Furtherne (amh) .Carassius is a highly diverse complex involving several taxa with different mitochondrial lineages [Carassius as an experimental fish model to study the evolution of the above-listed unusual and incredible phenomena using already well-established molecular cytogenetics coupled with perspective genomics and genetic engineering.lineages , severallineages , dual molineages ,77, bothlineages , intersplineages  and hermlineages . In thisCarassius involves two diametrically distinct entities: the first one is invasive and gynogenetic C. gibelio, and the second one is vulnerable sexual C. carassius. In the wild, C. carassius is rapidly becoming an endangered species in many regions [Carassius species, such as C. gibelio, which has colonized novel ecological niches enabled by the mode of asexual reproduction, avoiding the costs associated with sexual reproduction. In order for conservation efforts to be directed to pure C. carassius lineages [Carassius (C. carassius but also to understand the evolution of the highly diverse Carassius complex.In Europe the genus  regions ,97,104, lineages , it is narassius , and all


Elderly hemodialysis patients have a higher rate of mortality than nonelderly hemodialysis patients. Recent studies shown that the serum uric acid to creatinine ratio (SUA/Scr) was associated with all-cause mortality in general adults. The purpose of the present study was to investigate the association between the SUA/Scr and all-cause and cardiovascular disease mortality among elderly hemodialysis patients.A total of 222 patients (≥ 60 years) who received hemodialysis more than 8 h per week at Taizhou Second People’s Hospital for at least 3 months were enrolled in the present study from January 2015 to December 2019. Clinical characteristics including age, sex and height et. al, were obtained from the hemodialysis database. The laboratory data, including albumin (ALB), total cholesterol (TC), serum uric acid (SUA), serum creatinine (Scr) and so on, were collected before hemodialysis and analyzed by automatic biochemical analyzer. Survival information was recorded during the follow-up period. Multiple Cox regression was carried out to analyze the association between SUA/Scr and all-cause mortality. The survival rate of each group was calculated by the Kaplan–Meier method, and the ratio of survival curves was analyzed by the log-rank test. The contribution of SUA/Scr for predicting all-cause mortality risk was evaluated by net reclassification improvement (NRI).P < 0.001), body mass index (BMI) (P = 0.004), serum creatinine (P = 0.005) and prealbumin (P = 0.006) than surviving patients. After adjusting for age, sex, BMI, prealbumin, dialysis vintage, dialysis frequency, single-pool Kt/V (spKt/V), DM, hypertension and comorbidities, a higher ratio of SUA/Scr was independently associated with a higher risk of all-cause mortality . The predict value on all-cause mortality of SUA/Scr was superior to SUA  and Scr  among elderly hemodialysis patients.During the 19-month observation period, 78 patients died. Individuals in the nonsurviving group had significantly older ages (The serum uric acid to creatinine ratio is strongly associated with all-cause mortality in elderly hemodialysis patients which is more predictive than SUA or Scr alone.The online version contains supplementary material available at 10.1186/s12882-022-02798-4. The difference between groups of normally distributed continuous variables was tested by Student’s t test. For nonnormally distributed data, the nonparametric Mann-Whitney U test was used. Chi-square  years old. During the 19-month observation period, 78 patients died.P<0.001). Furthermore, the patients who died had lower BMI (P=0.004), serum creatinine (P=0.005) and prealbumin (P=0.006). Other baseline characteristics did not show significant differences between groups. Apparently, patients in the nonsurviving group had a higher SUA/Scr than surviving patients (P=0.001).The demographic and baseline characteristics of surviving and nonsurviving patients are shown in Table P=0.002). The SUA/Scr was significantly negatively correlated with diastolic blood pressure (P=0.012), albumin (P<0.001), prealbumin (P=0.001), serum calcium (P=0.002) and serum phosphorus (P=0.037).Table P=0.011). Furthermore, we found that significant NRI was achieved when compared SUA/Scr with Scr  and SUA separately .Patients were divided into four groups according to SUA/Scr quartile. The Kaplan-Meier curve was used to compare the accumulated mortality among the four groups. As shown in Figure P<0.001= in the crude model. Furthermore, SUA/Scr was an independent risk factor for all-cause mortality  after adjusting for age, sex, BMI, prealbumin, dialysis vintage, dialysis frequency, spKt/V, DM, hypertension and comorbidities. Additionally, SUA/Scr was also strongly associated with cardiovascular disease mortality . Additionally, SUA/Scr can reduce the interference of sex and renal function abnormalities . Our preThe SUA/Scr was calculated using SUA and Scr. It is need to clarify whether SUA/Scr was superior to SUA or Scr on predicting all-cause mortality in elderly hemodialysis. NRI results showed that predict value on risk of all-cause mortality SUA/Scr was superior to SUA or Scr. Thus, we concluded that SUA or Scr alone was weaker than SUA/Scr as predictors of all-cause mortality among hemodialysis patients.The present study has several limitations. First, the present study is a single-center study, which might cause selection bias. We minimize bias through quality control as far as possible. Second, the study did not distinguish the effects of residual renal function (RRF). It is attributed to the limitation of retrospective study we are unable to obtain accurate urine collection. Further multicenter studies are needed to clarify the relationship between SUA/Scr and all-cause mortality.In summary, the present study demonstrated that the serum uric acid to creatinine ratio is strongly associated with all-cause mortality in elderly hemodialysis patients. Further verification using multicenter studies with diverse population is needed.Additional file 1:Supplemental Table 1. Association of SUA/Scr with cardiovascular disease mortality by multiple Cox hazards regression analysis.


Saccostrea is very confused, however, there is relatively little molecular information available on Saccostrea. In this study, we determined and described for the first time the complete mitochondrial genome of Saccostrea malabonensis. The complete mitogenome of S. malabonensis is 16,204 bp in length, containing 12 protein-coding genes (lack of atp8 gene), two rRNA genes, 23 tRNA genes. The overall nucleotide composition of S. malabonensis has an AT content of 61.94% . Phylogenetic analyses showed that S. malabonensis is first clustered with S. cucullata then united with Saccostrea kegaki.The taxonomy of the genus  Saccostrea are well known for high plasticity in shell with different habitat conditions, which leads to taxonomic problems , and preserved in 95% ethanol. Voucher specimens are deposited in the Fourth Institute of Oceanography, Ministry of Natural Resources, and the deposited numbers are IDNW8 . Total genomic DNA was extracted from the ethanol-preserved tissue using TIANGEN marine animal DNA kit . The complete mitochondrial genome of S. malabonensis was sequenced by Illumina NovaSeq6000 platform with 350 bp paired-end. The reads were assembled using SPAdes (v3.14.1). ORFfinder (http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to identify protein encoding genes and rRNA genes. The tRNA genes were identified by the program MITOS webserver  were aligned by MAFFT v7.037b , two rRNA genes and 23 tRNA genes on the same strand (GenBank accession: ON649706). Previous studies on oysters  is split into two fragments that occurs only in Ostreidae. S. malabonensis has an AT content of 61.94% . Phylogenetic trees were constructed using the concatenated set of 12 protein-coding gene amino acid sequences of S. malabonensis and 20 published complete mitogenomes of mollusca. Aplysia californica was used as outgroup. Phylogenetic analyses showed that S. malabonensis is first clustered with S. cucullata then united with S.kegaki (Crassostrea, Ostrea and Saccostrea in Ostreidae, and the relationships have also been verified by other molecular phylogenetic studies (Volatiana et al. S. malabonensis will provide important genome information for molecular phylogenetic studies on Bivalvia.The complete mitogenome of S.kegaki . Our phy


Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates  and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections.IMPORTANCEFusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle. Fusobacterium is a Gram-negative, anaerobic, non-spore-forming bacillus commonly found in the oral, intestinal, respiratory, and genital tracts of animals and humans. Fusobacterium produces volatile fatty acids, including butyrate, acetate, and propionate, as major metabolic end products  and F. necrophorum subsp. funduliforme (biotype B), based on differences in cellular morphology, biochemical characterization, and differences in DNA level in 16S rRNA and DNA repeats . We concentrated on understanding the phylogenetic relationship, evolution pattern, and potential virulence genes of F. necrophorum at the subspecies level.With rapid advances in sequencing methods, whole-genome sequencing technology has been widely used to predict pathogens’ phenotypic traits, such as virulence and antimicrobial resistance. Sequence analysis offers better insight into strain-specific genetic variation, disease propensity, phylogenetic relationships, virulence patterns, potential virulence genes, and diversity among subspecies and strains , 16. Thibscesses . TherefoFusobacterium genus.(i) Phylogenetic distribution and relationship of  We studied genomic evolution by assembling dissimilarity matrices within the core genomes of 167 Fusobacterium strains (162 latest [July 2019] RefSeq sequences plus five test strains) and illustrated the results in Leptotrichia buccalis and Cetobacterium somerae, were used as an outgroup , F. mortiferum (dark green), F. ulcerans (blue), and F. varium (dark red). Furthermore, the data revealed that several uncategorized Fusobacterium species  formed a separate clade  Phylogenic relationships among  We used the whole-genome sequence to perform phylogenetic analysis based on a concatenated bacterial core gene set in a UBCG pipeline. The phylogeny implemented using RAxML revealed that all test strains  clustered according to their respective subspecies  (F. necrophorum subsp. necrophorum or F. necrophorum subsp. funduliforme on the phylogenetic tree based on the core genome (F. necrophorum KG35. F. necrophorum BL and F. necrophorum FDAARGOS_565 clustered with the F. necrophorum subsp. necrophorum ATCC 25286 (the only complete published F. necrophorum subsp. necrophorum genome) and test strains 8L1 and 212 . Therefore, UBCG analysis showed that out of 46 strains, 12 strains belonged to F. necrophorum subsp. necrophorum, while 34 belonged to F. necrophorum subsp. funduliforme. clades) . These fe genome , except (iii) Average nucleotide identity analysis. To confirm our phylogenetic analysis, we also used average nucleotide identity (ANI) analysis, which gives the mean of nucleotide identity values for F. necrophorum strains. Using the FastANI results, we calculated pairwise ANI values among the F. necrophorum strains, which ranged from 94.64% to 99.99%. Next, we generated a heat map to visualize RefSeq and test strain clustering based on the distance matrix of ANI values  were ≥95%, suggesting that the strains belong to F. necrophorum. However, the heat map showed similarities ranging from 94.645% to 96.028% for F. necrophorum KG35. The homology with ANI values <95% between the F. necrophorum strains KG35 and BFTR-2 (94.688%), KG35 and ATCC 25286 (94.739%), and KG35 and NCTC13726 (94.645%) is represented in the red grid in the heat map , and we included two outgroups to evaluate genus-level diversity. The Fusobacterium genus pangenome contained 52,441 gene clusters  to compare unique genes present at the species level. This pangenome contained 8,136 gene clusters. Of these 8,136 gene clusters, a total of 826 (10.15%) constituted the core genes and 404 (0.0004%) the soft core genes. Notably, the proportions of shell and cloud genes were higher, 1,856 (22.81%) and 5,050 (62.06%), respectively. Using whole-genome alignment with comparisons of core and ancillary genes (generated using Roary), we examined the genetic relatedness of the 46 strains  and metabolic pathways (transferases and dehydrogenases). There are some unique genes in F. necrophorum subsp. necrophorum. These genes are for iron transport and biosynthetic pathways. Overall, 19 of 40 genes have not been annotated and are labeled as hypothetical genes.Based on the pangenome-wide association analysis, (i) Virulence genes. Because the widely used virulence factor database did not have the Fusobacterium reference genome  Antimicrobial resistance genes. To identify the presence of ARGs, we screened both the RefSeq sequences and the test strains using ABRicate. This prediction tool identified several acquired ARGs in 38 Fusobacterium genomes, and the list of Fusobacterium species with different ARGs harbored by them is shown in Fusobacterium strains . A total of 38 genomes carried at least one of the 19 acquired ARGs available in the ABRicate databases. Most of these genes were harbored by F. ulcerans, F. varium, F. mortiferum, F. periodonticum, F. nucleatum, F. perfoetens, F. equinum, F. naviforme, and F. necrogenes. However, F. necrophorum subsp. funduliforme B35 was the only F. necrophorum strain that carried ARGs.lsaE gene, which confers resistance to pleuromutilin, lincosamide, and streptogramin A, in 13 genomes of F. ulcerans and F. varium except for one genome (Fusobacterium sp. strain HMSC073F01). Also, we identified the presence of vatB, which confers streptogramin resistance, in F. ulcerans. Furthermore, F. necrophorum subspecies funduliforme B35 harbored both macrolide [msr(D)] and tetracycline [tet(T)] resistance genes.We found the F. necrophorum subspeciation results from recombination events, we applied the ClonalFrameML model. Genome-wide recombination events in F. necrophorum, based on 46 genomes representing carriage and reference isolates assigned to F. necrophorum assembly genomes, are represented in Recombination events can occur from one lineage to another within a single species and can lead to subspeciation. To investigate whether F. necrophorum core genome (Data Set S8). This analysis yielded the following metrics: the relative rate of recombination with mutation (R/θ) was 0.30455, the mean length of the recombined DNA (δ) was 286, and the mean divergence of imported DNA (ν) was 0.0371. Each recombination event generated approximately 10.61 substitutions . The ratio of changes introduced by recombination to changes introduced by mutation (r/m) was 3.226   . These fain KG35 , and theFusobacterium. Our phylogenetic analysis  and a group of uncharacterized F. necrophorum strains (clade II). Later, clade II evolved to form a distinct clade of F. necrophorum subsp. necrophorum (clade IIa), which includes test strains 8L1 and 212 and reference strains BL and FDAARGOS 565. When comparing the genomic size with respect to recombination, we found that the genomic size of the strains in clade II is greater than that of F. necrophorum subsp. funduliforme  for our analyses. The Fusobacterium phylogenic topology published by McGuire et al. is comparable to the phylogeny in our study  analysis confirmed genetic dissimilarity among subspecies, this analysis did not provide any definite correlation between GC content and source  had a pairwise ANI of >96% , hemin receptor, and FadA adhesin were found only in the F. necrophorum subspecies necrophorum. On the other hand, YadA-like family proteins, hemagglutinin, OmpA, and Fic toxin, were only found in F. necrophorum subsp. funduliforme. Also, a set of genes, including ompH and those encoding virulence-associated protein (vapD), hemolysin III family protein, cell envelope integrity protein (creD), and leukotoxin, were found in both subspecies. However, there were notable exceptions for creD and leukotoxin A genes, as creD was absent in F. necrophorum subsp. funduliforme F1365 and leukotoxin A was absent in F. necrophorum DJ1.With an increase in fadA gene in F. necrophorum subsp. necrophorum. Because fadA is a virulence gene involved in host cell attachment and encodes a vital invasion factor for active invaders in F. nucleatum infections , indicating the chances of antimicrobial resistance emergence in Fusobacterium species. Therefore, it is necessary to develop novel and effective therapeutic and prevention strategies, such as vaccination, before the development of resistance in this bacterial species. It is of the utmost importance to search for potential virulence targets that could be used to develop therapeutic and prevention strategies against fusobacterial infection.Fusobacterial infections are commonly treated with antibiotics such as penicillin, amoxicillin, clindamycin, and imipenem. Although antimicrobial resistance is not commonly found in this genus, in contrast to other pathogens, the emergence of resistance is inevitable. Our genome sequence analysis showed few ARGs distributed among all F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, as their biological activities differ. We investigated recombination events that might have influenced evolutionary diversification of the F. necrophorum subspecies. The results revealed that F. necrophorum subsp. funduliforme may have undergone a genomic expansion during evolution, leading to a clade of a virulent form of F. necrophorum subsp. funduliforme and another unclassified clade of F. necrophorum. Later, this unclassified F. necrophorum subsp. necrophorum clade could have evolved to more virulent forms of F. necrophorum subsp. necrophorum. Similarly, some of the virulence genes found only in F. necrophorum subsp. necrophorum reflect evidence of recombination during evolution in the F. necrophorum subsp. necrophorum genome. Our data demonstrate that the difference observed is evidence for the effect of recombination events on the evolutionary relationship with other Fusobacterium necrophorum strains . Ta. TaF. neF. necrophorum, including four bovine isolates  and one human isolate (MK12), were used in this study (g) and resuspended in TES buffer . The cells were then pretreated with lysozyme  and lysed using Sarkosyl NL  with proteinase K (Sigma-Aldrich) . The crude lysate was resuspended in Tris-buffer-saturated