Thymus. It has been used as a therapeutic agent since ancient times. Thymus comprises numerous molecular species exhibiting diverse therapeutic properties that are dependent on their biologically active concentrations in the extracted oil. It is therefore not surprising that oils extracted from different thyme plants present different therapeutic properties. Furthermore, the phenophase of the same plant species has been shown to yield different anti-inflammatory properties. Given the proven efficacy of TO and the diversity of its constituents, a better understanding of the interactions of the various components is warranted. The aim of this review is to gather the latest research findings regarding TO and its components with respect to their immunomodulatory properties. An optimization of the various components has the potential to yield more effective thyme formulations with increased potency.Thyme oil (TO) is derived from the flowers of various plants belonging to the genus  Thymus serphyllum) is the wild relative of all cultivated species [Thymus vulgaris L.). Leaf morphology is one of the distinct features unique to the genus Thymus. The leaves are typically tiny (less than 1/8 inch long and 1/16 inch wide), narrow and elliptical, greenish-gray in color, and grouped in whorled phyllotaxy. Thyme flowers are white, yellow, or purple whorls that terminate branches. Some of the widely used cultivars of thymes for TO are Thymus rumidicus hispánicos, Thymus zygis, Thymus vulgaris, Thymus hyemalis, Thymus mastichina, Thymus citríodotus, Thymus corydothymus, Thymus loscossi, Thymus pipirella, and Thymus communis.Thymus is a perennial evergreen herb of the angiosperm plant family Lamiaceae (mint plant family) that has 350 species and 36 subspecies and is native to Europe, North Africa, and Asia . Because species . Garden Thymus vulgaris and Thymus magnus Nakai (Thyme oil (TO) is a complex mixture of at least 70 different species . Thymol us Nakai . It is aThymol is one of the main constituents of TO  within 8 days. In a different study, a combination of thymol and carvacrol reduced production of the inflammatory cytokines IL-25, IL-33, and Thymic Stromal Lymphopoietin (TSLP) in BEAS-2B-transformed human bronchial epithelial cells stimulated with chitin [Multiple studies highlight the anti-inflammatory properties of thymol ,19,20. Ind MCP-1 . Thymol h chitin . SHIP-1 A recent study utilizing U87 human malignant glioblastoma cells has shown that thymol induces apoptosis, increases Reactive Oxygen Species (ROS), and increases levels of the proapoptotic factors Bax and p53 . SimilarStreptococcus mutans by 50% [S. mutans by inducing autolysis, stress growth inhibition, and reduced biofilm formation. Currently, thymol-rich extracts are generally used as an expectorant in coughs associated with cold and also in dentistry as a disinfectant [E. coli. However, for E. faecalis and S. aureus, growth inhibition required a higher concentration. Antibacterial properties of thyme oil rich in thymol was also tested against the methicillin resistant (MRSA) strain [T. vulgaris to study the antimicrobial properties using MRSA strain S. aureus isolated from the oral cavity of patients suffering from periodontitis and pharyngitis. Thyme oil concentration of 0.01% (v/v) and 0.05% effectively reduced the growth up to 53% and 76%, respectively. The main components of volatile oil used in this research were phenolic monoterpenes, including thymol. In a similar study by Tohidpour et al. (2010), thymol extracted from T. vulgaris was more effective than essential oil from Eucalyptus globulus Labill in controlling the growth of MRSA [Pseudomonas aeruginosa even though the thymol level difference was nonsignificant . Recently, thymol-rich Thyme species are gaining attention for their antifungal properties. For example, studies on antifungal properties of thyme oil extract rich in thymol has been shown to be effective against growth of the fungal genus Cryptococcus, which is a pathogen responsible for cryptococcosis [Thymol has also been evaluated for its potential application as an antimicrobial agent. A thymol concentration of 100 μg/mL reduced viability of the dental caries causing bacteria s by 50% . Thymol nfectant . Thymol nfectant . The stu) strain ,30. Kryv of MRSA . Surpriscoccosis . In thiscoccosis .Recently, Liggri et al. (2023) have shown that both thymol and carvacrol bind to the novel mosquito protein AgamOBP5, an odorant-binding protein that is involved in the odorant perception process of mosquitoes . This waThymus capitatus plants was shown to be very high in carvacrol (79.9%) [Thymus vulgaris plants was shown to be much lower in carvacrol (1.56%) [2 and COX-2 levels were reduced with carvacrol.Carvacrol content in TO also varies significantly depending on the thyme species. TO derived from  (79.9%) . On the  (1.56%) . Similar (1.56%) . Mechani (1.56%) . IL-6, ICarvacrol showed analogous antitumoral properties as thymol in a study involving oral squamous carcinoma cells . Keap1/NThymus vulgaris L. has been shown to exhibit potent antibacterial activity against several strains of bacteria, including Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa [T. vulgaris L. had a minimum inhibitory concentration (MIC) of 0.5–1.5 mg/mL against these bacterial strains. Similar results have been observed by Gedikoğlu et al. (2019) in evaluating the antimicrobial properties of T. vulgaris L. on a series of gram-positive and gram-negative bacteria  [p < 0.05) higher antibacterial activity against bacteria than did the hydrodistilled essential oil extraction. Antibiotic-resistant pathogenic bacteria are gaining more attention and various plant derived species are being explored as alternative antimicrobial agents. A comparative study on essential oil extract from different herbs including T. vulgaris reported the synergistic effectiveness of carvacrol against 32 erythromycin-resistant Group A Streptococci [Streptococci. The same study evaluated the antibacterial properties of carvacrol alone and in combination with erythromycin. Surprisingly, carvacrol from T. vulgaris alone was effective in both treatment types’ ability to inhibit growth of Group A Streptococci, further enhancing the potential use of carvacrol as an effective phytotherapeutic agent against antibiotic-resistant bacteria [In addition to its anti-inflammatory properties, carvacrol has been extensively studied for its antibacterial properties. Several studies investigating the effects of carvacrol extracted from different Thyme species on various bacterial strains have been carried out. Carvacrol extracted from ruginosa . In theiimurium) . Surprisptococci . The Minbacteria . CarvacrThymus spp. show the lowest antibacterial activity compared to oxygenated compounds, especially phenol-type compounds such as thymol and carvacrol [Thyme spp. were also evaluated. Carvacrol extracted from T. zygis L. was found to be effective against several bacterial strains, including S. aureus and E. coli. An MIC of 0.16–0.63 mg/mL carvacrol extracted from T. zygis L. was found to be effective against controlling the growth of these bacterial strains. The antimicrobial properties of essential oil from the genus Thymus could be explained by their high percentage of phenol components. Carvacrol might interfere with the activity of cell wall enzymes, thus inhibiting bacterial growth by damaging their cell wall. A recent report investigating the antibacterial properties of carvacrol provided the possible mechanism of carvacrol against Streptococcus pyogenes [Among different chemotypes, hydrocarbon monoterpenes in essential oils extracted from arvacrol . The antpyogenes . CarvacrThymus cultivars as antifungal agents has been reported recently [T. vulgaris against eight yeast species and eight filamentous fungi isolated from mucocutaneous fungal infection pathogens for skin diseases. For this study, TO was extracted from fresh leaves and floral heads. Out of 25 bioactive compounds in the thyme oil, carvacrol (56.8%) was detected in high amounts, followed by p-cymene (12.8%), γ-terpinene (11.17%), and thymol (3.99%). A series of fungal species were used for the sensitivity assay, which included different strains of yeast , and filamentous fungus . Out of three concentrations , TO concentration of 60 μL was effective against all tested yeast strains as compared to the positive control (1% Hexamidine). Candida tropicalis and C. parapsilosis were the most vulnerable strains to the TO, with zones of inhibition of 50 mm and 60 mm. Among filamentous fungi, TO was more effective against Aspergillus terreus and A. fumigatus . Similar antifungal properties of carvacrol against Candida tropicalis were reported by other studies as well [Thymus rich in monoterpenic alcohols and/or volatile phenols [Botrytis cinerea, a primary pathogen causing stem and fruit rot during pre- and postharvest of ornamental crops, fruits, and vegetables [Thymus species such as T. zygis and T. vulgaris, with higher quantities of phenols, also demonstrated a wide spectrum of inhibitory growth against a range of pathogenic filamentous fungi and yeasts, with decreased sensibility to antifungal drug [The potential application of carvacrol from various recently ,48,49,50 as well ,52. In a phenols ,54. Anotgetables . Out of gal drug ,54,55. HP-cymene is typically found at lower concentrations in TO compared to thymol and is a component of other essential oils including cinnamon and cumin . P-cymenP-cymene’s antitumoral effects were reported in a high-fat diet colorectal cancer rat model . A signiThymus vulgaris have a much lower content in relation to the Thymus capitatus species [γ-Terpinene belongs to a group of isomeric compounds referred to as monoterpenes. Their difference lies in the position of the two carbon–carbon double bonds see . γ-Terpi species ,34. It iThymbra capitata TO and, by extension, its components were mostly attributed to p-cymene and carvacrol where strong synergism was reported against Gardnerella spp [2. Inhibition of the COX-2 enzyme with nimeluside (selective COX-2 inhibitor) abolished the IL-10 increase. Furthermore, with diminished IL-10 production, the inhibition of IL-1β and IL-6 was lost. γ-Terpinee in IL-10-deficient mice did not produce any inhibition on IL-1β and IL-6, suggesting the PGE2/IL-10 pathway as a mechanism for its anti-inflammation. This pathway is not utilized by carvacrol, thymol, and p-cymene and makes γ-terpinene unique particularly in terms of the induction of IL-10.There is much less research on γ-Terpinene compared to thymol, carvacrol, and p-cymene. The antimicrobial properties of ella spp . Nonetheella spp . InteresLinalool is another constituent of TO, but with lower prevalence than thymol, carvacrol, and p-cymene. A terpene alcohol, linalool is found in a variety of flowers and seeds and their essential oils . LinalooMultiple studies have investigated linalool’s anti-inflammatory qualities in a variety of systems. Cigarette smoke-induced inflammation was inhibited by linalool in mice . ReductiProtective effects of linalool have been reported in neurodegenerative animal models as well . The aniThyme oil (TO) has a proven record of therapeutic properties and characterized in detail anti-inflammatory mechanisms see . HoweverIn addition, future research directions relating to TO and its derivatives may include synergistic formulations with other drugs. For example, the anti-inflammatory properties of thymol, carvacrol, p-cymene, γ-terpinene, and linalool can be used in conjunction with widely used corticosteroids. Formulations composed of synthetic corticosteroids and TO constituents may allow for lower concentrations of the synthetic component and achieve a reduction in side effects typically seen when using only the synthetic drugs. A similar approach may be employed with antibiotics where the use of TO components along with antibiotics may permit a reduction in concentration of the synthetic antibiotic and a reduction in side effects and antibiotic resistance. This strategy has been used for many years now with clavulanic acid and amoxicillin. Another future direction worth investigating is the use of carvacrol and thymol in combination with known synthetic insect repellants where the formulation will permit a lower concentration of the synthetic repellants. The natural scent of carvacrol and thymol will be an added benefit.In conclusion, there is ample scientific evidence that TO and its constituents have diverse therapeutic properties. However, it is unlikely that they can compete or replace synthetic drugs with similar and often more potent properties. Future research should focus on optimizing formulations of TO constituents by themselves and with synthetic drugs to maximize their potency while reducing their toxicity.