phenol-chloroform. DNA was extracted by precipitation in 2.5 mL ice-cold ethanol, and the pellet was resuspended in TE buffer . Subsequently, the DNA was purified with a cesium chloride density gradient followed by dialysis against double-distilled water. The purity and concentration were checked spectrophotometrically. Purified DNA was used for paired-end sequencing at HiSeq using the Illumina platform.Five strains of is study . These sis study . For allis study . BrieflyFusobacterium sequence files and 41 F. necrophorum RefSeq assemblies were downloaded from the NCBI database (Data Sets S1 and S2). The sequences downloaded for this study were selected to generate a diverse data set for comparative genome analysis. Furthermore, genomes of strains belonging to two different genera, Leptotrichia buccalis DSM 1135 and Cetobacterium somerae ATCC BAA-474, were included in the data as an outgroup.Furthermore, 162 (i) Data preprocessing. All raw read preprocessing was performed on the Galaxy platform . For quality control, raw sequences were preprocessed using FastQC software v0.11.7. Based on the FastQC report, quality trimming was performed using Trimmomatic to filter and trim low-quality reads (ty reads . The pro(ii) Genome assembly, quality check, and annotation. The quality trimmed reads were assembled on the Galaxy platform  using SPAdes with default parameters  and the average nucleotide identity (ANI) values.(i) Up-to-date bacterial core gene. To predict genomic relationships at the genus and species levels, a UBCG approach with default parameters was used . The sets of 167 Fusobacterium genus genomes (162 NCBI RefSeq sequences plus five test strains) were used for the analysis of evolutionary relationships at the genus level between different species. A similar phylogenetic analysis was performed for 46 (41 NCBI RefSeq sequences plus five test strains) F. necrophorum genomes. For each UBCG, the core gene sequences were aligned with multiple-sequence alignment. The resulting alignments were concatenated, and phylogeny was inferred using the maximum-likelihood (ML) algorithm in the RAxML v 7.8.6 tool using the GTR+I+G4 model of nucleotide substitution (https://itol.embl.de/) (was used . These ttitution . All trembl.de/)  (Data Se(ii) Average nucleotide identity analysis. The DNA-DNA relatedness and orthologous genes shared between species of F. necrophorum were analyzed using the FastANI algorithm by constructing an ANI heat map  and detection of accessory or ancillary genes (genes common to only a subset of the strains and strain-specific genes). Comparative genomic analysis was performed for the five curated genome assemblies using the published NCBI RefSeq database for e PROKKA  pipelinee PROKKA  were useF. necrophorum subspecies. The comparison was performed in the Scoary program to identify the uniqueness of the gene via evaluation of the highest-scoring nonintersecting gene pairs, sorted by P value . These genes were checked against the strains used for comparative analysis at the genus and species levels.We methodically searched the literature to identify potential virulence genes in Fusobacterium and the contigs of the five test strains. The ABRicate v0.8.10 search engine (https://github.com/tseemann/ABRicate) was used to screen ARGs with the following databases: ResFinder  for the assemblies analyzed in this manuscript are provided in the supplementary material see Data Set S1.Sequencing raw data are available from the latest version of RefSeq:


Enterobacter hormaechei organisms belong to the Enterobacter cloacae complex (ECC) and are commonly recognized as causative agents for hospital infections. Recently, a few E. hormaechei MDR strains associated with infection in piglets, calves, and a fox were reported, highlighting the important role of animals and livestock in the emergence and spread of antimicrobial resistance. In this study, the vaginal swab sample from a 5-year-old cow with multiple anamnestic infectious abortions was carefully investigated. The animal was unresponsive to antibiotic therapy recommended by the veterinarian. The MDR bacterial strain isolated from the bovine sample, designated as the Saratov_2019, belonged to Enterobacter hormaechei. The genome-based phylogenetic analysis identified the isolate to be Enterobacter hormaechei subsp. xiangfangensis. The genome of the Saratov_2019 contained a 6364 bp plasmid. Importantly, we revealed the novel sequence type ST1416 and 13 MDR genes correlating with the MDR phenotype in only the chromosome but not the plasmid. These findings indicate that the potential spread of this strain may pose a threat for both animal and human health. The data obtained here support the notion of the important role of livestock in the emergence and spread of antimicrobial resistance, promoting careful investigation of the MDR spectra for livestock-related bacterial isolates. To the best of our knowledge, this is the first report on the association of E. hormaechei subsp. xiangfangensis with the infection of the reproductive system in cattle.The emergence of multidrug-resistant (MDR) bacterial strains is one of the significant global challenges with regard to bacterial drug-resistance control.  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been included in the WHO global priority pathogens list [The emergence and the increase in the finding of multidrug-resistant (MDR) strains is one of the current threats to both human and animal health worldwide . Accordiens list . These bens list .Enterobacter spp. has been reported to be one of the leading causes of MDR hospital infections [Enterobacter spp. organisms belong to the family Enterobacteriaceae and are recognized as common causative agents in a variety of bloodstream and intraabdominal infections [Enterobacter (the Enterobacter cloacae complex (ECC)) such as Enterobacter asburiae, Enterobacter carcinogenus, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter nimipressuralis, and Enterobacter mori [E. hormaechei isolate demonstrated a hypervirulent phenotype that was comparable with a hypervirulent K. pneumoniae-type strain in a Galleria mellonella-infection model [As members of the ESKAPE group, fections . Enterobfections . Currentter mori . Importater mori . Moreoveon model .Enterobacteriaceae isolated from farm animals may serve as reservoirs of AMR genes [E. hormaechei, have recently been identified as the causative agents of uterine infection in a dead fox [Initially, these ESKAPE pathogens were associated with the most recurrent global cause of hospital infections. However, the emergence of MDR strains in animal husbandry indicates the important role of livestock in the emergence and spread of antimicrobial resistance (AMR) and highlights the necessity of the accurate identification of species, subspecies and spectra of AMR and MDR for outbreak-related isolates ,13,14 asMR genes . Howeverdead fox , diarrhedead fox , and resdead fox .E. hormaechei subsp. xiangfangensis strain Saratov_2019 with MDR to eight groups of tested antibiotics, which was isolated from a cow with a reproductive system infection. Typically, Enterobacter spp. obtained from animals possess limited resistance to carbapenems [E. hormaechei subsp. xiangfangensis with such an extensive MDR phenotype has not been previously found in cattle.This study aimed to investigate the n = 3) for the study were obtained in April 2019 from a 5-year-old cow with multiple anamnestic infectious abortions. The vaginal swab was collected from a cow after thoroughly cleaning the vulva. The specimens were submitted to the diagnostic laboratory of the Saratov State Agrarian University in order to identify the possible cause of infection. The animal was kept at a small farm in the Saratov Region, Russia. The accompanying report for these animal specimens described the clinical signs of bovine genital tract inflammation such as mucopurulent or purulent vaginal discharge, fever, decreased milk yield, reduced appetite, tiredness, no sign of estrus, and several miscarriages in the past two years. The veterinarian who treated this cow reported that antibiotic therapy including oxytetracycline and third-generation cephalosporins had failed to improve the animal’s health. Thus, the animal was unresponsive to the antibiotic therapy recommended by the veterinarian. These clinical samples were studied bacteriologically using Endo agar , and cultivated at 37 °C in an aerobic environment for 3 days, which resulted in the isolation of the bacterial strain from the vaginal specimen only.The vaginal, blood, and uretus swab samples  . The teshttps://www.genoanalytica.ru/ (accessed on 15 May 2022)) and MinION . The DNA library preparation for Nanopore sequencing was undertaken with the 1D Genomic DNA by Ligation (SQK-LSK109) protocol  including DNA end repair, dA-tailing, and DNA clean-up steps. The final DNA library was sequenced using a FLO-MIN-106 R9.4 flow cell and the MinKNOW software (https://nanoporetech.com/community (accessed on 15 May 2022)).The DNA from the bacterial isolate derived from the corresponding vaginal sample was extracted using the DNeasy Blood and Tissue Qiagen Kit , and concentrations were measured with a spectrophotometer . To preliminarily identify the type of microorganism, the isolated DNA from the bacterial culture was amplified by the 16S rRNA method followed by sequencing, as described previously . The whohttps://www.ezbiocloud.net (accessed on 15 May 2022)) for subspecies-level identification of the relevant microorganism. Hybrid assembly de novo was generated with Unicycler v 0.4.7 (https://github.com/rrwick/Unicycler (accessed on 15 May 2022)). Contig alignments were performed using Mauve software (http://darlinglab.org/mauve/mauve.html (accessed on 15 May 2022)). Primary metagenomic data analysis was carried out with the help of the Metagenomics Analysis Server MG-RAST (https://www.mg-rast.org (accessed on 15 May 2022)). Genome-based classification and identification were carried out with the help of the Type (Strain) Genome Server (https://tygs.dsmz.de/ (accessed on 15 May 2022)). The whole genome de novo assembled sequence data were deposited in the NCBI GenBank as E. hormaechei subsp. xiangfangensis strain Saratov_2019 (Acc. No. JAHFZP000000000.17).The preliminary DNA identification of the bacterial isolate was conducted through an analysis of the 16S rRNA gene amplicon sequencing and the EzBioCloud database (https://pubmlst.org/ (accessed on 15 May 2022)). The ‘house-keeping genes‘-derived sequences were deposited in the PubMLST database (https://pubmlst.org/ (accessed on 15 May 2022)), with the access number—ST1416 (https://pubmlst.org/bigsdb?page=profileInfo&db=pubmlst_ecloacae_seqdef&scheme_id=1&profile_id=1416 (accessed on 15 May 2022)).The identification of allele profiles of multiple contigs and multi-locus sequence typing (MLST) was performed using a PubMLST database ).The identification of antibiotic-resistance genes was carried out by using the RGI Resistance Gene Identifier (E. hormaechei subsp. xiangfangensis strain Saratov_2019), CP024908.1 (E. hormaechei subsp. xiangfangensis strain OSUKPC4_L), CP029246.1 (E. hormaechei subsp. xiangfangensis strain OSUVMCKPC4-2), CP043382.1 (E. hormaechei subsp. xiangfangensis strain WCHEX045001), CP023430.1 (E. hormaechei subsp. xiangfangensis strain UM_CRE-14), CP012165.1 (E. hormaechei subsp. xiangfangensis strain 34978), CP053103.1 (E. hormaechei subsp. xiangfangensis strain Ec61), NZ_CP017183.1 (E. hormaechei subsp. xiangfangens is strain LMG27195), CP010384.1 (E. hormaechei subsp. xiangfangensis strain 34399), NZ_CP061744.1(E. hormaechei strain NJGLYY90-CR), CP030007.1 (E. hormaechei subsp. xiangfangensis strain Pb204), NZ_CP017179.1 , NZ_CP017180.1 (E. hormaechei subsp. oharae strain DSM 16687), NZ_CP017186.1 (E. hormaechei subsp. hoffmannii strain DSM 14563), NZ_MKEQ00000000.1 (E. hormaechei ATCC 49162), NZ_CP043318.1 (Enterobacter chengduensis WCHECl-C4), NZ_CP011863.1 (Enterobacter asburiae strain ATCC 35953), VTTY00000000.1 (Enterobacter dykesii strain E1), NZ_CP017184.1 (Enterobacter roggenkampii strain DSM 16690), LFDQ00000000.1 (Enterobacter quasiroggenkampii strain WCHECL1060), NZ_POVL00000000.1 (Enterobacter sichuanensis strain WCHECL1597), NZ_CP017181.1 (Enterobacter kobei strain DSM 13645), NZ_LT992502.1 (Enterobacter bugandensis isolate EB-247), NZ_QZCS00000000.1 (Enterobacter. chuandaensis strain 090028), NZ_MTFV00000000.1 (Enterobacter cloacae strain ATCC 13047), NZ_WJWQ00000000.1 (E. cloacae subsp. dissolvens ATCC 23373), QZCT00000000.1 (Enterobacter huaxiensis 090008), NZ_RXRX01000051.1 (Enterobacter quasimori 090044), SAMEA2548140 (Enterobacter taylorae NCTC 12126), NZ_FYBA01000003.1 (Enterobacter cancerogenus ATCC33241), PRJNA332046 (E. kobei ATCC BAA-260), NZ_BCTM00000000.1 (Kluyvera cryocrescens NBRC 102467), and BAFF01000003.1 (Escherichia hermannii NBRC 105704T).The following GenBank accession numbers were used for phylogenetic analysis in this study: NZ_JAHFZP000000000.1 (https://www.ezbiocloud.net (accessed on 15 May 2022)). The results showed that the relevant microorganism was related to Enterobacter hormaechei. No microorganisms grew on the Endo agar plates from the blood and uretus specimens.A single vaginal swab yielded a bacterial strain that was identified by the 16S rRNA gene sequence on the EzBioCloud Server  with the size of 6364 bp showed high homology (98.84%) with the plasmid replicon pECL-90-4 that was recently  found in the strain E. hormaechei NJGLYY90-CR from China (Acc. No. in NCBI CP061745.1). The chromosomes of the strains Saratov_2019 and NJGLYY90-CR also demonstrated high homology , and these strains were located in close proximity on the phylogenetic tree  of the total DNA derived from the Saratov_2019 strain, of which 66 contigs belonged to the chromosomal DNA, and a single contig was identified as a circular plasmid replicon. The Genome BLAST Distance Phylogeny (GBDP) method based on the comparison of the Saratov_2019 strain with the TYGS database currently consisting of a comprehensive collection of 14,927 microbial type-strain genomes revealed that our strain had the highest homology  and formed a phylogenetic cluster with the reference strains ter spp. . Notablytic tree . These dde novo assembled whole genome, which was generated with the use of the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed the presence of more than 4000 coding sequences (CDSs) and 77 pseudo genes as a result of either the frameshift mutations or premature stop codons in the relevant DNA sequences ).Importantly, the Saratov-2019 strain had a novel sequence type (ST), ST1416 . The relE. hormaechei subsp. xiangfangensis strains  leuS, rplB and rpoB in the strain 34399 of ST114; (iii) fusA, gyrB and leuS in the strains 34978, OSUKPC4_L, OSUVMCKPC4-2, UM_CRE-14, and WCHEX045001 of ST171; and (iv) fusA, leuS, rplB and rpoB in the strains Ec61 and NJGLYY90-CR of ST418 and ST451. Nevertheless, the E. hormaechei strain of ST1348 had an identical MLST profile in six alleles , and differed from Saratov_2019 by a single SNP for the allele 45 at position 48, which displayed a substitution C->T compared with that of allele 13 of Saratov_2019 corresponding to the pyrG gene  [E. hormaechei unlike the majority of the E. hormaechei spp. clinical isolates obtained from hospital infections worldwide [At least 13 of them were successfully identified in the genome of this strain . Interes1–mcr-9) ,30,31,32orldwide ,34,35,36E. hormaechei subsp. xiangfangensis strain from a cow with a reproductive system infection. This MDR strain had a novel ST1416 and showed no carbapenem resistance. Future prospective research is critical to reveal the actual prevalence of MDR microorganisms in animal husbandry worldwide. A certain limitation of our study was that the panel of antibiotics used only the most common veterinary drugs. Additionally, we plan to significantly extend the number and species of animals investigated in our future research. Due to the potential risk to the health of both animals and humans who are professionally employed in animal husbandry, effective control over the spread of both the AMR and MDR bacterial strains has to be implemented.According to our results, we report the first case of isolating the


Cutibacterium acnes in sarcoid granulomas suggests its potential role in granuloma formation. C. acnes is the sole microorganism ever isolated from sarcoid lesions. Histopathologic analysis of some sarcoid lymph nodes reveals latent infection and intracellular proliferation of cell-wall-deficient C. acnes followed by insoluble immune-complex formation. Activation of T helper type 1 (Th1) immune responses by C. acnes is generally higher in sarcoidosis patients than in healthy individuals. Pulmonary granulomatosis caused by an experimental adjuvant-induced allergic immune response to C. acnes is preventable by antimicrobials, suggesting that the allergic reaction targets C. acnes commensal in the lungs. C. acnes is the most common bacterium detected intracellularly in human peripheral lungs and mediastinal lymph nodes. Some sarcoidosis patients have increased amounts of C. acnes-derived circulating immune complexes, which suggests the proliferation of C. acnes in affected organs. In predisposed individuals with hypersensitive Th1 immune responses to C. acnes, granulomas may form to confine the intracellular proliferation of latent C. acnes triggered by certain host-related or drug-induced conditions. Current clinical trials in patients with cardiac sarcoidosis are evaluating combined treatment with steroids and antimicrobials during active disease with continued antimicrobial therapy while tapering off steroids after the disease subsides.The immunohistochemical detection of  Mycobacterium tuberculosis as a causative agent, but no acid-fast bacilli (including M. tuberculosis) have been cultured from sarcoid lesions . In. In38]. rculosis ,42.C. acnes-specific antibodies (PAB-antibody-detecting cell-membrane-bound LTA and TIG-antibody-detecting ribosome-bound trigger factor protein) revealed that the PAB antibody signals distribute around the periphery of the HW bodies, whereas TIG antibody signals distribute in a dot-like pattern over the entire internal region of the body [C. acnes  [Electron microscopy shows small round bodies extruding from the HW bodies in macrophages that are indicative of a dividing cell-wall-deficient bacterium . Immunoethe body . The disC. acnes . HW bodictively) .C. acnes, while the small round bodies filling the cells are thought to represent intracellularly proliferating C. acnes. Unlike extracellular cell-walled C. acnes in the hair follicles of the skin, the intracellular C. acnes lack a