Patients with peripheral artery disease (PAD) are at an increased risk of major adverse cardiovascular events, and those with disease in the lower extremities are at risk of major adverse limb events primarily driven by atherothrombosis. Traditionally, PAD refers to diseases of the arteries outside of the coronary circulation, including carotid, visceral and lower extremity peripheral artery disease, and the heterogeneity of PAD patients is represented by different atherothrombotic pathophysiology, clinical features and related antithrombotic strategies. The risk in this diverse population includes systemic risk of cardiovascular events as well as risk related to the diseased territory . Moreover, until the last decade, clinical data on antithrombotic management of PAD patients have been drawn from subanalyses of randomized clinical trials addressing patients affected by coronary artery disease. The high prevalence and related poor prognosis in PAD patients highlight the pivotal role of tailored antithrombotic therapy in patients affected by cerebrovascular, aortic and lower extremity peripheral artery disease. Thus, the proper assessment of thrombotic and hemorrhagic risk in patients with PAD represents a key clinical challenge that must be met to permit the optimal antithrombotic prescription for the various clinical settings in daily practice. The aim of this updated review is to analyze different features of atherothrombotic disease as well as current evidence of antithrombotic management in asymptomatic and secondary prevention in PAD patients according to each arterial bed. Peripheral artery disease (PAD) encompasses a variety of non-coronary artery diseases, and its prevalence varies based on screening approaches and clinical features. Recent data reveal a global prevalence of 80 million strokes, the majority 87%) of which are ischemic . Estimat% of whicAtherothrombosis represents the common pathophysiologic process in coronary and non-coronary artery disease in patients affected by atherosclerotic damage. PAD involves the same CV risk factors as coronary artery disease (CAD), including arterial hypertension, diabetes mellitus, dyslipidemia, smoking, history of CV disease, chronic kidney disease, life habits, history of radiation therapy, psycho-social and genetic factors ,5. TheseExtracranial carotid atherosclerosis can be readily detected with non-invasive assessment such as high-resolution B-mode carotid ultrasonography (Duplex US), CT and MRI scan also able to detect subclinical atherosclerosis ,5. CarotAtherosclerosis is frequently associated with abdominal aortic disease, especially in polyvascular disease ,12. WhilAtherosclerosis is a common LEPAD feature that can explain symptoms and signs related to different clinical presentations, from claudication to ALI/CLTI . Lower eGuideline-directed medical therapy (GDMT) includes antithrombotic therapy as one of the cornerstones of multidimensional management, which includes structured exercise and lifestyle modification in order to reduce MACE and MALE ,5,6. TheGDMT includes antithrombotic therapy in carotid artery disease in the presence of a ≥50% stenosis since no RCT has assessed antithrombotic therapy in non-significant carotid stenosis . In asym12 receptor inhibitors. The benefit of ticagrelor compared to aspirin in patients with acute cerebral ischemia (non-severe IS or high-risk TIA) was assessed among 13,199 patients enrolled in the Acute Stroke or Transient Ischemic Attack Treated with Aspirin or Ticagrelor and Patient Outcomes (SOCRATES) trial. Compared with aspirin, ticagrelor monotherapy (90 mg twice daily) was not superior to aspirin with respect to the primary outcome of stroke, MI or death at 90 days . No differences were detected in major bleeding (according to PLATO classification) comparing the two study groups  . A . A p = 0p = NS), endoleak  and MI  [After a peripheral embolic event from abdominal aortic plaque, GDMT recommends SAPT (aspirin or clopidogrel), whereas little evidence is available for DAPT in this clinical scenario ,19. Due  p = NS) . Very li p = NS) . Moreove p = NS) . An operAntithrombotic management recommendations in patients with isolated asymptomatic LEPAD are conflicting. ESC guidelines and a recent ESC position paper do not recommend SAPT in asymptomatic LEPAD, whereas ACA/AHC guidelines acknowledge SAPT as a reasonable option in asymptomatic patients with abnormal ABI (≤0.90) to reduce MI, IS and vascular death risk ,5,19. Twp < 0.05) with no differences in intracranial hemorrhage  [p = 0.022) counterbalanced by increased TIMI major bleeding of 2.0% vs. 1.1%, p < 0.0001. The overall benefit was greater among PAD patients compared to those without [p < 0.001) without differences in severe bleeding  according to the GUSTO classification [p = 0.013) with no differences in bleeding adverse events [p < 0.001) and placebo (65 ± 135 m with p < 0.001) with more side effects as headache, diarrhea and palpitation in cilostazol group (p < 0.001) [p = 0.04) but this benefit disappeared by 1 year . Moreover, the bleeding complication rate at 6 months after LER was comparable among groups  [p = NS) [GDMT supports antithrombotic therapy for the secondary prevention of LEPAD with SAPT (aspirin or clopidogrel) with a class I recommendation ,5,59. AC p = NS) . In the  p = NS) . However p = NS) . As in c p = NS) . Unless  p = NS) . A DAPT  p = NS) . DAPT wi p = NS) . In the  without . Ticagre without . Unless  without . Overall without . Subsequ without . Further without . Vorapax without . However without . In the  without . Antithr without . There wfication . Cilostae events . Cilosta< 0.001) . In subs< 0.001) . However p = NS) . The Edo p = NS) . The eff p = NS) . No diff p = NS) . Subsequ p = NS) . In the  p = NS) . It was  p = NS) . It is i[p = NS) . While t[p = NS) . Conside[p = NS) . Althoug[p = NS) . An oper12 inhibitors in daily practice. Until last years, after DAPT discontinuation, the SAPT strategy included aspirin but new data suggest P2Y12 monotherapy as an effective antithrombotic strategy after short DAPT (up to 6 months) in ACS patients with high bleeding risk features [p = 0.02. There were fewer life-threatening and major bleeding adverse events in the rivaroxaban arm  and in the rivaroxaban plus vitamin K2 arm  compared to the VKA arm [Rx and Xisomab 3G3) were conducted in dialysis patients with promising results on FXI inhibition with no evidence of impaired major bleeding rates compared to placebo [12, such as clopidogrel and ticagrelor, there are few available data, particularly on safety profiles in PAD patients. Balancing thrombo/hemorrhagic risk, an SAPT strategy with aspirin or clopidogrel is recommended according to Kidney Disease Improving Global Outcomes guidelines in patients with end-stage renal disease/dialysis with concomitant ASCVD [Antithrombotic therapy in PAD patients affected by polyvascular disease and CV comorbidities may represent a challenge in daily practice. Considering patients with previous MI, the benefit of a DAPT strategy, including aspirin and ticagrelor, provided an absolute risk reduction of 4.1% for MACE and a 35% of MALE reduction counterbalanced by an increase of TIMI major bleeding  in the PAD subgroup of PEGASUS-TIMI 54 RCT . A seconfeatures . DAPT wifeatures . Atrial features . In the features . Howeverfeatures . The efffeatures . More refeatures . Hence, features . In casefeatures . Patientfeatures . Moreovefeatures . These p VKA arm . Further placebo . Other o placebo . Hence, nt ASCVD .PAD patients are at increased risk of MACE and MALE events that can be reduced by antithrombotic therapy. We have provided an overview of current evidence in different clinical settings in PAD and propose an algorithm for antithrombotic therapy management in daily practice. In patients undergoing revascularization, evidence supports more aggressive antithrombotic therapy, specifically, dual pathway inhibition after LER, irrespective of the type of intervention. The optimal management of patients undergoing revascularization for carotid and abdominal aortic disease is less clear. The development of newer antithrombotic strategies  may play an important role in the future.


Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) among HPV-positive women undergoing colposcopy at the Second Xiangya Hospital of Central South University, Hunan, China. Additionally, we aimed to assess the impact of C. trachomatis or M. genitalium co-infection with HPV on the severity of cervical lesions.The aim of this study was to determine the prevalence of C. trachomatis and M. genitalium. Multivariate logistic regression analyses were performed to examine the association between sexually transmitted pathogens and cervical lesions.We collected HPV data, cervical cytology results, and demographic information from 439 women attending colposcopy. Cervical swabs were obtained for simultaneous amplification testing (SAT) of C. trachomatis was detected in 17 (3.87%) individuals, and M. genitalium in 16 (3.64%) individuals. There was no co-infection of C. trachomatis and M. genitalium. The highest prevalence of M. genitalium was observed in women aged 19–30 years , with a subsequent decline in prevalence with increasing age (Ptrend = 0.014). The most common HPV subtype in our study was HPV52 (30.79%), followed by HPV16 (18.62%), HPV58 (16.95%), and HPV53 (10.02%). Infection with HPV16 , HPV31 , and HPV33  was associated with an increased severity of cervical lesions, while HPV53 infection was not likely to lead to advanced cervical lesions . The leukocyte level in vaginal secretions (P = 0.042) and cervical cytology results (P < 0.001) showed associations with the degree of cervical lesions. However, there was no significant association between C. trachomatis or M. genitalium infection and the severity of cervical lesions, nor with their co-infection with HPV16.Among the participants, Chlamydia trachomatis or Mycoplasma genitalium and the degree of cervical lesions in HPV-positive population in Hunan, China. Our findings emphasized the need to pay more attention to M. genitalium infection among young women. Increased levels of leukocytes in vaginal secretions may be linked to cervical lesions. HPV16, HPV31, and HPV33 in Hunan province, China, may exhibit higher cervical pathogenicity.There was no correlation between co-infection of  In early 2021, the GLOBOCAN 2020 database, produced by the International Agency for Research on Cancer (IARC), revealed that cervical cancer (CC) ranks as the fourth most common cancer worldwide among women in terms of both incidence and mortality . While tChlamydia trachomatis (CT) is the most common causative agent of bacterial sexually transmitted infections (STIs) and can result in various complications in women, including pelvic inflammatory disease (PID), hydrosalpinx, and infertility [C. trachomatis infection and HPV infection, indicating they are mutual risk factors [C. trachomatis infection rates between HPV-infected and non-HPV-infected groups or between groups with different cervical biopsy results [C. trachomatis infection and high-risk HPV infection [C. trachomatis and HPV infection remains controversial and requires further investigation. Additionally, most existing studies focus solely on the relationship between C. trachomatis infection and HPV infection, disregarding the fact that HPV infection does not necessarily lead to cervical lesions [C. trachomatis infection and cervical lesions directly reflects the impact of C. trachomatis on female cervical health, yet few relevant studies have been conducted.Sexually transmitted pathogens pose a significant public health concern worldwide, particularly for women’s health. ertility . Some st factors , 8. Howe results . Similarnfection . Therefo lesions . TherefoMycoplasma genitalium (MG) is an emerging sexually transmitted pathogen that causes genitourinary tract diseases and has recently gained attention [M. genitalium can cause cervicitis [M. genitalium infections are asymptomatic. The increasing prevalence and antimicrobial resistance of M. genitalium are concerning [M. genitalium is not commonly conducted in most hospitals in China, and epidemiological data on M. genitalium in Hunan Province is lacking. The relationship between M. genitalium, HPV progression, and cervical lesions remains unknown but is of significant importance.ttention . Researcrvicitis  and is arvicitis  and infervicitis . Furtherncerning . HoweverC. trachomatis infection and HPV infection. Moreover, epidemiological data on M. genitalium and its correlation with HPV infection are limited. Therefore, we conducted a study among HPV-positive individuals who underwent colposcopy in Hunan Province, China. This study aimed to investigate the distribution of HPV subtypes, as well as the prevalence and age distribution of C. trachomatis and M. genitalium. Additionally, it analyzed the risk factors for cervical lesions and examined the influence of co-infection with C. trachomatis or M. genitalium on cervical lesions in HPV-infected individuals. These findings have important implications for the prevention and control of infections caused by these pathogens, as well as the management of cervical lesions.Currently, there is inconsistency in the association between Trichomonas vaginalis (TV) at the time of examination, (2) women had sexual activity or gynecological examinations within the past 24 h, (3) women who are menstruating, (4) pregnant women, (5) women who had vaginal douching or used vaginal medications within the past 48 h, (6) women who had undergone hysterectomy, (7) women who had undergone cervical loop electrosurgical excision procedure (LEEP), laser treatment, or cold knife conization (CKC) within the past year, and (8) women with incomplete laboratory measurements. Following the application of these criteria, a total of 439 women (aged 19 to 74 years) were included in the final analysis. The study protocol was approved by the institutional review board of the Second Xiangya Hospital of Central South University  and by the Chinese medical research registration information system (Ref number: MR-43-23-027426). Written informed consent was ensured from all study participants to take part in the study voluntarily after they get informed about the objective and purpose of the study.A cross-sectional study was carried out at the Second Xiangya Hospital of Central South University from July to September 2022. A population of 500 women visiting the hospital’s gynecology department and undergoing colposcopy was invited to participate in the study. The inclusion criteria were as follows: (1) women with a history of sexual activity, (2) positive HPV test results, and (3) informed consent. The exclusion criteria included: (1) women infected with bacterial vaginosis (BV), fungi, or C. trachomatis and M. genitalium. The cervical swab was then placed into 2 ml of a transport and preservation medium  and immediately frozen at -80℃. Acetic acid and Lugol iodine were applied to examine the cervix and vaginal walls. Any suspicious areas were biopsied for histopathological assessment. The histopathological grading included no squamous intraepithelial lesions (NO SIL), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cervical cancer. If multiple biopsy samples were taken simultaneously, the sample with the highest grade was considered for analysis. The researchers will track and record the biopsy results of each patient in the hospital’s medical record system using the patient’s unique card number.The study participants underwent colposcopy performed by a single trained gynecologist. After inserting a vaginal dilator to fully expose the cervix, a flocked swab  was inserted into the cervix and rotated several times to collect cervical secretions for the detection of Before undergoing colposcopy, the patient’s laboratory measurements, including HPV types, cervical cytology tests, and the level of vaginal leukocytes, as well as demographic information such as age, pregnancy history, reproductive history, and menopause, were recorded.Laboratory examination data were collected when the subjects underwent colposcopy.HPV testing was performed using the Human Papillomavirus nucleic acid typing test kit , which allowed for the detection of 21 HPV types. Among these, 15 HPV types were classified as high-risk HPV , and 6 HPV types were classified as low-risk HPV .The pathologists categorized the results of the cervical cytology as follows: Negative for intraepithelial lesion or malignancy (NILM), Atypical squamous cells of undetermined significance (ASC-US), Atypical squamous cells: cannot exclude high-grade squamous intraepithelial lesion (ASC-H), Low-grade squamous intraepithelial lesion (LSIL), High-grade squamous intraepithelial lesion (HSIL), Atypical glandular cells (AGC) and Squamous cell carcinoma (SCC). None of the patients participating in this study had been diagnosed with AGC. Therefore, the AGC cytological grade is not listed in Table The levels of leukocytes in the vaginal secretions of the participants were examined by gynecologists. Based on the number of leukocytes observed in each high-power field, the examination results were classified into three categories: 0–15/HP, 16–30/HP, and > 30/HP.All laboratory measurements were obtained from the Second Xiangya Hospital, Central South University.C. trachomatis and M. genitalium commercial test kits . The magnetic beads containing the target nucleic acid were mixed with the prepared amplification detection liquid. A 30µL magnetic bead suspension was placed into each microreaction tube, and 10µL of preheated enzyme solution was added to start the reaction. The reaction process was set at 42℃ for 1 min per cycle, with a total of 40 cycles. The fluorescence signals in the fluorescein FAM channel were recorded every minute. A Ct value of ≤ 35 indicated a positive result for C. trachomatis RNA or M. genitalium RNA. For samples with Ct values between 35 and 40, retesting was required. A Ct value of < 40 in the retesting was considered positive.In a biosafety cabinet, the swab soaked in the preservation solution was fully ground and then discarded. The same volume of normal saline was added and thoroughly mixed. The target RNA was extracted using magnetic beads by adding nucleic acid extraction solution to the appropriate samples, following the instructions provided by the C. trachomatis and M. genitalium by age. A multivariate logistic regression model was employed to evaluate factors associated with cervical lesions using variables with a P-value of less than 0.1 in univariate analyses. Throughout the study, results were considered statistically significant when P ≤ 0.05.All statistical analyses were conducted using SPSS version 25.0 . The Shapiro-Wilk test was employed to assess the normal distribution of variables. Continuous variables that were not normally distributed are presented as the Median , while categorical variables are presented as percentages. The Chi-square test or nonparametric Kruskal-Wallis test was used to compare the three groups statistically. Chi-square trend tests were used to examine changes in the prevalence of . The median age of the participants was 45 years (IQR 36–53). Based on cervical biopsy results, the 439 HPV-positive patients were categorized into three groups: no squamous intraepithelial lesions group , low-grade squamous intraepithelial lesion (LSIL) group (n = 127), and high-grade squamous intraepithelial lesion (≥ HSIL) group (n = 108). Table C. trachomatis was detected in 17 individuals (3.87%), M. genitalium in 16 individuals (3.64%), and there was no co-infection of C. trachomatis and M. genitalium. Significant differences were observed in the leukocyte level of vaginal secretions among the groups (P = 0.042), with the highest leukocyte level (> 30/HP) observed in the HSIL group (12.38%). Cervical cytology results also showed significant differences among the groups (P < 0.001), with the NILM grade being more common in the group without complicated lesions (81.86%), while the ASC-H (10.19%) and HSIL (15.74%) grades were more common in the HSIL group. No significant differences were found in age, pregnancy history, reproductive history, menopause, HPV single or mixed infection, or the positive rate of C. trachomatis or M. genitalium among the groups.According to the inclusion and exclusion criteria, a total of 439 out of the 500 individuals who underwent colposcopy were included in our study Fig. . The medroup n = 27, and hC. trachomatis and M. genitalium with respect to age. The overall prevalence of M. genitalium was 3.64% , with statistically significant differences observed among different age groups. The highest prevalence was found in women aged 19–30 years , and it steadily decreased as women aged, reaching 1.94%  in women over 50 years old  and in women over 50 years old , and it did not show a trend with increasing age (Ptrend = 0.301).Subjects in our study were stratified by age (ranging from 19 to 74 years), and we examined the changes in the prevalence of C. trachomatis or M. genitalium and the severity of cervical lesions (Table ). The results showed that neither LSIL nor HSIL phenotypes were associated with either C. trachomatis or M. genitalium in any of the models, using the NO SIL phenotype as the reference. After adjusting for all confounding factors, the prevalence of C. trachomatis or M. genitalium was not found to be associated with the severity of cervical lesions .Multivariate logistic regression analyses were conducted to investigate the association between ns Table . The resOut of the 439 women included in our study, 20 had untyped HPV test results, so we analyzed accurate HPV typing results for 419 women. The most prevalent HPV genotype was HPV52 in 129 subjects (30.79%), followed by HPV16 in 78 subjects (18.62%), HPV58 in 71 subjects (16.95%), and HPV53 in 42 subjects (10.02%). In the univariate analysis, HPV genotypes associated with cervical lesions were HPV16 , HPV31 , and HPV33 , indicating that they increased the severity of cervical lesions. Surprisingly, patients infected with HPV53 were less likely to develop advanced cervical lesions , we further explored the combined effects of sexually transmitted infections (STIs) and HPV genotypes on the severity of cervical lesions. Due to the small number of positive cases of C. trachomatis and M. genitalium, and the fact that none of the patients infected with HPV31 and HPV33 were infected with C. trachomatis or M. genitalium, we only investigated the relationship between HPV16 or HPV53 and C. trachomatis or M. genitalium . Since patients infected with HPV53 rarely had C. trachomatis infection, and co-infection was only present in the NO SIL group but not in the LSIL and HSIL groups, statistical analysis could not be performed. Furthermore, no significant interaction was found between HPV53 and M. genitalium (P > 0.05).Having identified the