cell wall. In culture medium containing a rich nutrient, these intracellular cell-wall-deficient C. acnes seem to revert to a conventional cell-walled form. Therefore, isolation of C. acnes from sarcoid lesions requires a longer incubation period than usual and a highly osmotic culture medium. In the early stages of culture, the organisms often appear round rather than coryneform.Based on histopathologic analysis, HW bodies are thought to represent latently infected C. acnes is most commonly linked to sarcoidosis [C. acnes in sarcoid lesions was demonstrated via quantitative polymerase chain reaction (PCR) using FFPE lymph node specimens [C. acnes DNA can also be detected in some control samples, sarcoid samples contain a larger number of C. acnes genomes—almost the same number of M. tuberculosis genomes detected in tuberculosis samples [C. acnes in sarcoid granulomas [C. acnes DNA, but M. tuberculosis DNA was almost undetected, which suggested the potential association of C. acnes with sarcoidosis even in European countries where M. tuberculosis has long been the suspected cause of sarcoidosis.A meta-analysis of 58 studies that included more than 6000 patients from several countries to investigate all types of infectious agents proposed to be associated with sarcoidosis revealed that coidosis . Intracepecimens ,45. Alth samples . In situanulomas . Lymph nanulomas  also conC. acnes is a commensal Gram-positive anaerobic bacterium that lives extracellularly on the skin and mucosal surfaces of the oral cavity and the gastrointestinal and genitourinary tracts [C. acnes detected via quantitative PCR in sebaceous materials aspirated from normal hair follicles begins to increase after the age of 10 years and reaches a maximum at the age of 15–19 years [C. acnes LTA  are raised in all adults with no significant difference between healthy controls and sarcoidosis patients [C. acnes exhibit increased production of a Th1-type cytokine interleukin-2 (cellular immune response) compared with PBMCs obtained from healthy controls [C. acnes trigger factor [C. acnes [y tracts . The num19 years . Serum apatients . In contcontrols . Cellular factor  or catalr factor  are usedC. acnes ,55,56. TC. acnes. The assumption of such a host factor is also based on the phenomenon of the Kveim reaction. Allergic cellular immune responses in sarcoidosis patients are assumed on the basis of experimental models of organ-specific autoimmune diseases such as experimental autoimmune encephalomyelitis [C. acnes is assumed to be an intracellular antigen like the organ-specific autoantigen. Furthermore, experimental autoimmune thyroiditis is always self-limiting when induced in healthy animals, and DA rats that have undergone spontaneous remission are resistant to re-induction of experimental autoimmune thyroiditis [Granuloma formation may occur only in so-called “high responders” to myelitis  and expemyelitis . For examyelitis . Such dimyelitis  and dissroiditis . These eC. acnes can be induced in healthy mice via subcutaneous immunization with C. acnes using complete Freund’s adjuvant. Some mice develop multiple granulomas in the lungs, which suggests that the target antigens are present in the peripheral lungs where the granulomas are formed [C. acnes in mouse lungs is 33% by culture [C. acnes in the peripheral lungs. In mice with latent infection, immunization with C. acnes using complete Freund’s adjuvant induces allergic cellular immune responses to C. acnes, and multiple foci of granulomatous inflammation are induced by targeting C. acnes that is infecting the lungs. On the other hand, in mice without latent infection, granulomatous inflammation does not occur even if an allergic reaction is induced because there is no target antigen in the lungs. In humans, C. acnes is cultured from peripheral lung tissue and mediastinal lymph nodes in adults at a frequency of 50–60% [Similar to the experimental models of organ-specific autoimmune diseases, allergic cellular immune responses to e formed ,63. The  culture ,62. Thesf 50–60% .C. acnes forms immune complexes extracellularly due to the presence of infectious antibodies raised by commensal C. acnes in the body. Many IgA/IgM-bound insoluble immune complexes derived from C. acnes in sarcoid lymph nodes are found in macrophages located in hyperplastic lymphatic sinuses [C. acnes and the infectious antibody against C. acnes LTA bind to an identical epitope of LTA on the bacterial surface. Due to epitope competition, PAB antibodies are unable to react with the immune complexes bound with immunoglobulins. After removal of the bound immunoglobulins via trypsin digestion, PAB antibodies can detect C. acnes-derived immune complexes [C. acnes that are mostly free from immunoglobulins, these immune-complexed C. acnes are originally extracellular C. acnes phagocytosed by macrophages  via dissemination through the lymphatic system and bloodstream, whereas local entry and subsequent latent infection of C. acnes through other than systemic spread may occur in some organs or patients. Latent infection in systemic organs can be simultaneously reactivated by additional triggering events, which leads to granuloma formation at all sites of latent infection in each organ of sarcoidosis patients.The assumed pathogenesis of sarcoidosis caused by n humans ,36,64. Sthe host  or by cathe host . Latent patients ,36,70. AC. acnes ; thus, i balance . It was  balance . The pros itself . SuccessC. acnes is classified as an endogenous hypersensitivity infection; granulomatous inflammation occurs only in patients with hypersensitivity responses against C. acnes. Diseases caused by a fungus such as candida or aspergillus are commonly encountered as opportunistic infections, but these microorganisms can also cause hypersensitivity pneumonitis with a background of a hypersensitivity predisposition and are thus classified as a both-mixed type endogenous infection.Diseases caused by commensal microorganisms are referred to as endogenous infections and are generally classified into three major types  accordiC. acnes. The first prerequisite is latent infection by cell-wall-deficient C. acnes, which occurs not only in macrophages and vascular endothelial cells but also in epithelial and mesenchymal cells [C. acnes, which may be caused by host-related conditions such as physical or mental stress [C. acnes. A hypersensitive Th1 immune response to C. acnes may be caused by a disruption of peripheral T-cell tolerance to certain C. acnes antigens . The hodiseases ,83. ThusC. acnes as the pathogen, reactivation of latent C. acnes triggered by environmental factors, and hypersensitive Th1 immune responses against the intracellular C. acnes as host factors.Sarcoidosis as an endogenous hypersensitivity infection develops only after these three factors are established; that is, latent infection by C. acnes causes granuloma formation and may provoke a new latent infection at the site of disease activity. Unless this latent infection is eliminated, recurrent inflammation can occur. With each recurrence, the affected tissue is destroyed by granulomatous inflammation in addition to postinflammatory fibrosis, resulting in gradual expansion of the lesion. Steroids have a therapeutic effect by suppressing allergic reactions. New granuloma formation can be prevented if the intracellular proliferation of C. acnes can be prevented by antimicrobial therapy. Tetracyclines, which are used for acne vulgaris [C. acnes is suggested [C. acnes infection . Becauscoidosis ; the effcoidosis  and an a samples . In the  systems .C. acnes as an endogenous hypersensitivity infectious agent that causes sarcoidosis. The concept of endogenous hypersensitivity infections is not limited to the etiology of sarcoidosis but should be considered as a general theory when investigating the etiology of other intractable diseases suspected to be caused by certain infectious microorganisms.The results of many studies suggest the potential association of


Clostridioides difficile infection is closely related to the intestinal flora disorders induced by antibiotics, and changes in the intestinal flora may cause the occurrence and development of Clostridioides difficile infection. Epigallocatechin-3-gallate (EGCG) is one of the major bioactive ingredients of green tea and has been suggested to alleviate the growth of C. difficile in vitro. EGCG can ameliorate several diseases, such as obesity, by regulating the gut microbiota. However, whether EGCG can attenuate C. difficile infection by improving the gut microbiota is unknown. After establishing a mouse model of C. difficile infection, mice were administered EGCG (25 or 50 mg/kg/day) or PBS intragastrically for 2 weeks to assess the benefits of EGCG. Colonic pathology, inflammation, the intestinal barrier, gut microbiota composition, metabolomics, and the transcriptome were evaluated in the different groups. Compared with those of the mice in the CDI group, EGCG improved survival rates after infection, improved inflammatory markers, and restored the damage to the intestinal barrier. Furthermore, EGCG could improve the intestinal microbial community caused by C. difficile infection, such as by reducing the relative abundance of Enterococcaceae and Enterobacteriaceae. Moreover, EGCG can increase short-chain fatty acids, improve amino acid metabolism, and downregulate pathways related to intestinal inflammation. EGCG alters the microbiota and alleviates C. difficile infection, which provides new insights into potential therapies. Clostridioides difficile infection (CDI) is a common result of antibiotics-induced diarrhea and may lead to death if left untreated. Health care costs and mortality increase as a result of frequent recurrence, as well as reduced quality of life [C. difficile can exploit this vulnerability [C. difficile infection is associated with dysbiosis of the gut microbiota [ of life ,2. When rability ,4. C. dicrobiota .C. difficile [C. difficile infection remains unclear.Epigallocatechin-3-gallate (EGCG) is an anti-inflammatory, antioxidant, antitumor, cancer chemopreventive, and antiaging factor ,7,8,9. Cifficile . A growiifficile , obesityifficile , and radifficile  by improifficile . The intOur study examined the survival of infected mice after EGCG administration, evaluated the protective effect of EGCG against intestinal infection, and examined the underlying mechanism of EGCG-mediated intestinal protection. We found that EGCG increased survival, reduced weight loss, improved intestinal pathological damage and barrier damage, restored the gut microbiota, and changed the metabolism in mice. In addition, we found that the protective mechanism may include inflammatory signaling pathways. In conclusion, our findings suggest the potential use of EGCG in clinical therapies, for example as a dietary supplement prebiotic during infection.C. difficile infection group , (3) oral administration of 25 mg/kg body weight EGCG , and (4) 50 mg/kg EGCG  from day -8 to day 5 daily. The EGCG  doses were based on the literature [C. difficile infection , the mice were administered a cocktail of antibiotics for 5 days [8C. difficile strain VPI 10463 (ATCC 43255) was gavaged into the mice on day 0. Feces samples were collected before the mice were sacrificed.C57BL/6 female mice aged 6 to 8 weeks were randomly grouped: (1) control group  was administered PBS orally once daily, (2) terature . For C. r 5 days  , congestion/edema (0–3 points), and neutrophil margination (0–3 points) . ImmunohCytokines in the mouse serum were detected using the Bio-Plex Pro Mouse Cytokine 23-Plex Panel (Bio-Rad). Mouse LBP ELISA kits  were used to measure the LPS binding protein (LBP) concentrations.Colon tissue was extracted with the RNeasy Plus Mini Kit to extract RNA . qPCR analysis was performed using Premix Ex Taq  and a ViiA7 real-time PCR system . β-actin acted as an internal control. A list of primer sequences can be found in Fecal DNA was collected from the mice prior to execution and was isolated using the DNA PowerSoil Kit . The V3-V4 variable region was amplified using universal primers . The PCRAs previously described , analysiThe concentration of short-chain fatty acids (SCFAs) in the cecum contents was quantified by GC–MS . Brieflyp values were adjusted. The KEGG pathway reference database  was used for the KEGG enrichment analysis. RNA sequencing raw data are available in the SRA database (PRJNA867511).Colon tissue was extracted with a TRIzol reagent to obtain the total RNA. cDNA libraries were purified, constructed, and sequenced using an Illumina NovaSeq. After filtering using Trimmomatic, sequencing reads were mapped to the reference genome (GRCm39) by hisat2 (v2.2.1.0) . The DESp < 0.05 was considered statistically significant.The data are presented as the mean ± SEM and were analyzed using GraphPad Prism 9.0.0.0 . In order to make multiple comparisons, we used one-way ANOVA and Tukey’s multiple comparison test. C. difficile-induced colitis. Mice were gavaged with two different doses of EGCG (EL and EH group) daily , weight loss  daily A. As shoght loss C, and thC. difficile infection.A retrospective histological analysis of the colon tissues demonstrated that EGCG protected against intestinal injury caused by infection. The results showed that CDI group exhibited various inflammatory features, such as a thickened colonic epithelium, inflammatory cell accumulation, structural incompleteness, ruptured crypt glands, and the infiltration of inflammatory cells after infection D. In conC. difficile spores sprout in the human gut, they produce toxins and destroy host cells [C. difficile infection. EGCG protected the mucosal barrier and ameliorated intestinal barrier damage, and the protective effect was more obvious in the high-dose group than the low-dose group. We also evaluated the intestinal inflammatory indices Ly6G and F4/80 through immunohistochemistry. The Ly6G and F4/80 indices represent neutrophil and macrophage infiltration, respectively  based on Bray–Curtis  suggested the CON, CDI, EL, and EH microbiota structures differ significantly  and immune regulation . Further KEGG pathway analysis of the differentially expressed genes suggested that the affected pathways between the CON and CDI groups included the intestinal immune network for IgA production, the TNF signaling pathway, Th17-cell differentiation, and the IL-17 signaling pathway  for transcriptomic analysis of the intestine to examine which mechanisms contribute to the protective effect of EGCG against  pathway C. The di pathway D.C. difficile infection [C. difficile infection [C. difficile infection shows that dysregulation of the gut microbiota is characteristic of infected mice, and we showed that EGCG regulated the structure of the intestine microbiota and its metabolites and increased the production of SCFAs, thereby promoting anti-inflammatory and antioxidant effects in the gut.Having a healthy gut microbiota contributes significantly to the development and progression of nfection . Furthernfection . Therapenfection . Daily cnfection . C. diffC. difficile infection, as indicated by a decrease in the incidence of diarrhea and an increase in survival. Previous studies have shown that EGCG improves intestinal barrier function [C. difficile, as evidenced by an increase in colonic mucus and enhanced mucosal junctions in the colon. EGCG also improved the proinflammatory serum markers that induced intestinal epithelial tight junction damage. EGCG attenuated colon inflammation by decreasing serum inflammatory cytokines such as IL-6 and TNF-α in CDI mice. Increased intestinal permeability due to C. difficile infection leads to robust translocation of the bacterial components, resulting in endotoxemia. Our results showed increased serum LBP levels in the CDI group, while oral EGCG decreased the levels of LBP. We hypothesize that these different experimental results are linked with the intestinal microbiota and metabolism.Our results showed that EGCG therapy remitted function . Our stuC. difficile infection. Through polyphenols, beneficial bacteria are promoted in the intestine, and pathogenic bacteria are indirectly reduced. We found that large amounts of endotoxin-producing bacteria Enterococcaceae and Enterobacteriaceae were reduced by EGCG supplementation, which were linked with intestinal inflammation. Additionally, the relative abundance of these conditionally pathogenic bacteria increased in C. difficile patients [Lactobacillaceae and Lachnospiraceae in EL and EH group with C. difficile infection. The limitation of the study is that no samples were collected before infection, which would rasie concerns about the similarity of the microbiota between the groups. However, we conducted randomization and control variables to reduce differences. Subsequent experiments should collect a series of consecutive samples.In addition to the protective effect on the intestinal barrier, microbes induced by EGCG promote intestinal microbiota composition and intestinal integrity . One of patients . After tC. difficile infection. EGCG improves the intestinal microbiota and its metabolites. As dietary proanthocyanidins bypass the intestine and reach the microbiota, they are biotransformed into metabolites that affect the body in a variety of ways [C. difficile infection. Arginine and aspartic acid are strong commensal agents associated with C. difficile spores [After EGCG intervention, alterations in the intestinal microbiota and its metabolites may be associated with improving  of ways . As a re of ways ,40. Addie spores . SCFAs, e spores . SCFAs ie spores ,44. In ce spores . It is uC. difficile infection. Transcriptomic analysis showed that the protective effect of EGCG may be associated with cytokine–cytokine receptor interactions, Th17-cell differentiation, and the PPAR signaling pathway. PPARs are a group of nuclear receptors that primarily regulate various metabolic processes  and have a variety of physiological activities such as inflammation, cell proliferation, reproduction, and cellular specialization [Although it has been previously shown that EGCG functions as a direct antioxidant to regulate intestinal health, our experimental results suggest that this is not the only mechanism through which EGCG ameliorates lization . The Th1lization ,48.C. difficile infection using a mouse model. Potential mechanisms for the effect of EGCG include the amelioration of C. difficile-induced alterations in inflammation pathways and the homeostasis of the intestinal microbiota and metabolome. In this study, we were able to learn more about the benefits of EGCG in intestinal inflammatory disease progression, and the bacterial taxa and genetic molecules identified may serve as potential disease biomarkers or even drug targets for future validation.In conclusion, we investigated the benefits of the intestinal microbiota and its metabolites regarding the effect of the prebiotic EGCG on