correlation between HPV16, HPV31, HPV33, and HPV53 with the extent of cervical lesions Table , we furtC. trachomatis and M. genitalium infections, as well as the impact of co-infection with HPV on cervical lesions. The aim of the study was to contribute to the existing research in this field. C. trachomatis was detected in 17 participants (3.87%), while M. genitalium was detected in 16 participants (3.64%). Co-infection of C. trachomatis and M. genitalium was not observed. The prevalence of M. genitalium was highest in women aged 19–30 years  and decreased steadily with age (Ptrend = 0.014). It was observed that HPV16, HPV31, and HPV33 may exhibit higher cervical pathogenicity in Hunan province, China. Additionally, a surprising finding was the association between elevated levels of leukocytes in vaginal secretions and cervical lesions. However, neither C. trachomatis nor M. genitalium infection, alone or in co-infection with HPV16, were found to be associated with the severity of cervical lesions. This study provided insights into the pathogenicity of different HPV subtypes on cervical lesions and explored the age distribution of M. genitalium and its relationship with cervical lesions in the HPV-positive population in Hunan province, China. These findings are expected to contribute to the understanding of the epidemic pathology of sexually transmitted pathogens and aid in the prevention, screening, and control of such pathogens.A cross-sectional study was conducted among 439 HPV-positive women who underwent colposcopy in Hunan province, China, to investigate the prevalence and age distribution of C. trachomatis or M. genitalium.The population included in our study had certain unique characteristics. Samples were collected from the colposcopy laboratory, and patients who underwent colposcopy were excluded if they had bacterial vaginitis, fungal vaginitis, trichomonas vaginitis, HIV, or syphilis. This exclusion was done to prevent worsening of the patients’ condition and to avoid the risk of infection for healthcare workers. Therefore, bacterial, fungal, trichomonas, HIV, and syphilis infections were not considered in our study population to eliminate any potential synergistic pathogenic effects they might have with C. trachomatis and M. genitalium was relatively low, with C. trachomatis often having a higher prevalence compared to M. genitalium. For instance, among the general female population visiting gynecology departments in Sichuan province, China, the infection rates of C. trachomatis and M. genitalium were reported as 6.5% and 2.6%, respectively [C. trachomatis and M. genitalium were 11.3% and 1.0%, respectively [C. trachomatis (3.87%) was slightly lower than that reported in other studies, while the infection rate of M. genitalium (3.64%) was slightly higher. Co-infection of M. genitalium with other sexually transmitted pathogens has been reported. In young high-risk women with asymptomatic bacterial vaginosis in the United States, the prevalence of M. genitalium co-infection with C. trachomatis was 29.9%, and co-infection with Neisseria gonorrhoeae was 23.6% [M. genitalium with C. trachomatis was only 0.6%, and with N. gonorrhoeae was 0.1% [C. trachomatis and M. genitalium was not observed, likely due to differences in the study population and the low positive rates of C. trachomatis and M. genitalium.The prevalence of both ectively . In a stectively . In our as 23.6% . Howeverwas 0.1% . In our M. genitalium was observed in women aged 19 to 30 years , and this prevalence declined steadily with age, consistent with previous research [C. trachomatis was higher among women over 50 years old , but not in young women under 30 years of age. This finding contradicts Chen’s research, as Chen et al. discovered that the prevalence of C. trachomatis infection was highest among the group aged ≤ 25 years and gradually decreased with age [C. trachomatis infection was higher in the group aged > 35 years compared to the group aged ≤ 35 years [C. trachomatis is not necessarily exclusive to young women. The inconsistent results may be attributed to differences in study areas and populations. In conclusion, our findings suggest that the growing openness towards sexual concepts and the younger age at first sexual intercourse among Chinese women may increase the likelihood of sexually transmitted pathogen infections in young women, but the health of elderly women should not be disregarded.Studies have indicated that sexually transmitted pathogen infections are more prevalent among young women . Our sturesearch , 17. HowC. trachomatis or M. genitalium were more likely to have elevated leukocyte levels in vaginal secretions [Few studies have explored the relationship between leukocyte levels in vaginal secretions and the severity of cervical lesions. A study conducted in southwest China found that patients infected with cretions . In our cretions , 20. Somcretions , 22. In Persistent infection with high-risk HPV can result in cervical lesions and potentially cervical cancer. The prevalence and distribution of HPV subtypes vary across countries, races, and populations. In our study, the most prevalent HPV genotype was HPV52 (30.79%), followed by HPV16 18.62%), HPV58 (16.95%), and HPV53 (10.02%). These results align with data from other Chinese populations. A study conducted in southern China identified HPV52, HPV16, and HPV58 as the three most common HPV subtypes .62%, HPV. This inC. trachomatis and M. genitalium infection, either alone or in co-infection with HPV16, and the severity of cervical lesions. Regarding C. trachomatis infection, its association with HPV infection and cervical lesions is currently controversial. Several studies have indicated an association between C. trachomatis infection and HPV infection as well as cervical lesions [C. trachomatis infection does not increase the risk of HPV infection and cervical lesions [M. genitalium infection, there is limited research on its association with HPV infection and cervical lesions. A study by A et al. found no association between M. genitalium infection and HPV infection or cervical lesions [Many previous studies have primarily focused on the relationship between cervical cytology results and sexually transmitted pathogens , 24. How lesions , 23. How lesions , 10, 25, lesions , which iC. trachomatis and M. genitalium, the number of positive cases was insufficient to conduct comprehensive stratified analysis. Additionally, the samples collected were primarily from Changsha city, Hunan Province, which may affect the generalizability of the findings regarding the prevalence of sexually transmitted pathogens. Future research should be conducted using large longitudinal cohorts, including women from different locations, to identify and validate risk factors for HPV infection and the progression of cervical lesions, ultimately aiming to prevent cervical cancer.This study had several limitations. Firstly, it was a cross-sectional study, which cannot accurately capture the dynamic changes in HPV infection and cervical lesions among the subjects. Secondly, the sample size was small, and due to the low infection rates of M. genitalium infection among young women, while C. trachomatis infection in older women should not be overlooked. Increased levels of leukocytes in vaginal secretions may be associated with cervical lesions. In Hunan province of China, HPV16, HPV31, and HPV33 appear to have higher pathogenicity in relation to cervical lesions. The findings of this study highlight the ongoing controversy regarding whether C. trachomatis and M. genitalium infection, either alone or in co-infection with HPV, contribute to an increased severity of cervical lesions. Further clarification is needed through large longitudinal cohort studies in the future.In conclusion, our study suggests that greater attention should be given to


The prognostic impact of intrahepatic lymphatic vessel invasion (LVI) in liver cancer has rarely been reported. We sought to clarify the prognostic impact of intrahepatic lymphatic system involvement in liver cancer. Tumor-associated lymphangiogenesis reportedly correlates with prognosis after HCC resection. A meta-analysis showed that overall survival was poorer in patients with positive LVI than with negative LVI after resection of ICC and colorectal liver metastasis. Lymphangiogenesis was also reported to predict unfavorable prognosis in ICC. A few reports showed correlations between LVI/lymphangiogenesis and LNM in liver cancer. LVI and lymphangiogenesis showed worse prognostic impacts for liver cancer than their absence, but further study is needed.Although several prognosticators, such as lymph node metastasis (LNM), were reported for hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), the prognostic impact of intrahepatic lymphatic vessel invasion (LVI) in liver cancer has rarely been reported. We sought to clarify the prognostic impact of intrahepatic lymphatic system involvement in liver cancer. We systematically reviewed retrospective studies that described LVI and clinical outcomes of liver cancer and also included studies that investigated tumor-associated lymphangiogenesis. We conducted a meta-analysis using RevMan software . The prognostic impact of intrahepatic LVI in HCC was not reported previously. However, tumor-associated lymphangiogenesis reportedly correlates with prognosis after HCC resection. The prognostic impact of intrahepatic LVI was reported severally for ICC and a meta-analysis showed that overall survival was poorer in patients with positive LVI than with negative LVI after resection of ICC. Lymphangiogenesis was also reported to predict unfavorable prognosis in ICC. Regarding colorectal liver metastases, LVI was identified as a poor prognosticator in a meta-analysis. A few reports showed correlations between LVI/lymphangiogenesis and LNM in liver cancer. LVI and lymphangiogenesis showed worse prognostic impacts for liver cancer than their absence, but further study is needed. The liver produces 25–50% of total lymph fluid in the body and includes a dense network of lymphatic vessels linking to the cisterna chyli and thoracic duct ,2,3. TheLiver cancer is the fourth leading cause of cancer-related deaths worldwide . In 2015One of the major concerns regarding intrahepatic LVI in liver cancer is the increasing incidence of LNM. The prognostic impacts of LNM such as hepatic nodes in HCC and ICC were widely reported, and a positive finding is considered a strong poor prognosticator ,49,50,51We systematically reviewed studies that provided information on both LVI and clinical outcomes in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . We alsoWe conducted a meta-analysis using RevMan software . Dichotomous outcomes are shown as risk differences and 95% confidence intervals. Heterogeneity among the included trials was evaluated using a forest plot. I-squared and chi-squared statistics were used to evaluate statistical heterogeneity ,58. A ra2 field) in several areas of highest vascular density identified in low-magnification (×25–40) views were used for assessing LVD .,79.62,79 0.0007) . Several 0.0007) ,82,83,84 0.0007) . Intratu< 0.001) .p < 0.00001 In terms of CRLM, several studies investigated the intrahepatic lymphatic system and clinical outcomes  34,35,3,37. Becap = 0.0001) [p = 0.010) [Schoppmann et al. reported significant correlations between LVI and lymphangiogenesis at both primary colorectal sites and liver metastatic sites . LVD was 0.0001) . They co 0.0001) . They al 0.0001) ,87,88,89= 0.010) . High ex= 0.010) .p = 0.039) [Two main routes of cancer metastasis were reported: hematogenous spread and lymphogenous spread . The rou= 0.039) . They su= 0.039) . They te= 0.039) .p < 0.001 and 59% vs. 38%, p < 0.001, respectively) [High-level lymphangiogenesis was also associated with a significantly higher incidence of LNM than low-level lymphangiogenesis in ICC in studies by Sha et al. and Thelen et al. (ctively) . Tumor cctively) . As mentctively) . Neverthctively) . In poolctively) . With ICctively) .A large-scale study of the intrahepatic lymphatic systems in terms of liver cancer prognosis was rarely reported. However, the invasion of lymphatics may have a clinical prognostic impact as strong as that of blood vessel invasion in liver cancer, since the liver manages almost half the lymph generated in the human body ,2,3,4. Fp = 0.0001) [With regard to HCC, no studies appeared to have evaluated LVI. This may be due to the scarcity of LVI itself in HCC as compared to ICC, because of the reduced invasiveness of HCC compared to ICC . However 0.0001) . Yokomor 0.0001) . Since t 0.0001) . The assSeveral reports found that microscopic serosal invasion in HCC resulted in poor prognosis ,91,92. TAlthough LVI is easy to consider as the main cause of LNM, the correlations between LVI and LNM were not clarified in HCC. This is largely attributable to the low incidence of LNM in HCC, at 0–7.45% in operable cases ,103,104.2 = 66%), LVI (+) showed significantly poorer 5-year OS than that of LVI (−)  showed significantly poor 5-year OS in LVI (+) patients than that of LVI (−) in CRLM patients (p < 0.00001, Regarding CRLM, two routes of metastasis are known: the lymphatic route, such as secondary to initial colonization of regional lymph nodes, and direct hematogenous spread from the primary tumor, which is independent of lymph node metastatic growth ,114. FirThis study showed several limitations. First, the number of patients was insufficient. Second, the accuracy of LVI determinations may have been lacking, as not all the included studies explained the details of the pathological diagnosis for LVI or lymphangiogenesis, such as the use of D2-40. Third, details of LVI, such as the types of lymphatic vessels involved, were also not described in the majority of papers. More precise evaluations of LVI and lymphangiogenesis are warranted in future research to develop more appropriate treatment strategies, including perioperative chemotherapy or lymphadenectomy.LVI and lymphangiogenesis have prognostic impacts for liver cancer, but the correlations with LNM remain unclear. Further large-scale studies are warranted. In other words, to reveal the association between LVI/lymphangiogenesis and LNM/prognosis, thorough pathological evaluation of LVI/lymphangiogenesis using D2-40 antibody should be performed in liver cancer as well as other gastroenterological cancer. However, the definition of LVI/lymphangiogensis needs further discussion. Nevertheless, if the prognostic role of intrahepatic lymphatic system and mechanism of lymphatic spread of carcinoma are fully investigated, the treatment strategy such as surgical procedure or perioperative chemotherapy will be better utilized.


Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the ‘starch and sucrose metabolism’, ‘biosynthesis of amino acids’ and ‘phenylpropanoid biosynthesis’ pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus. This Whole Genome Shotgun (WGS) project has been deposited at DDBJ/ENA/GenBank under accession JAHTMV000000000. The version described in this paper is version JAHTMV010000000. The WGS submission is associated with NCBI BioProject: PRJNA744025. The datasets of transcriptomic analyses used in this study are associated with NCBI BioProject: PRJNA772366. Alignments for phylogenetic analysis were submitted to TreeBASE (ID: 28495). The sequenced strain ZRL20160001-S65 was deposited at the China General Microbiological Culture Collection Center , with accession number CGMCC 14343.Agaricus sinodeliciosus as a rare, mild saline-alkali-tolerant mushroom, and may have great potential to provide a resource for meeting food needs and improving traits associated with mild saline-alkali tolerance in Agaricus. Little was known regarding the mechanisms of saline-alkali adaptation of rotting fungi. Here we sequence the genome of A. sinodeliciosus, and perform comparative genomic analysis to reveal the genome evolution of A. sinodeliciosus after a solitary evolutionary history under saline-alkali environments. Through saline and alkali tolerance tests, we illustrate the effect of mildly alkaline conditions for the growth and fruiting body formation of A. sinodeliciosus. Then combining transcriptomic analyses, we illustrate the mechanism of mildly alkaline tolerance of A. sinodeliciosus. Our research gives important clues for understanding of A. sinodeliciosus genome evolution and adaptive mechanisms under mild saline-alkali conditions.Agaricus sinodeliciosus  is a wild edible mushroom which originates from northwest China blast  estimated by Varga et al.  and trehalose 6-phosphate phosphatase (TPP) gene [EC:3.1.3.12], which are involved in trehalose synthesis from UDP-glucose, were also up-regulated at pH 8.0. Gene expression of trehalose synthase (treT) [EC:2.4.1.245], which is involved in trehalose synthesis from UDP-glucose and d-glucose, were up-regulated at pH 8.0.For the reconstructed ‘starch and sucrose metabolism’ pathway in A. sinodeliciosus has 20 amino acid biosynthetic pathways, of which lysine synthesis through the a-aminoadipate (AAA) pathway, and the AAA pathway are present only in euglenoids and higher fungi [N-acetylglutamate synthase (NAGS) [EC:2.3.1.1] involved in arginine biosynthesis, pyrroline-5-carboxylate reductase (proC) [EC:1.5.1.2] involved in proline biosynthesis, and aromatic amino acid aminotransferase I (ARO8) [EC:2.6.1.57] involved in lysine biosynthesis were additionally up-regulated at pH 8.0. The glutamine synthetase (glnA) [EC:6.3.1.2] gene involved in glutamine biosynthesis was highly expressed in A. sinodeliciosus .er fungi . A gene A. sinodeliciosus, two genes related to caffeic acid 3-O-methyltransferase (COMT) [EC:2.1.1.68], which is a key enzyme for the synthesis of phenolic acids  and monolignol  derived from caffeic acid, were down-regulated at pH 8.0. A gene related to coniferyl alcohol acyltransferase (CFAT) [EC:2.3.1.-], which is involved in chavicol and isochavicol synthesized from 4-coumaryl alcohol, and eugenol and isoeugenol synthesized from coniferyl alcohol, was also down-regulated in response to pH 8.0.In the reconstructed ‘phenylpropanoid biosynthesis’ pathway in A. sinodeliciosus is a mild saline-alkali-tolerant mushroom in nature, closest to A. bisporus in molecular phylogeny with an estimated divergence time of 8.89 Mya. A 32.37 Mb high-quality genome was assembled from a monokaryotic strain of A. sinodeliciosus, using long reads (PacBio) and rectified using accurate reads (Illumina). The genome evolution of A. sinodeliciosus was explored based on comparative genomic analyses of 44 edible and medicinal mushrooms, and of four closely related taxa.A. sinodeliciosus, we first performed saline and alkali tolerance tests. Although the composition of saline-alkali soil is complex, our study showed that pH is a key factor affecting the growth and development of A. sinodeliciosus. In practice, the mycelium of A. sinodeliciosus can grow within the pH range of 7–8 with significant growth rate differences, while only at pH 8 does it starts to form numerous ‘hyphal knots’, which are thought of as the starting points of fruiting body formation  transporters and ATP-binding cassette (ABC) transporters, and extracellular peptides and amino acids are transported into the cell by amino acid permease. These transmembrane transporters in reatment  and S1. reatment . GlucoseA. sinodeliciosus under mild alkali conditions  were enhanced to hydrolyse and synthesize triglycerides formed by glycerol and free fatty acids in  pathway ; and traA. sinodeliciosus increased the absorption and biosynthesis of soluble sugars  under mild alkali conditions  after mild alkali exposure , polyphenols , amino acids, lectins and terpenoids (such as sesquiterpene) were all increased under mild alkali conditions compared with neutral conditions (Pleurotus eryngii [A. bisporus [Lentinula edodes [A. sinodeliciosus under mild alkali conditions (A. sinodeliciosus too.In mushrooms, post-harvest lignification has been widely recognized to be a leading cause of quality deterioration in storage, because of the induction of increased toughness and leathery flesh . Post-ha eryngii , A. bispbisporus  and Lenta edodes . The phenditions , suggestA. sinodeliciosus has good prospects to provide resources for meeting food needs and improving traits associated with mild saline-alkali tolerance in Agaricus. The variation in genomic architecture among the three closely related taxa of Agaricus opens a new avenue for elucidation of genome evolution under environmental stress for Agaricus. While using a single individual it is difficult to capture all genomic and transcriptomic variation, especially in light of physiological responses to soil environmental conditions, population genetics studies on these species would be of significance to generalize the present study from a single strain of A. sinodeliciosus to the species level. Furthermore, rapidly evolved genes and hub genes under mild alkali conditions in A. sinodeliciosus can be candidate resistance genes, to further study the molecular mechanisms under mild saline-alkali soils.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


A. baumannii infections. Peptide vaccines offer an attractive and promising preventive strategy against A. baumannii.The lack of appropriate vaccines is an obstacle to the effective management of A. baumannii outer membrane protein K (OMPK) using comprehensive bioinformatics and detailed molecular docking analysis.In this study, we identified specific T cell epitopes of A. baumannii OMPK were predicted by three tools namely IEDB, SYFPEITHI, and ProPred. The predicted epitopes were shortlisted based on several analyses including prediction scoring, clustering, exclusion of human similarity, considering immunogenicity and cytokine production, and removal of toxic and/or allergen epitopes. The epitopic peptides with high prediction scores and appropriate properties containing both class-I and class-II T cell epitopes were selected. Two of these class I/II epitopic peptides were chosen for molecular docking studies and assessing their physicochemical properties as vaccine candidates.Both class-I and class-II T cell epitopes of The results showed many T-cell epitopes of OMPK that could be evaluated for possible immunogenicity. Two of these epitopes (containing both class-I and II epitopes) had high prediction scores, were predicted by several tools, attached to several HLAs, and had the best docking score. They had different physicochemical properties and were conserved among Acinetobacter species.A. baumannii OMPK high immunogenic class-I and class-II T cell epitopes and introduced two promising high immunogenic peptides as vaccine candidates. It is recommended to perform in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.We identified the The online version contains supplementary material available at 10.1186/s13104-023-06409-9. Acinetobacter baumannii, an opportunistic pathogen responsible for hospital-acquired infections, is considered a serious threat to public health [A. baumannii is responsible for approximately 20.9% of all hospital-acquired infections in Europe, the Eastern Mediterranean, and Africa [A. baumannii infections and occurs mostly in patients receiving mechanical ventilation in ICUs [A. baumannii was present in 79.9% of all cases of acquired pneumonia and ventilator-associated pneumonia (VAP) [Bacteria of the genus Acinetobacter are non-motile Gram-negative coccobacilli that are ubiquitous in nature but are mainly found in soil, water, and sewage . Acinetoc health . This bac health . In inted Africa . Hospita in ICUs . A recenia (VAP) .Acinetobacter baumannii can be spread through respiratory droplets such as respiratory secretions, sneezes, and saliva, as well as through person-to-person contact, skin picking, and contact with contaminated surfaces [A. baumannii ability to propagate easily is mainly due to three characteristics: desiccation resistance, the ability to form biofilms on nonliving surfaces, and a propensity to adhere to host cells [A. baumannii has made it difficult to control using common antibiotics. Therefore, the development of effective vaccines is an alternative means to prevent infections caused by A. baumannii [A. baumannii as a key priority for the development of new medical countermeasures, including vaccine development [surfaces . A. baumst cells . The incaumannii . As a reelopment .A. baumannii [A. baumannii infection. However, none of them have yet received approval for use in humans, and further research is still needed to develop a suitable vaccine candidate against A. baumannii [Over the past decade, research efforts have identified several promising immunomodulatory strategies that could lead to a safe and effective vaccine against aumannii . Severalaumannii .A. baumannii [A. baumannii is the presence of a dense polysaccharide capsule that shields most of the outer membrane antigens from immune recognition [A. baumannii vaccines are often composed of antigens present on the bacterial outer membrane, such as OMPA, OMP22, and OMPK [Outer membrane proteinsaumannii . One of ognition . OMPs arognition , 14. Theand OMPK .A. baumannii OMP that is specific for the nucleoside-forming ion channels. This protein is equivalent to OMPK in the fish pathogen Vibrio harveyi [A. baumannii OMPK has significant immunogenic properties and is considered a promising vaccine candidate [A. baumannii, although no