Helicobacter species in experimental infection and routine health monitoring of laboratory mice. Therefore, fluorescent bead-based hybridization assays for Helicobacter sp. DNA and serology were developed. Multiplex PCR amplicons  and antibodies against H. pylori, H. hepaticus, H. bilis were assessed in naturally and experimentally infected mice, and results compared to conventional PCR. Species-specific and sensitive detection of seven Helicobacter spp. <100 copies/PCR, and of two species <1000 copies/PCR was successfully established in the Helicobacter multiplex DNA finder. The novel assay was highly comparable with conventional PCR . Antibody detection of H. hepaticus and H. bilis showed low sensitivity  and cross-reactivity in H. typhlonius-infected mice. Infection experiments showed that antibodies develop earliest two weeks after DNA detection in feces. In conclusion, detection of Helicobacter antibodies showed low sensitivity depending on the timing relative to infection. However, Helicobacter multiplex DNA finder is a sensitive and specific high-throughput assay applicable in routine health monitoring for laboratory animals.High-throughput multiplexed assays are needed to simplify detection of  Helicobacter includes over 30 formally assigned species infecting a variety of hosts, including rodents and humans. The genus is subdivided into two groups, enterohepatic and gastric Helicobacter spp., depending on their properties to colonize the respective organs . Al. AlH. pygy assay . BrieflyHelicobacter multiplex DNA finder was indicated by chi-square test of independence and by kappa statistics (k), where a value of one represents complete, zero represents no agreement.The agreement of conventional PCR and the p-value < 0.05 was considered statistically significant.Continuous MFI values obtained in multiplex serology assays were compared between groups by Wilcoxon–Mann–Whitney test. A All graphical presentations and statistical analyses were carried out using GraphPad Prism 8 .Helicobacter and one Campylobacter species plus a genus-specific Helicobacter sequence were applied to Helicobacter multiplex DNA finder resulting in an analytical sensitivity below 100 copies/PCR for all Helicobacter/Campylobacter spp. but 1000 copies/PCR for H. typhlonius and H. heilmannii and 10,000 for C. jejuni when targeting the gyraseA gene , all could be detected, indicating a high reproducibility.Ten-fold dilution series of plasmid DNA containing the selected target sequences of eight seA gene . Despite6 plasmid copies/PCR that contained the selected target sequences of nine Helicobacter and Campylobacter spp. plus one genus-specific Helicobacter sequence to the Helicobacter multiplex DNA finder. Additionally, specificity of polA gene detection was tested by applying 50 ng of fecal DNA. Detection of all Helicobacter and Campylobacter spp.  and the polA-specific quality control was highly specific. Only H. cinaedi and H. typhlonius, H. cinaedi and H. pylori showed weak expected cross-reactivities  due to the high homology of probe and primer sequences, with only four to six mismatches in their nucleotide sequence  of H. canis, C. lari, and C. coli were not detected in the Helicobacter multiplex DNA finder, although sequence homology of the selected Helicobacter and Campylobacter sequences were between 88 and 90%. Specificity was analyzed by applying 10sequence . The uneHelicobacter multiplex DNA finder, the detection of H. hepaticus, H. typhlonius, H. bilis and of the Helicobacter genus were analyzed. Since H. typhlonius showed weak cross-reactivities with polA, H. typhlonius was defined as positive where the polA and H. typhlonius netMFI ratio was below 100.For a direct comparison of the conventional PCR and the novel H. typhlonius and H. cinaedi (0.5%) with a low netMFI value.DNA was extracted from mouse samples (n = 241) including liver (n = 16), duodenum (n = 16) and pooled fecal samples (n = 209) of colony and sentinel mice from routine health monitoring at the DKFZ animal facility and applied to both assays. Of all included negative PCR and hybridization controls (n = 208), one was positive for Helicobacter/Campylobacter (n = 191) was detected. The Helicobacter multiplex DNA finder detected Helicobacter spp. in 191 samples (79%) with 93 single infections with either H. hepaticus as the most prevalent type, followed by H. typhlonius and H. bilis  and/or any H. bilis . The HelHelicobacter spp. and/or genus-negative by the conventional PCR and the Helicobacter multiplex DNA finder and 189 were concordantly positive for any Helicobacter spp. and/or genus, resulting in a kappa of 0.98 (95%CI 0.94–1.00).Fifty samples were concordantly Helicobacter-positive samples, 48 samples were excluded from further analyses since conventional PCR detected Helicobacter genus only without subsequent species-specific analysis.Of all 189 concordantly H. bilis, H. hepaticus and H. typhlonius identified infections ) with 185 concordantly and 41 discordantly fections . In totaH. hepaticus and H. bilis, as well as 13 H. pylori proteins. Four serum samples showed invalid serology results and were excluded from further analysis (Helicobacter-negative mice (n = 57) as well as H. bilis (n = 26), H. hepaticus (n = 86), and H. typhlonius (n = 23) single DNA-positive mice to assess sensitivity and species-specificity of Helicobacter multiplex serology.In total, sera of n = 338 mice from the health monitoring of animals housed at DKFZ were analyzed for antibody responses to each of the seven antigens of analysis . Among tH. hepaticus proteins except HH0407 were significantly higher in the H. hepaticus single DNA-positive group compared to the Helicobacter genus DNA-negative group. However, for proteins HH0713, HH0966, and HH1201 only, these antibody responses did not show cross-reactive responses in H. bilis or H. typhlonius DNA-positive mice (Continuous antibody responses (in MFI) to all ive mice A.H. bilis proteins, only antibody responses to P167C and D were significantly higher in the H. bilis DNA-positive mice compared to DNA-negatives. Antibody responses to P167D were also found to be significantly higher in the H. typhlonius DNA-positive group  and HH0713 (64%). However, 44% of H. typhlonius and 35% of H. bilis single-positive mice were also sero-positive to HH0435 . Sero-prevalence to these two proteins was low in H. hepaticus  and H. typhlonius  single-positive mice . The expected Helicobacter spp. infection status of all donor mice was confirmed by the Helicobacter multiplex DNA finder and remained positive at wpe 0, 2 and 16. In total, 178 of 180 (98.9%) bi-weekly collected fecal samples of exposed mice had a valid DNA quality. H. typhlonius DNA was identified two wpe in all five single-infected mice and in nine out of ten mice with dual infection. H. typhlonius DNA-positivity was detected until 16 wpe in single-infected mice and in mice dual-infected with H. hepaticus. However, in mice dual-infected with H. bilis, H. typhlonius DNA was not detectable after six wpe , and had a good DNA quality and were negative for Helicobacter genus but positive for H. hepaticus (n = 3) and H. typhlonius (n = 1).Three negative control mice co-housed with a H. bilis and H. hepaticus proteins with multiplex serology. In the case of H. hepaticus and H. typhlonius single- and dual-infection, the animals developed an antibody response to proteins HH0435 and HH0713 at the earliest four wpe and two weeks after DNA detection in feces. H. hepaticus single-infected mice reached a median plateau of ~8000 and ~6000 MFI to antigens HH0435 and HH0713 eight and 14 wpe, respectively   B. H. hepectively C. None oH. bilis and H. typhlonius dual-infected group developed antibody responses to HH0435 four wpe and two weeks after the first H. typhlonius DNA detection in feces ), with a more sensitive detection of H. hepaticus, H. bilis, and H. typhlonius, analyzing 241 samples with both assays.The imilated . The intRNA copy ,39,40. WHelicobacter spp. are not routinely applied in the health monitoring of laboratory animals. Previous attempts to detect antibodies to different Helicobacter spp. often applied whole or membrane protein extracts that may result in the detection of cross-reacting antibodies and consequently low specificity. The application of individual proteins might increase specificity and a multiplex approach could thereby allow high sensitivity to be maintained [H. pylori multiplex serology, we attempted here to re-assess the performance of serology in the routine health monitoring of laboratory animals [H. hepaticus and H. bilis and applied these in a multiplex serology assay. Comparing antibody responses to results from the Helicobacter multiplex DNA finder in murine samples from routine health monitoring showed, however, a low sensitivity (maximum 71% for H. hepaticus and 62% for H. bilis) of the newly developed serological assay. Additionally, we detected a substantial cross-reactivity with H. typhlonius-infected mice, resulting also in low species-specificity.So far, serological assays for the detection of intained ,26,43. B animals . To do sHelicobacter antibodies are indeed detectable