accurate information about OMPK epitopes vaccine potential can be found in the literature.Outer membrane protein K harveyi . Previouandidate –17. BothA. baumannii-related diseases. Today, in addition to molecular approaches, computer calculations and bioinformatics studies have provided a new method for vaccine creation through in silico epitope prediction. Numerous studies, similar to the present study, have utilized bioinformatics and sometimes laboratory analysis to investigate suitable vaccine candidates for various bacteria [A. baumannii OmpK through comprehensive bioinformatics and detailed molecular docking analysis, based on cell-dependent immune responses. In addition, this study introduces two T-cell epitopic peptides derived from OMPK, which exhibit suitable immunological and physicochemical properties, as potential A. baumannii vaccine candidates.The development of epitopic (peptide) vaccines could offer an attractive and viable treatment option for bacteria –23. The A. baumannii OMPK was retrieved from the protein database of NCBI .The sequence of http://www.allelefrequencies.net/), the prediction servers, or using our previous experiences on K. pneumoniae antigens [The most frequent human leukocyte antigenantigens –26 (Addihttp://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm), IEDB (http://tools.iedb.org), and ProPred (http://crdd.osdd.net/raghava/propred/). For the prediction of class-I and class-II epitopes, the percentile ranks of ≤ 1 and ≤ 10, respectively, were selected as the thresholds in IEDB. In ProPred-I and SYFPEITHI servers, the score of ≥ 10 was used as the threshold, whereas in ProPred-II all the predicted epitopes were chosen. Epitopes of 9 and 15 residues were chosen for the prediction of class-I and class-II epitopes, respectively.Similar to our previous studies , 27, thehttp://tools.iedb.org/cluster/), and an epitope of each cluster was selected for advanced analyses based on its higher prediction score compared to the others. The immunogenicity of class-I epitopes was predicted by a specific IEDB tool (http://tools.iedb.org/immunogenicity/). The potency of class-II epitopes in interferon-gammahttp://crdd.osdd.net/raghava/ifnepitope/).Regarding ≥ 70 similarities, the predicted epitopes were clustered with the IEDB clustering tool . The percentage of human similarity was estimated by two indicators of coverage and identity in the BLASTP server using the human proteome (taxid 9606) as the reference organism. The ≥ 90% similar epitopes to human proteome were excluded from further analyses. The AllerCatPro [The T cell epitopes were assessed for possible toxicity using the ToxinPred server .Epitopes were searched for any experimental record using the IEDB home page  used as a template.The structure of the OMPK protein was generated using the SWISS-MODEL server , with tThe PEP-FOLD server  was applhttps://saves.mbi.ucla.edu/, [https://prosa.services.came.sbg.ac.at/prosa.php, [http://molprobity.biochem.duke.edu/, [The accuracy of the protein structure model was evaluated using ERRAT , the Z-osa.php, ), and thke.edu/, ).http://haddock.science.uu.nl/services/HADDOCK2.2). Consensus prediction of interface residues in transient complexes (CPORT) (https://milou.science.uu.nl/services/CPORT/) was applied to accurate prediction of amino acid residues incorporated in the binding [For binding of class-I and -II epitopes to their HLA alleles, molecular docking studies were performed by HADDOCK 2.2 tool ( binding . Dockinghttps://web.expasy.org/protparam/) [https://pepcalc.com/) and the epitopes’ hydrophobicity was predicted by PEPTIDE 2.0 (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php). Epitope conservancy was checked via BLASTP and NCBI conserved domain search.The ProtParam tool (tparam/)  was usedhttp://webclu.bio.wzw.tum.de/cgi-bin/stride/stridecgi.py). The structures and location of the epitopes were depicted by the Web3DMol server (http://web3dmol.net/). The simplified molecular-input line-entry systemhttps://www.novoprolabs.com/tools/convert-peptide-to-smiles-string) and used to depict 2D structures of the peptides.The secondary structure prediction was done by the Stride Web interface  web servers.Antigenicity prediction of the final epitopes was carried out by the Vaxijen v2.0 , and ANT cell epitopes were predicted and several analyses were conducted to screen the predicted epitopes as follows: (1) excluding duplicate epitopes and choosing one epitope from an epitope cluster, (2) choosing high-scored epitopes and/or the epitopes predicted by more prediction tools, (3) weighing positivity in IFN-γ production and higher immunogenicity score for class-II and class-I epitopes, respectively, as preferred criteria of epitope selection, and (4) excluding allergens, toxic epitopes, and the epitopes with human similarity of 90% or more. By employing these analyses, the dominant class-I and class-II epitopes were chosen, and also their class II/I windows were found by comparing both classes with each other.The number of initially predicted class-I and class-II epitopes were 608, and 441, respectively, which were reduced to 92 and 62 following clustering and 22 and 27 after screening strategies  were modeled. The overall quality factor for Ep1, Ep2, and OMPK, as determined by ERRAT, was 79.21, 73.13, and 89.67, respectively. The Ramachandran plot showed that most amino acid residues in all three molecules  were located in the allowed and/or favored regions  and HLA molecules (receptors), ligand-receptor docking was performed by the HADDOCK tool. The docking scores of our class I epitope-HLA and class II epitope-HLA were higher than the docking scores of HLA combined with their original peptides (controls), showing the high affinity of our epitopes to their specific HLA molecules Table .E. coli, in vivo. The peptide is poorly water-soluble, but it is stable with a net charge of zero. The Ep2 (GKHISPDTRLYLGIE) peptide  had 33.33% hydrophobicity and a pI of 6.75. Its estimated half-life was > 30 h in mammalian reticulocytes in vitro and > 10 min in E. coli, in vivo. The peptide is good water-soluble, but it is unstable with a net charge of 0.1.Table Both Ep1 and Ep2 peptides were predicted to be antigens by the Vaxijen server, while neither of them was predicted to be an antigen by the ANTIGENpro server Table .Klebsiella pneumoniae.Both peptides are mainly composed of turns and they were both located on the lateral surfaces of the OMPK structure Fig. . The pepA. baumannii infections through both cell-mediated and humoral immunity. OMPK was able to trigger both humoral (IgG antibodies) and cytokines responses, which provided defense against a fatal challenge with A. baumannii [Previous research on OMPs has shown that these proteins can trigger defense against aumannii , 16, 38.aumannii , 16. MF5aumannii –41. In aaumannii –46.A. baumannii infections likely involves significant contributions from humoral immune responses, including IgM, IgG, and IgA [A. baumannii infections are still poorly understood. According to a small number of experimental studies, A. baumannii infection severity was linked to higher levels of Th1 inflammatory cytokine responses and lower levels of IL-10, suggesting a protective effect of Th2 immune responses [A. baumannii infections are still unknown [A. baumannii vaccine candidates. They were both immunogenic, non-toxic, non-allergen, and non-similar to the human proteome. In addition, they contain both class I and II T cell epitopes that were found to bind to their HLAs with high affinity. Ep1 was predicted to be able to produce IFN-γ while Ep2 could not, but it should not be assumed as a negative criterion since as mentioned above, the role of Th1 cytokines such as IFN-γ is not clearly understood in protection against A. baumannii [Host defense against  and IgA . The T cesponses , 49. How unknown , demandiaumannii .In the present study, the modeled 3D structures underwent verification using in silico tools. The majority of amino acid residues in our modeled protein (OMPK) and its final epitopic regions (Ep1 and Ep2) were located in the allowed/favored regions of the Ramachandran plot, indicating the high quality of our models. Furthermore, we utilized the ERRAT assessment to identify any potential anomalies in the structure , which sA. baumannii [The results of the present study showed that Ep1 and Ep2 peptides have different physicochemical properties so Ep1 is stable, poor water-soluble, more hydrophobic, and with a lower estimated half-life than Ep2. Being more hydrophilic is indicated by the negative GRAVY , as seenaumannii , 52, 53.A. baumannii-related diseases. The epitopes may be chemically synthesized or cloned/expressed and utilized as radiochemical probes in designing diagnostic kits [Both epitopes identified in this study are located on the surface of the OMPK protein, making them easily accessible to antibodies. This feature, in addition to their potential as vaccine candidates, also makes them suitable candidates for the diagnosis of tic kits .K. pneumoniae, so they may induce immune responses against K. pneumoniae as well.The conservancy study of the Ep1 and Ep2 peptides showed that they do not belong to a conserved domain superfamily, but they are highly conserved among Acinetobacter species, hence it may induce immune responses against many species of this genus. Also, both peptides had 100% similarity to a sequence of ion channel protein TSX of A. baumannii vaccines has increased significantly. OMPA [A. baumannii that have been studied in silico. Collectively, the results of our study, along with previous research provide promising prospects for the development of a suitable vaccine based on OMPs to combat A. baumannii infections in the future.In recent years, the number of bioinformatics studies on ly. OMPA , 56, Dcaly. OMPA , NucAb [ly. OMPA , and Tonly. OMPA  can be mA. baumannii OMPK high immunogenic class-I and class-II T cell epitopes, which can be utilized in future research. Additionally, two high immunogenic peptides containing both T cell epitope classes as well as the necessary physicochemical properties for the development of an A. baumannii vaccine were introduced. It is recommended to do an in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.Altogether, we identified the Limited immunity due to several factors such as easy degradation and their recognition difficulty is linked to epitope-based vaccinations, such as our suggested Ep1 and Ep2 peptides. Conjugating them with conventional adjuvants and/or novel built-in adjuvants, such as new biomaterials or carriers, is one efficient technique to bypass this limitation . AnotherAdditional file 1: Table S1. The list of HLA alleles used for A. baumannii OMPK T cell epitope prediction.


Taenia solium taeniasis/cysticercosis (TSTC) is a foodborne, zoonotic neglected tropical disease affecting predominately low- and middle-income countries. Humans are definitive hosts for T. solium, whereas pigs act as intermediate hosts. Taeniasis, i.e. intestinal infection with adult T. solium in the human host, occurs through ingestion of undercooked pork infected with the larval stage . Human cysticercosis occurs after humans ingest T. solium eggs, acting as accidental intermediate hosts. Migration of cysticerci to the human brain results in neurocysticercosis (NCC), manifesting in a variety of clinical symptoms, most notably epilepsy. NCC is the leading cause of acquired epilepsy cases in endemic areas. PCC results in reduced pork value because of condemnation or the risk of condemnation of the meat. Available serological diagnostic tests for porcine and human cysticercosis are characterized by low sensitivity and are not cost-effective. An effective vaccine for T. solium cysticercosis in pigs has been developed, although it is not yet commercially available in all endemic countries, and still no vaccine is available for use in humans. This primer highlights the recent development in the field of diagnostic tests and vaccine production and explores possible strategies for future control and eradication of T. solium. In the absence of highly specific diagnostic tests and human vaccines, treatment of infected pigs and tapeworm carriers and prevention of disease transmission remain the principal means to interrupt the zoonotic cycle of T. solium in endemic countries.The online version contains supplementary material available at 10.1186/s13071-023-05989-6. T. solium involves humans as definitive hosts and pigs as intermediate hosts  through stool into the environment. Several risk factors, like poor sanitation, open-air defecation, bio-fertilizer preparation from human stool and free-roaming pigs, help to transmit T. solium eggs in new areas and contaminate surface water as well as vegetation ), 13.03% (95% CI [9.95–16.88]) and 15.68% (95% CI [10.25–23.24]), respectively [T. solium results in PCC. It is widely prevalent in sub-Saharan Africa with a meta-analysis reporting a high-pooled prevalence using available diagnostic tests  across the continent [ast Asia . Importeast Asia , though ast Asia –13. A meectively . NCCs af CI 9.95–.88) and ectively . Infectiontinent  but hypeontinent –24. TaenWhile taeniasis itself has little impact on human health despite rare sequelae, cysticercosis not only has direct effects on human and animal health but also on the socioeconomic status of the former. NCC-associated epilepsy has direct effects on human health-related expenses like diagnosis, treatment and medicine cost for patients during hospitalization. Besides, indirect effects of NCC are linked to people who either become unemployed or are unable to work because of epilepsy . PCC resT. solium transmission persists, ultimately increasing the global burden of this NTD and affecting the livelihood of the pig-raising community in endemic regions.TSTC is a focal disease, affecting the poorest communities of developing countries. In many of these communities pigs are raised in free-roaming systems Fig. , and lowT. solium infection, which has shown promising capability in recent times using recombinant antigens [As cysticercosis is mainly a problem in LMICs, diagnostic technologies not requiring specialized equipment or highly trained personnel would be advantageous. This has prompted the development of rapid detection technologies for antigens .T. solium, whose signal is then transferred to a biosensor for analysis. This technology has higher sensitivity and specificity compared with ELISA, with a sensitivity of 93.59% and 97.44% for TSOL18 and GP50, respectively, and a specificity of 100% for both antigens [A lateral flow assay-based diagnostic tool was developed using up-converting phosphor (UCP) nanoparticles to detect binding of UCP to TSOL18 (oncosphere-stage protein) and GP50  antigen of antigens . HoweverT. solium in NCC patients with a mobile phone reader. The assay specificity was 99% (95–100%) while sensitivity was 89% (79–95%) in patients with two or more viable cysts [Another study developed a point-of-care (POC) assay using quantum dot nanoparticles in a lateral flow assay format and detected rT24h antigen of le cysts .T. solium-specific) and r2B2t (Echinococcus granulosus-specific) for simultaneous and differential diagnosis of NCC and cystic echinococcosis. For the diagnosis of NCC, the sensitivity and specificity of this assay ranged from 57.94 to 63.49% and 90.87–91.30%, respectively [Multiplex Bead Assay is one of the recombinant antigen-based diagnostic tools, which can estimate the intensity of the antibody response and allow direct comparison of antibody levels between samples. In one study, this assay was used for evaluating two recombinant antigens, rT24H  is a DNA-based isothermal PCR technique, which has emerged as a good diagnostic tool in the last decade for the detection of different taeniids at species level. A multiplex LAMP (mLAMP) assay uses mitochondrial cytochrome oxidase subunit 1 (COX1) marker in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). This assay was successfully used to identify Taenia species from genomic and copro-DNA samples with 100% specificity, making it the first field-based test for sensitive and specific identification of human Taenia species [T. solium in endemic countries [T. solium sequences. In the T. solium genome, TsolR13 sequence has shown high sensitivity (97.3%) and specificity (100%) for diagnosis of NCC [Taenia solium, T. saginata and T. asiatica in human stool. This study targeted the internal transcribed spacer I (ITS-1) gene of Taenia solium and COX1 gene of T. saginata and T. asiatica. For the diagnosis of taeniasis, this study showed 94% sensitivity and 98% specificity [DNA-based diagnostic methods have been developed to identify  species . Lyophilountries , 36. Recs of NCC . Anothercificity .T. solium antigens have been explored for their suitability, for instance, T. solium scolex protein antigen [T. solium paramyosin, a muscle and tegument protein [T. solium. This combined therapy increases the protection level up to 99.7% of all animals exposed to subsequent infection and results in significant reduction in prevalence of PCC in endemic countries [T. solium SP3VAC  and a modified parenterally administered SP3Vac-phage version have undergone trials in Mexico [Vaccination of pigs is an efficient method to reduce human exposure to infected pork, which ultimately reduces both taeniasis and cysticercosis. For pig vaccine development, different  antigen ; TSOL16, antigen , 41; T.  protein . Among t protein . TSOL18  protein . The vac protein  and Nepa protein  evaluateountries –50. Anotn Mexico . Whilst n Mexico .T. solium to date has primarily focused on identifying candidate vaccines for pig to control PCC in endemic countries. Animal vaccines are comparatively easier and cheaper to develop than human vaccines. Therefore, until now human vaccination has not been considered an effective control strategy for TSTC in humans [Taenia solium calreticulin, a tegument protein of the parasite, is essential for embryogenesis, oogenesis and spermiogenesis. This protein has been explored as a candidate vaccine target, and several studies showed reduced worm burden in an experimental hamster model of taeniasis [T. solium showed only 45% reduction of parasite load against an intraperitoneal challenge with T. crassiceps cysts in a murine model of cysticercosis [T. solium still need to be explored to identify a reliable and affordable vaccine for human use. While there is currently little willingness to pay by farmers for the highly effective TSOL18 vaccine for pig, the development and incorporation of a human vaccine into childhood vaccination schedules may be an option to significantly reduce infection in exposed populations.Most vaccine research work on n humans . Taenia aeniasis –53. In acercosis . More caT. solium. Among four TPPs, two were classified for taeniasis-specific test and POC test development, one for NCC POC test development and one for porcine cysticercosis-specific diagnostic test development [Taenia species or other parasites and not show any positive response in the absence of viable cysts. In addition, the cost of this test should be 0.5–2 USD per test with 24–36 month stability at 2–40 °C temperature [The available diagnostic tests are still not fully suitable for sensitive and specific field level diagnosis of TSTC. To help overcome this gap, WHO defined four new target product profiles (TPPs) for researchers, diagnostic developers and manufacturers to develop effective and useful diagnostic tests for elopment . TPPs maperature .T. solium infections in both humans and animals. Moreover, specific methods and diagnostic tools are still unavailable for inspecting vegetables or other possible transmission vehicles  for T. solium eggs [The current reference methods for PCC and NCC are the full carcass muscle/brain dissection and imaging techniques, which are not feasible economically in developing countries. There is no dry LAMP format for the detection of ium eggs .T. solium taeniasis and cysticercosis in endemic countries, the WHO aims to scale up the intervention methods used to control T. solium. WHO projected that 27% of endemic countries will achieve intensified T. solium control in hyperendemic areas by 2030 [T. solium, including risk factor identification in both host and environment. In endemic countries, spatial distribution and risk factor analysis for human and porcine cysticercosis is one of the best options to unravel the transmission patterns of T. solium. A new agent-based model, CystiAgent, has created a model for analysing spatial and behavioural features of T. solium transmission in northern Peru. This model has the ability to represent key spatial and environmental features of transmission and simulate spatially targeted interventions, such as ring strategy [T. solium (EPICYST) has also been developed [ex ante assessment of control options for T. solium but maximising the utility of these models will require continued collaborative efforts between modelling teams to improve and harmonise models and model parameters [T. solium, a novel metric, zoonotic disability-adjusted life years (zDALY), may be more appropriate [T. solium control milestones [T. solium prevalence data and the PCC risk factors to obtain a complete insight into the T. solium landscape and subnational variation of indicators [T. solium biology with identification of environmental dynamics of eggs and risk factors analysis of pig-to-people and environment-to-pig transmission will be paramount to achieving WHO 2030 targets.To tackle  by 2030 . This tastrategy . Anotherstrategy . A detereveloped . These mrameters , 60. As ropriate . zDALYs ropriate . Approprlestones . A recendicators . Better Taken together, although eradication of TSTC appears possible in principle, it is still far from being achieved. Some very effective components, such as combined treatment/vaccination with oxfendazole and TSOL18, are available, but there are still economic and sociocultural factors hindering their implementation in many endemic countries, and affordable, effective diagnostic technologies still lag behind. In the meantime, an achievable, immediate objective will be to implement highly targeted, small-scale control programmes in the most impacted areas by taking advantage of the improved new mapping tools combined with better transmission models.Additional file 1: Biological characteristics and life cycle of Taenia solium. Humans harbor adult T. solium parasites in their small intestine. Adult parasites are hermaphrodites and release fertilised eggs or egg-containing mature proglottids, each containing up to 50,000–100,000 eggs, which are excreted with faecal material, contaminating the environment. Pigs are infected by oral ingestion of this faecal material or contaminated vegetation or feed. Upon ingestion, eggs hatch and oncospheres penetrate the gut wall, migrating to the musculature where they encyst, leading to pig cysticercosis (PCC). Human ingestion of undercooked or raw contaminated pork leads to the release of larval stages in the GI tract with subsequent development to an adult tapeworm (taeniasis). Accidental ingestion of mature eggs leads to human cysticercosis (HCC) and neurocysticercosis (NCC). Pigs do not harbor adult parasites; thus, infection is spread from humans to animals (anthropozoonosis). Created with BioRender.com.