two weeks after Helicobacter DNA is identified, and that a plateau of maximum antibody response was observable six weeks after DNA detection. However, this infection experiment of immune-competent mice and sampling at defined time-points after infection does not reflect real-life scenarios in routine health monitoring.The low sensitivity of the antibody assay in comparison to DNA detection in fecal samples likely results from the differential ability of the infected mice to build an antibody immune response due to their genetic background and genetic modification and/or the timing of sampling in relation to time-point of infection. Using experimentally infected CD1 outbred mice, we were able to show that Helicobacter multiplex DNA finder when it comes to sensitivity and species-specificity. However, due to low-costs and high-throughput application in combination with screening for other serologically assessed infectious agents [Helicobacter serology could give the opportunity to monitor more frequently than the FELASA-recommended quarterly diagnostics. A possible screening scenario could also be to run the Helicobacter multiplex DNA finder as triage test for serologically positive mice [In summary, the serological assay developed here is inferior to the s agents  the multive mice .Helicobacter multiplex DNA finder as well as multiplex serology, could be in human samples. In humans, H. pylori was classified as a carcinogen for the development of gastric cancer in 1994, and since then multiple biomarker studies have been conducted to identify individuals at risk for developing cancer [Helicobacter spp. other than H. pylori in human disease is less well described. H. hepaticus and H. bilis have also been isolated from the human enterohepatic tract, but their prevalence and specific disease associations in humans remain unclear [Helicobacter multiplex serology described here has already been applied in a study assessing the association of different Helicobacter spp. with non-alcoholic fatty liver disease, a precursor of liver cancer, and indeed sero-positivity to H. hepaticus protein HH0713 was associated with disease [Helicobacter multiplex DNA finder and the Helicobacter multiplex serology can in future be applied to large epidemiological studies with human samples to obtain more knowledge about the prevalence of H. hepaticus and H. bilis and their potential association with cancer. However, DNA extraction methods might be different in human samples and could require the establishment of new protocols.An additional application of interest, for both the g cancer . A causa unclear ,51,52,53 disease . Both thHelicobacter spp. by the detection of DNA in the Helicobacter multiplex DNA finder and antibodies in the Helicobacter multiplex serology. While the Helicobacter multiplex serology lacks species-specificity and sensitivity, the Helicobacter multiplex DNA finder can be readily implemented in the routine health monitoring of laboratory animals. Due to the high-throughput applicability, however, both assays are promising tools to be used in large epidemiological studies investigating Helicobacter infection and disease correlation in humans.We have developed two multiplex assays identifying infection with different


Giardia duodenalis is the underlying cause of a significant number of outbreaks of gastrointestinal illness in humans and animals worldwide. The purpose of this study was to elucidate the prevalence and genetic diversity of G. duodenalis in captive alpine musk deer (Moschus chrysogaster) in China. A total of 202 fecal samples were collected from three farms in Gansu Province, China. Identification of G. duodenalis was conducted by nested PCR targeting the genes coding for SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi). The overall prevalence of G. duodenalis in captive alpine musk deer in surveyed area was 19.3% (39/202). Two G. duodenalis genetic assemblages were identified, namely assemblage A and E. Mixed genotype infections (A+E) were found in 15.4% (6/39) of positive samples. Multilocus genotyping (MLG) analysis of G. duodenalis isolates revealed six novel assemblage A MLGs formed by two newly-described MLG-subtypes which belonged to sub-assemblage AI. To the best of our knowledge, this is the first report on MLG of G. duodenalis isolates in captive alpine musk deer in China. The presence of zoonotic assemblages and sub-assemblages of G. duodenalis in deer species suggests that these animals may potentially act as a reservoir of this protozoan for humans. Giardia duodenalis  is the most prevalent protozoan pathogen, commonly found in the intestinal tract of humans and animals worldwide  or indirectly   . Approxiountries . Giardiaountries . Nitroimountries .Giardia species have been recognized  considering different host distribution  in the world, which are mainly distributed in the Qinghai Tibet Plateau, Sichuan and Gansu Province . Farm owners’ consent was obtained prior to proceeding to fecal sample collection from selected animals.In September 2020, 202 fecal samples were collected from three farms in Gansu Province, China , according to manufacturer’s instructions. The extracted DNA was stored at -20°C until PCR assay. Four genes were used for G. duodenalis genotyping by nested PCR, namely SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) . Nucleotide sequences were aligned and edited with DNAstar Lasergene Editseq 7.1.0 (https://www.dnastar.com/software/lasergene/) and Chromas Pro 2.1.10 (http://technelysium.com.au/wp/chromaspro/). Genotypes and subtypes of G. duodenalis were determined by aligning reference sequences available in NCBI GenBank database using ClustalX 2.1 . To determine genetic diversity among the isolates, concatenated sequences (bg-tpi-gdh) from each isolate at the three analyzed loci were aligned with reference sequences. Neighbor-joining (NJ) analysis was performed using MEGA 7.0 (http://www.megasoftware.net/) to infer the phylogenetic relationships of concatenated sequences based on the Kimura-2 parameter model.All positive secondary PCR products from SSU rRNA, bg and tpi genes of G. duodenalis are available in the NCBI GenBank database under the accession numbers OM273018-OM273020, respectively.Representative nucleotide sequences of G. duodenalis by PCR at the SSU rRNA locus  and assemblage E . Mixed genotype infections (A+E) were found in 6 of 39 samples.Overall, a total of 39 samples  were confirmed to be bg gene was obtained from 18 of 39 G. duodenalis-positive isolates; among these, 13/18 (72.2%) of isolates were identified as belonging to genetic assemblage A and 5/18 (27.8%) of assemblage E  and A3 (n=9) sequences were identical to the sequences MK639171 and MK639172, respectively. In addition, A2 (n=2) sequences showed a SNP (C21T) compared to the sequence MK639173. At the gdh locus, seven and three isolates were successfully amplified and identified as assemblage A and E, respectively. The subtypes A1 (n=3) and A2 (n=4) were consistent with the sequences MN047217 and the MK645799, respectively. Moreover, the three assemblage E sequences were identical to the sequence MK645786.Sequence analysis of the bg, tpi and gdh loci, and formed six assemblage A MLGs after removal of sequences of mixed infection samples  have historically been bred in China, which include siberian musk deer (M. moschiferus), forest musk deer (M. berezovskii), black musk deer (M. fuscus), alpine musk deer (M. chrysogaster), himalayan musk deer (M. leucogaster), and anhui musk deer (M. anhuiensis) (Six species of musk deer (uiensis) . In prevuiensis) . In partuiensis) .Giardia spp. infects a broad range of hosts including humans, livestock, companion animals, wildlife and birds  in Croatia  in alpine musk deer, which is higher than that reported in sika deer (0.6% and 0.8%) and forest musk deer (2.2%) in China. The discrepancies in infection rates of G. duodenalis may be related to the differences in geographical location, sampling season, animal species, and sample size. To the best of our knowledge, this is the first study to isolate and characterize G. duodenalis from alpine musk deer in China using MLG.nd birds . However Croatia . In the gdh, bg and tpi genes have currently been widely used for genotyping G. duodenalis isolates from human and animal samples in order to obtain high-sequencing resolution  and D (mainly found in canines) have also been reported occasionally in these animal hosts  in the genomes of assemblages A and E  can be found in the article/supplementary material.The animal study was reviewed and approved by Research Ethics Committee of Tarim University. Written informed consent was obtained from the owners for the participation of their animals in this study.ZC, QW, XH, JB, BZ, BW, and XG performed the experiments. ZC drafted the manuscript. MQ and JL critically revised the manuscript. All authors contributed to the article and approved the submitted version.The research was funded by the National Natural Science Foundation of China (32102689), Science and Technology Planning Project of Henan Province (222102110240), the Program for Young and Middle-aged Leading Science, Technology, and Innovation of Xinjiang Production & Construction Group (2018CB034), and Scientific research project of Xuchang University (2022GJPY009 and 2022YB034).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.