Pudu puda), have been reported as hosts for novel pathogens such as Mycoplasma ovis-like and a novel ecotype of Anaplasma phagocytophilum. A better understanding of the epidemiology of this group and other intracellular bacteria that might have cervids as hosts would enlighten their population relevance. This study aimed to determine the occurrence and genetic diversity of Bartonella spp., hemotropic mycoplasmas, and Coxiella burnetii in pudus from Chile.Recent evidence shows a high diversity of infectious agents in wildlife that represent a threat to human, domestic, and wild animal health. In Chile, wild populations of the most common cervid species, pudu (Bartonella and IS1111 element for C. burnetii) and conventional PCR  was used for pathogen screening and molecular characterization.The DNA was extracted from the blood samples of 69 wild free-ranging and 30 captive pudus from Chile. A combination of real-time (nouG gene for Bartonella spp. was detected in 10.1% [95% CI (5.2–18.2%)] samples, hemotropic Mycoplasma spp. in 1.7% [95% CI (0.08–10.1%)], and C. burnetii in 1.0% [95% CI (0.05–6.3%)] samples. Two sequenced samples were identified as Mycoplasma ovis-like, and one free-ranging pudu was positive for C. burnetii. While one captive and two free-ranging pudus were positive for Bartonella henselae, one wild pudu was co-positive for B. henselae and Bartonella sp., similar to Bartonellae identified in ruminants.DNA of B. henselae in wild ungulate species, and C. burnetii and Bartonella spp. in wild ungulate species in South America. Further research will be necessary to evaluate the potential role of pudu as reservoirs of infection and identify the sources for disease transmission among humans and wild and domestic animals.To the best of our knowledge, this is the first report of  The recognition of the role of wildlife as reservoirs of pathogens that threaten the health of humans and/or livestock species has increased in the past several decades; accordingly, the relevance of infectious agents in the wildlife conservation field has also amplified , 2. As eBartonella spp., Coxiella burnetii, and hemotropic Mycoplasma spp. are intracellular bacteria that infect a wide range of animals , and the Centers for Disease Control and Prevention (CDC) has classified this microorganism as a potential bioterrorism agent , and alpacas (Vicugna pacos) (Pudu puda) has been identified as the potential host species of several hemoplasmas  . Finallyoplasmas , includiBartonella spp., hemotropic mycoplasmas, and C. burnetii in free-ranging and captive pudus from Chile.Pudu is the most common cervid in Argentina and Chile and is considered threatened in both countries , 33, as Blood samples from frozen banks in rescue centers and zoos/breeding centers were used. The frozen bank samples were opportunistically collected from 69 free-ranging pudus between 2016 and 2022 on admission day from two wildlife rehabilitation centers in the template forest ecosystem of southern Chile in Los Lagos District, one  located in the continental area and the other (Ch. S: Chiloe Silvestre NGO) in Chiloe island. Additionally, blood samples from 30 captive pudus were collected between 2017 and 2021 during preventive medicine procedures in two facilities, one located in the Mediterranean ecosystem of Central Chile in the Metropolitan District and the other in Los Lagos District, and do not have contact between centers. Blood samples were obtained by venipuncture of the jugular vein using an evacuated tube system  and stored at −20°C within 6 h of collection. For extensive sampling details, refer to the study mentioned in the reference .®, Norcross, GA, U.S.A.), according to the manufacturer's instructions, to obtain a concentration between 20 and 50 ng/μl of purified DNA. Concentration and purity of DNA were measured . The 260/280 nm absorbance ratio (OD260/OD280) provided an estimate of sample purity, accepting a ratio of 1.8 ± 0.2 as pure. DNA was stored at −20°C before performing PCR assays.The 99 frozen EDTA-blood samples were thawed at room temperature and vortexed at the UACh Veterinary Clinical Pathology Laboratory, Valdivia, Chile. DNA extraction from 200 μl of blood was performed using an E.Z.N.Z. Tissue DNA Kit (E.Z.N.A. Omega BioTekDNA samples were subjected to qPCR targeting the irbp gene (interphotoreceptor retinoid-binding protein) using the primers IRBP-CF_FWD (5′-TCCAACACCACCACTGAGATCTGGAC-3′) and IRBP-CF-REV (5′-GTGAGGAAGAAATCGGACTGGCC-3′), with the aim to check DNA template integrity and discard the presence of PCR inhibitors, as previously described . All cPCBartonella spp., the DNA of all irbp cPCR-positive samples were subsequently subjected to an initial screening by quantitative real-time PCR (qPCR) targeting the nuoG gene of Bartonella spp. (83 bp), using primers  as previously described , encoding the nuoG B. henselae sequence (insert containing 83 bp). Bartonella henselae genomic DNA from a cat tested in a previous study was used as a positive control  as a negative control. Replicates showing a Cq difference higher than 0.5 were retested.To detect and quantify escribed . qPCR am control . All PCRBartonella spp. nuoG-qPCR positive samples were subjected to cPCR amplification of a fragment of three loci . Only three samples had consistent Cq, and the quantification of Bartonella spp. was 18.5 ± 14.02 nuoG-copies/μl .Molecular occurrence of Bartonella spp. gltA, ITS, and rpoB genes were deposited in GenBank  was deposited in GenBank and used for phylogenetic analysis.Representative sequences of  GenBank  under thBartonella spp. DNA was successfully amplified by cPCR in 60% (6/10) of qPCR-positive samples, and six sequences were obtained  from four pudus [one captive  and three free-ranging ]. The rpoB sequences were 100% similar to each other and showed 98.2% similarity with B. henselae from cats in Brazil (MN107418), 99.7% identity with B. henselae from a cat from Paraguay (MW514669), and 100% identity with B. henselae from Urva auropunctata from St. Kitts (MW728257). The gltA sequence showed 95.05% identity with uncultured Bartonella sp. from a cattle tail louse from Israel (KJ522487), and the ITS sequence showed 93.1% identity with Bartonella sp. from deer ked (DQ485307). As such, wild pudu #6235 was co-positive to B. henselae and Bartonella sp., similar to Bartonellae identified in ruminants.rpoB sequences of the present study were allocated in the same taxa, sharing a clade with B. henselae Houston 1 (AF171070), B. henselae from a cat from Paraguay (MW514660), and B. henselae from Urva auropunctata from St. Kitts and Nevis (MW728257) (The W728257) . The rpogltA phylogenetic reconstruction evidenced that the sequence of the present study was allocated to the same clade with Bartonella sp. from a Cervus from Japan (CP019781), Bartonella sp. from a cattle tail louse from Israel (KJ522487), and B. capreoli, B. schoenbuchensis, and B. chomeli (The  chomeli .Bartonella sp. sequence from a deer-ked (DQ485307), B. schoenbuchensis , B. chomeli (KM215718), B. melophagi (JF834886), and B. bovis   .C. burnetii DNA in pudu detected by qPCR  was 1.0% (1/99) [95% CI (0.05–6.3%)].Molecular occurrence of Mycoplasma spp. in pudu by cPCR was 1.7% (1/60) [95% CI (0.08–10.1%)]. The sequence of the 16S rRNA fragment showed 100% BLASTn identity with Mycoplasma ovis-like amplified previously from Chilean pudu (MW532816) (Molecular occurrence of hemotropic W532816) .B. henselae in a wild ungulate species and Bartonella spp. and C. burnetii in wild ungulate species in South America. The circulation of Mycoplasma ovis-like in free-ranging pudu in Chile is also confirmed  plays a role in the sylvatic cycle of Q fever (C. burnetii DNA in blood samples of wild boar (Sus scrofa), marsh deer (Blastocerus dichotomus), brown brocket deer (Mazama gouazoubira), small red brocket deer (Mazama bororo), red brocket deer (Mazama americana), and pampas deer (Ozotocerus bezoarticus) (C. burnetii by an indirect immunofluorescence assay (IFA) for IgG antibodies (anti-phase I); to date, it is the only evidence of exposure to this pathogen in deer in the region.utbreaks . The liv America , includi America . In the  Q fever . In Soutarticus) , 68. A rarticus)  found thC. burnetii have been reported in samples of animal origin only in bats and bulk tank milk from cows  in the southern macrozone found no evidence of C. burnetii infection  as the main vector  (B. melophagi (JF834886)  , B. meloLycalopex fulvipes) present a high prevalence of M. haemocanis causing enzootic and asymptomatic infections (Mycoplasma spp. have been recently reported in llamas (12.8%) and alpacas (6.3%) (Candidatus Mycoplasma haemolamae) (Mycoplasma ovis-like in free-living pudu in southern Chile (14%)  and exotic ungulates  in the Los Lagos District is recommended to understand the epidemiology of these infectious agents and the possible role of pudu as a host. It is also recommended to evaluate the pathogenicity of Mycoplasma ovis-like in pudu.Hemoplasma bacteria have been extensively studied in wild and domestic carnivores in Chile during the last decade. Darwin foxes   and Mycole (14%) . MoleculB. henselae and C. burnetii, both zoonotic pathogens, could be relevant to public health. Both B. henselae and C. burnetii are pathogens related to occupational diseases, with evidence of health risks for those working with infected species through occupational exposure in rehabilitation centers, breeding centers, and zoological parks  can be found below: Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because we only use samples from Frozen Banks of the rehabilitation centers and zoos. We don't have involved in any managements animals procedure.EH-H: conceptualization, supervision, funding acquisition, investigation, resources, data curation, writing—original draft preparation, and writing—reviewing and editing. PS-G: methodology, investigation, resources, data curation, writing—original draft preparation, and writing—reviewing and editing. JC, CV, FV, and IK: resources and data curation. SC and CO: resources. DM-A: writing—original draft preparation, and writing—reviewing and editing. MO and NC: methodology and investigation. AG: investigation and writing—reviewing and editing. AM: conceptualization, supervision, funding acquisition, methodology, investigation, writing—original draft preparation, and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.